Veterinary Clinical Pathology ISSN 0275-6382
ORIGINAL RESEARCH
Determination of hematology and plasma chemistry reference
intervals for 3 populations of captive Atlantic sturgeon
(Acipenser oxyrinchus oxyrinchus)
Mark A. Matsche1, Jill Arnold2, Erin Jenkins2, Howard Townsend1, Kevin Rosemary1
1
Cooperative Oxford Laboratory, Oxford, MD, USA; and 2National Aquarium, Baltimore, MD, USA
Key Words
Blood values, chondrostei, fish,
hemacytometer
Correspondence
Mark A. Matsche, Cooperative Oxford
Laboratory, 904 South Morris Street, Oxford,
MD 21654, USA
E-mail: mmatsche@dnr.state.md.us
DOI:10.1111/vcp.12174
Introduction
The Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus),
class Osteichthes, is a large, long-lived, anadromous
fish found along the Atlantic coast of North America.
Adults migrate up rivers to spawn in freshwater.1 After
hatching, juveniles move downstream and may reside
within the river or estuaries for one to 6 years
before emigrating into coastal waters.2 Adult fish
spend the majority of their lives in coastal waters
where they may migrate over considerable distances,
but will return to their natal rivers to spawn.1
A major fishery for sturgeon along the Atlantic
coast of North America followed the establishment of a
Vet Clin Pathol 43/3 (2014) 387–396 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
Background: The imperiled status of Atlantic sturgeon (Acipenser oxyrinchus
oxyrinchus), a large, long-lived, anadromous fish found along the Atlantic
coast of North America, has prompted efforts at captive propagation for
research and stock enhancement.
Objective: The purpose of this study was to establish hematology and
plasma chemistry reference intervals of captive Atlantic sturgeon main-
tained under different culture conditions.
Methods: Blood specimens were collected from a total of 119 fish at
3 hatcheries: Lamar, PA (n = 36, ages 10–14 years); Chalk Point, MD
(n = 40, siblings of Lamar); and Horn Point, Cambridge, MD (n = 43,
mixed population from Chesapeake Bay). Reference intervals (using robust
techniques), median, mean, and standard deviations were determined for
WBC, RBC, thrombocytes, PCV, HGB, MCV, MCH, MCHC, and absolute
counts for lymphocytes (L), neutrophils (N), monocytes, and eosinophils.
Chemistry analytes included concentrations of total proteins, albumin, glu-
cose, urea, calcium, phosphate, sodium, potassium, chloride, and globulins,
AST, CK, and LDH activities, and osmolality.
Results: Mean concentrations of total proteins, albumin, and glucose were
at or below the analytic range. Statistical comparisons showed significant
differences among hatcheries for each remaining plasma chemistry analyte
and for PCV, RBC, MCHC, MCH, eosinophil and monocyte counts, and N:L
ratio throughout all 3 groups. Therefore, reference intervals were calcu-
lated separately for each population.
Conclusions: Reference intervals for fish maintained under differing con-
ditions should be established per population.
caviar market in 1870, but by the turn of the century,
the fishery had largely collapsed.3 While only a rem-
nant fishery continued along parts of the Atlantic
coast, the Atlantic sturgeon populations remained
depressed. A number of factors, such as pollution and
dam construction blocking access to upstream spawn-
ing habitat, are likely to have contributed to the
decline of this species.1 In 1998, a coast-wide morato-
rium on Atlantic sturgeon fisheries was imposed by the
Atlantic States Marine Fisheries Commission and, in
2012, the mid-Atlantic and other population segments
of this species were listed as endangered by the
National Oceanic and Atmospheric Administration.4
As a result of the imperiled status of Atlantic sturgeon,
387
Atlantic sturgeon hematology and plasma chemistry
a number of restoration strategies have been proposed
including protection and restoration of critical habitat1
and reintroduction or stock enhancement with cul-
tured fish.5,6
Culture techniques have been developed and con-
tinue to be refined for Atlantic sturgeon, but little work
has been done to evaluate the health of captive fish.7
Successful maintenance of captive animal populations
often requires that individuals undergo health assess-
ments for the determination of the physiologic condi-
tion and detection of disease. Changes in hematologic
and plasma biochemistry variables can be an important
indicator of changes in health status, but are not com-
monly used in fish because species-specific reference
intervals (RI) may not be available. Also, a wide vari-
ety of biologic and environmental factors can affect RI
including temperature, age, and culture conditions.8–14
In recent years, there has been increased interest
in determining normal blood values from sturgeon for
reference purposes.15–21 The Clinical and Laboratory
Standards Institute (CLSI) recommends establishing RI
using the nonparametric ranking method with at least
120 subjects.22 Achieving such a sample size, however,
is often not feasible with large, endangered fish species
such as Atlantic sturgeon. Robust techniques are rec-
ommended when sample size is ≥ 40 and < 120 sub-
jects.22 The proximity of 3 area hatcheries provided a
sufficient number of test subjects for RI determination
using nonparametric techniques, but the fish at each
facility were maintained under different husbandry
conditions. The primary goal of this study was to deter-
mine hematologic and biochemical RI for healthy, sub-
adult Atlantic sturgeon maintained in captivity. The
variability of blood values among the 3 populations
was evaluated to determine if data could be pooled for
nonparametric analysis, or if separate RI were appro-
priate for each population.
Materials and Methods
Specimen collection
Atlantic sturgeon used in this study consisted of F1
progeny of wild Hudson River broodstock
Table 1. Water temperature, salinity, and body weights of Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus, at the time of blood collection. Salinity
and temperature are presented as actual values measured on the day of sampling with the minimum and maximum (min, max) values observed over
30 days prior to sampling. Fish weights are presented as mean  standard deviation (SD). Values with different superscripts are significantly different
(ANOVA F = 60.39, P < .0001).
Fish Hatchery n Confinement
Horn point 43 Outdoor earthen ponds
Lamar 36 Concrete raceway
Chalk point 40 Indoor fiberglass tanks
388 Vet Clin Pathol 43/3 (2014) 387–396 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
Matsche et al.
(1994–1998 year classes) produced by the United
States Fish and Wildlife Service, Northeast Fishery
Center, Lamar, Pennsylvania (Lamar). Fingerlings
were maintained both at Lamar and at the GenOn
Energy Chalk Point Aquaculture Facility, Aquasco,
MD, USA (Chalk Point). At Lamar, fish were
housed in outdoor, covered, concrete raceways
(30 9 2.5 m), containing spring water (average hard-
ness = 70 mg/L) that was partially recirculated and
oxygenated by injection of O2. At Chalk Point, fish
were maintained in indoor, fiberglass tanks (6 m
diameter) containing ambient Patuxent River water
using a flow-through system and aeration. A third
population of Atlantic sturgeon used in this study
consisted of fish captured from the Chesapeake Bay
between 2001 and 2006 and maintained at the Uni-
versity of Maryland Center of Environmental Science,
Horn Point Laboratory, Cambridge, MD, USA (Horn
Point). Fish consisted of sexually immature subadults
and were maintained at Horn Point in earthen ponds
containing aerated Choptank River water. All fish
were fed daily with a commercial pelleted diet
consisting of 45–50% protein, 15–25% fat, 1–2%
fiber, and 8–9% ash (Nelson’s and Sons, Murray, UT,
USA [Horn Point] or Zeigler Brothers, Gardners, PA,
USA [Lamar and Chalk Point]). For a listing of the
water temperatures and salinity conditions in the 3
study environments please consult Table 1.
This study was conducted on 3 days over a 5-day
period at the end of October, 2008. Fish were not fed
on sampling days. The animals were handled in accor-
dance with the established policies of the University of
Maryland and the US Fish and Wildlife Service. A total
of 119 fish were restrained individually on a rubber
stretcher with no anesthetic. Blood was collected from
the caudal vessels immediately posterior to the anal fin
using a 5-mL syringe with a 20-Gauge, 1-inch needle.
Blood was immediately transferred to 0.6-mL heparin-
ized tubes (Becton-Dickinson, Franklin Lakes, NJ,
USA) and placed in a cooler for transport to the on-site
laboratory at each field station. Total handling time of
fish to obtain blood specimens was typically < 1 min.
Following phlebotomy, fish were weighed and
released.
Water Source Salinity (ppt) Temperature (°C) Fish Weight (Kg)
Choptank river 13.0 (11, 14) 11.0 (11, 21) 8.3  5.5a
Spring 0 8.5 (8, 11) 11.2  3.7b
Patuxent river 9.0 (8.5, 13) 12.5 (11, 24) 19.8  5.9c
Matsche et al.
Specimen processing and analysis
Blood specimens were processed for determination of
PCV, HGB concentration, manual cell counts, and
preparation and staining of blood smears for WBC dif-
ferential counts no later than 1 h following collection.
The PCV was determined by microcentrifugation (IEC
Micro-MB; Thermo Fisher Scientific, Waltham, MA,
USA) of 2 tubes for 5 min at 13,700 rpm (12,162g).
HGB concentration was determined using a spectro-
photometer (Hb 201+; HemoCue Inc, Lake Forest, CA,
USA). Total RBC and WBC counts were determined by
loading 1:100 dilution of whole blood in Natt–Herrick’s
diluent23 into an improved Neubauer hemacytometer
(Brightline, Hausser Scientific, Horsham, PA, USA)
and enumerating cells according to standard formulas
for total RBC, WBC, and thrombocytes.24 Four blood
smears were prepared, air-dried, and 2 smears were
stained with PROTOCOL HEMA-3 (Fisher Scientific,
Fairlawn, NJ, USA) following manufacturer’s instruc-
tions. Leukocytes were identified per cell descriptions
published for the Shortnose sturgeon (Acipenser breviro-
strum),18 and included small lymphocytes (< 10 lm),
large lymphocytes (≥ 10 lm), neutrophils, monocytes,
and eosinophils. A thrombocyte-like cell described ear-
lier in the Shortnose sturgeon was rarely observed in
this study, and therefore this cell type was not
included. A 400-cell WBC differential count was per-
formed for each blood film by the authors (1,0009
magnification). The absolute value of each cell type
was calculated by standard methods. Neutrophil:lym-
phocyte ratios (N:L ratio) were calculated using the
sum of small and large lymphocytes.
Also within 1 h of collection, heparinized blood
was centrifuged at 3,500 9 g for 5 min and an ali-
quot of collected plasma was frozen at 20°C for
osmolality determination (performed within one
month of collection) using a vapor pressure osmom-
eter (Vapro 5520, Wescor Inc., Logan, UT, USA).
Daily quality control was performed for PCV and
HGB concentration (tri-level control set, R&D Sys-
tems Inc., Minneapolis, MN, USA), and osmolality
(Wescor 100, 290, and 1,000 mmol/kg standards).
There are no commercial products for quality control
for the WBC, RBC, or thrombocyte manual counts
for fish; for consistency, 3 analysts were used, where
one analyst was assigned to count one cell type for
all 119 specimens. At least 3 analysts performed the
WBC differential counts; all counts between analysts
agreed within a 95% confidence interval (CI).25 The
remainder of the heparinized plasma (0.6 mL), after
removal of the aliquot for osmolality analysis, was
submitted in the gel-separator tube to a commercial
Vet Clin Pathol 43/3 (2014) 387–396 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
Atlantic sturgeon hematology and plasma chemistry
veterinary laboratory (Antech Diagnostics, Inc., Lake
Success, NY, USA) for clinical chemistry analysis
(AU-5400 automated chemistry analyzer, Olympus
America, Inc., Center Valley, PA, USA).
Test methods performed by the chemistry ana-
lyzer are provided in Table S1 with the coefficients of
variation (CV) from the manufacturer’s precision data
at the mean closest to that found in the 3 sturgeon
populations. In addition, Antech Diagnostics performs
quality control testing on analyzers after every 200–
400 patients as well as before and after each shift.
During the year of this study, Antech performed vali-
dations from May–June, 2008 where studies complied
with manufacturer’s performance specifications on all
assays.
Statistical analysis
For each analyte, data were graphically plotted and
examined, and tested for outliers using the Dixon
Q test.26 Robust procedures were used to calculate
95% RI for hematologic and plasma chemistry val-
ues27, and 90% CI were calculated for each lower and
upper estimate.22 Values detected as outliers were
included in the robust calculations. The Kruskal–Wal-
lis test followed by sequential Bonferroni correction28
was used to compare hematologic and plasma chemis-
try analytes among the fish hatcheries.
One-way ANOVA and subsequent Bonferroni
pairwise comparisons were used to test for differences
in weight of Atlantic sturgeon among Horn Point,
Lamar, and Chalk Point. Differences were considered
significant when P < .05. Reference intervals and
ANOVA were determined using SAS software (v.9.2 or
Enterprise Guide 4.1).29
Results
Mean weights of Atlantic sturgeon used in this study
from Horn Point, Lamar, and Chalk Point were sig-
nificantly different (Table 1). Significant differences
were detected among hatcheries for PCV, RBC
count, MCHC, MCH, counts of eosinophils and
monocytes, N:L ratios, and each plasma chemistry
analyte. Because of the numerous significant differ-
ences in blood variables, robust RI were estimated
separately for each hatchery population. Variability
in hematologic RI among the fish populations was
highest for eosinophil and monocyte counts
(Table 2). Blood cells observed for Atlantic sturgeon
in this study were comparable in size and morpho-
logic appearance as described earlier for Shortnose
389
Atlantic sturgeon hematology and plasma chemistry Matsche et al.

Table 2. Hematology reference intervals for captive Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus, located at Horn Point (H), Lamar (L), and
Chalk Point (C) fish hatcheries. Reference intervals were determined using robust techniques.

90% CI
Reference
Analyte FH* N Mean SD Median Min Max Interval Lower Limit Upper Limit D**

PCV H 43 25 4 25 18 34 24–26 23–24 25–26

L 36 22 4 23 11 29 21–24 20–22 23–24 g
C 40 25 4 25 19 34 24–26 23–24 25–27
Hemoglobin (g/L) H 43 58 9 57 40 80 54–61 53–55 60–62
L 36 57 13 59 30 80 55–62 54–56 61–63 n
C 40 59 12 56 40 90 50–61 49–51 60–62
RBC count (91012 cells/L) H 43 1.15 0.21 1.18 0.51 1.51 1.14–1.24 1.13–1.15 1.23–1.24
L 36 0.94 0.25 0.98 0.48 1.42 0.90–1.05 0.89–0.92 1.00–1.09 g
C 40 1.11 0.25 1.09 0.63 1.70 0.99–1.19 0.92–1.03 1.15–1.24
MCV (fl) H 43 220 45 214 154 373 198–230 189–207 222–241
L 36 246 68 224 160 449 196–252 190–207 245–259 g
C 40 238 58 234 142 397 212–256 204–220 248–263
MCH (pg) H 43 53 14 51 37 120† 48–54 46–50 52–57
L 36 64 18 58 37 106 49–68 47–52 66–71 n
C 40 56 18 52 32 103 45–60 42–47 58–63
MCHC (g/L) H 43 240 30 240 190 320‡ 240–250 235–245 245–255
L 36 260 30 260 180 330 250–270 236–245 264–274 g
C 40 240 50 240 150 350 220–260 216–225 255–264
Total WBC count (9109 cells/L) H 43 30.0 10.7 30.0 5.3 55.9 26.4–33.7 23.8–28.9 30.8–32.6
L 36 24.2 7.7 23.9 10.3 42.0 21.0–26.8 19.1–23.2 25.2–29.0 g
C 40 29.0 11.4 27.7 11.0 57.6 23.2–32.2 20.7–25.5 29.6–35.3
Total lymphocytes (9109 cells/L) H 43 18.2 6.3 18.0 4.0 34.1 16.0–20.3 13.3–19.1 18.0–22.7
L 36 15.2 6.5 14.9 4.3 32.2 12.5–17.4 10.8–14.1 15.3–19.4 g
C 40 20.1 7.4 20.1 6.4 36.9 17.4–22.7 16.0–18.9 21.2–24.3
Small lymphocytes (9109 cells/L) H 43 17.0 6.0 16.8 3.6 32.1 14.8–19.0 13.2–16.3 17.6–20.4
L 36 13.8 6.2 13.6 3.6 29.8 11.4–15.9 10.0–12.7 14.5–17.4 g
C 40 18.5 6.8 18.0 5.9 34.7 15.5–20.6 14.0–16.9 19.2–22.0
Large lymphocytes (9109 cells/L) H 43 1.2 0.7 1.1 0.3 3.1 0.8–1.4 0.1–1.5 0.6–2.2
L 36 1.4 0.7 1.3 0.5 4.4§ 1.1–1.6 0.5–1.6 1.0–2.1 n
C 40 1.6 0.9 1.4 0.2 4.3 1.0–1.7 0.4–1.5 1.2–2.2
Neutrophils (9109 cells/L) H 43 7.4 3.7 6.1 0.7 16.8 4.5–7.9 4.0–5.1 7.4–8.5
L 36 7.7 4.6 6.4 2.8 22.3 4.8–7.9 4.4–5.3 7.4–8.4 n
C 40 6.8 5.2 4.4 1.3 21.1 1.6–7.1 1.0–2.1 6.6–7.6
Monocytes (9109 cells/L) H 43 0.4 0.3 0.4 0 1.5 0.3–0.5 0.3–0.4 0.4–0.6
L 36 1.0 0.8 0.8 0.1 3.5 0.6–1.0 0.5–0.7 0.9–1.1 g
C 40 0.7 0.5 0.7 0.9 1.9 0.5–0.9 0.4–0.6 0.8–1.0
Eosinophils (9109 cells/L) H 43 4.0 2.5 3.5 0.3 12.7¶ 2.8–4.6 2.6–3.0 4.4–4.8
L 36 0.3 0.3 0.2 0 1.3 0–0.3 0–0.1 0.3–0.3 n
C 40 1.4 2.1 0.8 0 12.4†† 0.5–1.1 0.4–0.6 1.0–1.2
Thrombocytes (9109 cells/L) H 43 35.5 10.6 36.0 13.0 58.0 32.5–39.5 31.1–33.9 38.2–41.0
L 36 36.1 15.1 34.5 13.0 73.0 28.3–40.7 26.8–29.9 39.2–42.2 n
C 40 35.4 8.8 37.0 14.0 55.0 34.8–39.2 33.7–36.1 38.0–40.4
N:L ratio‡‡ H 43 0.41 0.16 0.37 0.18 0.76 0.33–0.45 0.29–0.35 0.43–0.47
L 36 0.70 0.87 0.40 0.11 2.08 0.19–0.58 0.15–0.24 0.54–0.62 n
C 40 0.36 0.29 0.24 0.07 1.32 0.12–0.37 0.09–0.15 0.34–0.40

*Fish hatchery.
**Distribution of data – Gaussian (g) or non-Gaussian (n).
†Detected as outlier. Second highest value was 75 pg.
‡Detected as outlier. Second highest value was 270 g/L.
§Detected as outlier. Second highest value was 2.5 9 109 cells/L.
¶Detected as outlier. Second highest value was 8.1 9 109 cells/L.
††Detected as outlier. Second highest value was 5.1 9 109 cells/L.
‡‡Ratio of total neutrophils to total lymphocytes.

390 Vet Clin Pathol 43/3 (2014) 387–396 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
Matsche et al. Atlantic sturgeon hematology and plasma chemistry

Table 3. Plasma chemistry reference intervals for captive Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus, located at Horn Point (H), Lamar (L), and
Chalk Point (C) fish hatcheries. Reference intervals were determined using robust techniques.

90% CI
Reference
Analyte FH* N Mean SD Median Min Max Interval Lower Limit Upper Limit D**

Total protein† (g/L) H 43 32 5 31 24 43 29–33 28–31 32–34

L 36 30 6 29 20 51 27–31 26–28 31–32 g
C 40 36 10 36 18 54 33–39 32–34 38–40
Albumin‡ (g/L) H 43 11 2 11 7 15 10–12 10–11 12–13
L 36 11 2 11 7 19 10–12 10–12 12–13 n
C 40 14 4 15 7 22 14–15 13–14 14–16
Glucose§ (mmol/L) H 43 0.15 0.32 0.06 0 1.32 0.61–0.68 0–0.66 0.30–0.75
L 36 0.15 0.03 0.15 0 3.47 0–0.30 0–0.13 0.18–0.46 g
C 40 1.38 0.76 1.43 0 3.58 1.24–1.62 1.02–1.33 1.49–1.70
Urea (mmol/L) H 43 2.9 0.7 2.5 2.1 4.6 2.4–3.0 2.1–2.8 2.6–3.2
L 36 3.9 1.1 3.9 2.1 6.4 3.5–4.3 3.1–3.9 4.1–4.8 g
C 40 6.4 2.0 6.1 2.5 11.4 5.2–7.0 4.8–5.4 6.6–7.3
Calcium (mmol/L) H 43 2.0 0.1 2.0 1.8 2.4 2.0–2.0 1.9–2.0 1.9–2.0
L 36 1.9 0.1 1.9 1.7 2.1 1.9–2.0 1.8–1.9 1.9–2.0 g
C 40 1.9 0.1 2.0 1.7 2.1 1.9–2.0 1.8–1.9 1.9–2.0
Phosphate (mmol/L) H 43 3.6 0.9 3.5 2.4 4.5 3.3–3.6 3.2–3.4 3.5–3.7
L 36 2.8 0.3 2.8 2.3 3.7 2.8–2.9 2.7–2.9 2.9–3.0 g
C 40 2.8 0.4 2.8 2.0 3.8 2.6–2.9 2.5–2.7 2.8–3.0
Sodium (mmol/L) H 43 147 3.8 146 139 156 144–148 143–146 147–148
L 36 150 2.5 150 146 158 149–151 148–150 149–152 g
C 40 135 1.8 135 131 142 135–135 134–135 135–136
Chloride (mmol/L) H 43 128 4.0 127 120 138 125–129 124–126 128–130
L 36 130 2.8 130 126 141 129–131 128–130 130–131 g
C 40 117 2.5 117 111 122 116–118 115–117 117–118
Potassium (mmol/L) H 43 3.1 0.4 3.1 2.1 4.0 3.0–3.2 2.9–3.0 3.1–3.2
L 36 3.3 0.5 3.3 2.4 4.8 3.1–3.4 3.0–3.1 3.3–3.4 g
C 40 2.7 0.3 2.7 2.3 3.2 2.6–2.8 2.5–2.6 2.7–2.8
Globulins (g/L) H 43 21 3 20 15 29 19–22 18–20 20–22
L 36 18 3 18 13 26 17–19 16–18 18–20 g
C 40 22 7 22 11 37 20–24 19–21 23–25
Osmolality (mmol/kg) H 43 285 11.1 284 257 304 280–288 278–282 286–290
L 36 291 38 281 216 395 263–299 259–266 294–303 g
C 40 259 16.1 262 218 286 258–266 255–261 264–268
AST (U/L) H 43 193 83 187 55 423 157–217 143–174 202–233
L 36 156 60 140 53 321 110–169 96–121 155–182 n
C 40 266 101 247 165 522 217–277 198–236 260–285
CK (U/L) H 43 248 219 183 60 1,062 149–219 126–174 201–235
L 36 112 179 46 13 1,479 10–81 1–22 70–90 n
C 40 679 539 632 35 6,292¶ 424–840 389–455 802–881
LDH (U/L) H 43 3,783 1,765 3,329 838 7,720 2,714–4,221 2,345–3,026 3,987–4,518
L 36 2,464 921 2,247 1,025 4,691 1,811–2,682 1,667–1,994 2,498–2,855 n
C 40 3,637 1,678 3,519 1,119 6,796 2,815–4,223 2,476–3,105 3,924–4,482

*Fish hatchery.
**Distribution of data – Gaussian (g) or non-Gaussian (n).
†Numerous concentrations were below the analyzer’s lower quantification limit (30 g/L).
‡Numerous concentrations were below the analyzer’s lower quantification limit (15 g/L).
§Numerous concentrations from Horn Point and Lamar fish were below the analyzer’s lower quantification limit (0.6 mmol/L).
¶Detected as outlier. Second highest value was 3,735 U/L.
LDH indicates lactate dehydrogenase.

sturgeon.18 For clinical chemistry, glucose concen- linearity for total protein (30 g/L), albumin (15 g/L),
tration and enzyme activity RI had the highest vari- and glucose (0.6 mmol/L) concentration, and there-
ability among the fish populations (Table 3). Mean fore these analytes were not included in the statisti-
concentrations were at or below the lower limit of cal tests to compare the 3 hatcheries.

Vet Clin Pathol 43/3 (2014) 387–396 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology 391
Atlantic sturgeon hematology and plasma chemistry
Discussion
Hematology
The PCV RI reported here for Atlantic sturgeon are
comparable to mean values in other sturgeons such
as 2-year-old Atlantic and Shortnose sturgeon (28–
28.5%)30, 16-month-old Shortnose sturgeon (20–
25%)31, juvenile Great sturgeon (Huso huso) (15–22%)14,
juvenile Bester sturgeon (H Huso) female 9 (A ruthe-
nus) male (18–27%)32, and young-of-year Siberian
sturgeon (A baerii) (24–28%).33 In contrast, the non-
parametric RI reported for cultured, subadult Short-
nose sturgeon were higher (26–46%).18 Among fish,
PCV typically varies between 20% and 45%, with stur-
geon and other less active species on the low end or
slightly below this range.24 A wide variety of factors
can affect PCV values in fish such as age, sex, maturity,
temperature, photoperiod, season, exposure to
hypoxia, and disease.24
The RBC count RI reported here were slightly
higher than those reported earlier for Shortnose stur-
geon18, particularly at the lower limit (RI for Short-
nose sturgeon: 0.65–1.09 9 106 cells/lL).18 Mean
RBC counts in other sturgeon species are typically
lower than the means reported here14,31,32, although
much higher RBC counts (2.3–2.9 9 106 cells/lL)
have been reported in juvenile Siberian sturgeon. 33
RBC counts typically range from 1 to 5 9 106 cells/lL
in fish, and as with PCV, sturgeon and other less
active species typically have values on the low end of
this range as a result of decreased oxygen demands.24
In Bester sturgeon, RBC count increased with age,
from 0.36 9 106 cells/lL in young-of-year fish to
0.83 9 106 cells/lL in > 2-year-old fish.32 In Persian
sturgeon (A persicus) and Great sturgeon, there was a
general increase in RBC count from one-year-old to
6-year-old fish.34 Disease, parasitism, poor nutrition,
exposure to toxicants, and hypoxia/anoxia may result
in anemia.35 Increased RBC counts can result from
mobilization of RBC from the spleen in stressed fish.36
The RI for MCV in this study are similar to the
range of values typically reported in fish (150–
350 fL).24 However, mean MCV values reported from
other sturgeon studies (325–448 fL)18,32 are often on
the high end or exceed this range, which may indicate
that RBC are generally larger in sturgeon compared
with other fish species. In contrast, markedly lower
MCV values (23.9–27.7 fL) have been reported in
Great sturgeon.14 Changes in MCV values can indicate
RBC swelling or shrinkage, which can be induced by a
wide variety of osmotic disturbances that result in a
net flux of water to or from RBC.37
392 Vet Clin Pathol 43/3 (2014) 387–396 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
Matsche et al.
Hemoglobin concentrations reported here for
Atlantic sturgeon are comparable to concentrations in
other reference populations of fishes, which typically
range from 50 to 100 g/L.24 Similar mean HGB con-
centrations have been reported elsewhere from juve-
nile sturgeon including Atlantic30, Shortnose18,30,
Stellate (A stellatus)32, Russian (A gueldenstaedtii)32,
Bester32, Siberian33, Persian34, and Great34 sturgeon.
In the Bester sturgeon, HGB concentration increased
with age, from a mean of 26 g/L in young-of-year fish,
to 83 g/L in 3rd year fish.32 Similarly, HGB concentra-
tion increased from one-year-old to 6-year-old Persian
sturgeon (mean HGB [g/L]: 46.6  3.5 SD, one year
old vs 65.8  2.4 SD, 6-year-old) and in Great stur-
geon (mean HGB [g/L]: 55.7  1.7 SD, one year old vs
78.6  4.9 SD, 6 year old).34 Mean HGB values were
not affected by daily temperature fluctuations in
young-of-year Siberian sturgeon33, but decreased,
along with PCV, with increasing salinity in Great stur-
geon fingerlings.14 In fish, MCH ranges from 30 to
100 pg as a result of variation in RBC size24, and the
MCH values reported here for Atlantic sturgeon are
consistent with this range. However, MCH values in
sturgeon are typically higher (eg, means range from 80
– 160 pg) as RBC tend to be larger than in other groups
of fishes.18,32 In contrast, markedly lower MCH values
(4.1–5.2 pg) have been reported in Great sturgeon
exposed to a range of salinities.14 MCHC values
reported in sturgeon in this and other studies (16–
30.5 g/L)14,18,30,32 are consistent with values reported
from teleosts.24
Total WBC counts are highly variable in fish (typi-
cally 30–150 9 109 cells/L) as a result of phylogenetic
differences and sensitivity to a wide variety of environ-
mental conditions and stressors.24,38 Total WBC counts
tend to be lower in sturgeon than in other groups of
fishes. The RI for Atlantic sturgeon reported in this
study were comparable to mean total WBC counts in
Siberian sturgeon (19.4–25.5 9 109 cells/L)33 and in
Great sturgeon (18.2–22.3 9 109 cells/L).14 In con-
trast, the nonparametric RI for WBC count in Short-
nose sturgeon was higher (28.4–90.8 9 109 cells/L).18
Lymphocytes are typically the most numerous
type of WBC in sturgeon and other fishes14,18,24,36, and
changes in leukogram can result from changes in envi-
ronmental conditions and in response to disease, expo-
sure to toxicants, handling/confinement, and other
stress factors.38 The functional significance of large vs
small lymphocytes, as evident in Atlantic sturgeon in
this study and previously described in Shortnose stur-
geon18, is unknown.
Elevated N:L ratios may reflect neutrophilia, with
or without an accompanying lymphopenia, which is
Matsche et al.
considered a classic stress response in fish and other
animals.38 The N:L ratios determined for Atlantic stur-
geon in this study were within the reported RI (0.07–
1.03) of cultured Shortnose sturgeon.18 It has been
suggested that N:L ratios > 0.6 may indicate a stress
response in sturgeon.18 In this study, N:L ratio was
highest in fish held in concrete raceways and lowest in
fish held in ponds, which may indicate that fish in this
study were subjected to different levels of stress related
to stocking density, substrate material of confinement
enclosures, water quality, or other factors that differed
among the hatcheries. In contrast, another study
reported slightly higher neutrophil fractions and lower
lymphocyte fractions in Persian and Great sturgeon
reared in ponds compared with fish of the same age
reared in tanks.34 The N:L ratio may also change with
age as neutrophil and eosinophil fractions in Persian
and Great sturgeon decreased with age.34
Changing salinity can be an important factor in
fish hematology, but response has not been consistent
among studies involving sturgeon. One study reported
decreasing RBC count and PCV with increasing salinity
(0–12 ppt) in Great sturgeon.14 In contrast, PCV
increased in juvenile Shortnose sturgeon after
10 weeks in hyperosmotic conditions31, but no differ-
ences were detected in Gulf sturgeon acclimated to
freshwater.39 During an experiment of gradual accli-
mation of Adriatic sturgeon (A naccarii) from 0 – 35 ppt
salinity over 47 days, PCV, RBC count, and HGB con-
centration increased up to 22 ppt salinity, and then
decreased to below initial values from 22 – 29 ppt.40
The authors attributed these hematologic variations to
changes in plasma water content. Following the salin-
ity acclimation of fish, blood values stabilized to initial
values in fish over 20 days at a constant salinity of
35 ppt.40 Changes in salinity may also affect the leuko-
gram, the lymphocyte fraction generally increased and
the neutrophil fraction decreased with salinity in juve-
nile Great sturgeon.14 Atlantic sturgeon maintained in
freshwater (Lamar) in this study had lower RBC counts
and PCV values compared with fish held in brackish
water (salinity = 8.5–14 ppt, chalk Point and Horn
Point), but salinity was not controlled and there were
substantial differences in husbandry conditions among
the hatcheries.
Plasma chemistry
The RI for total protein reported in this study are gen-
erally consistent with the range of mean values
reported in other sturgeon studies (18–55 g/L), but it
should be noted that the lower limit of linearity of the
analyzer used in this study is 30 g/L.15,17–19,21,30 Total
Vet Clin Pathol 43/3 (2014) 387–396 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
Atlantic sturgeon hematology and plasma chemistry
protein concentration in Atlantic sturgeon from mixed
culture conditions reported here was higher than
mean values in 2-year-old Atlantic sturgeon main-
tained in freshwater (18 g/L  1 SE).30 Plasma protein
concentrations in sturgeon are often on the low end
among fish in general (20–80 g/L), and fluctuations in
plasma protein concentrations are often indicative of
changes in plasma volume.41 Albumin concentrations
reported here for Atlantic sturgeon are consistent with
mean values reported in other sturgeon species (9–
13 g/L)15,17–19, but the means reported here are below
the analytic range (15 g/L) for the analyzer used in this
study. Low values were also reported for Shortnose
sturgeon (8–17 g/L)18 and hybrid sturgeon A naccarii
female 9 A baerii male (8–25 g/L)17 maintained under
relatively uniform conditions. The RI for plasma globu-
lins (calculated) in Atlantic sturgeon in this study were
lower at the upper limit than in Shortnose sturgeon
(18–37 g/L).18 Globulins are often higher in wild fish
and may increase with fish size and age.41
Plasma glucose concentrations vary in fish by size,
age, sex, maturity, diet, nutritional status, and stress,
and are therefore highly variable.41 In this study, the
mean values for all 3 hatchery populations were near
or below the analyzer lower limit of linearity
(0.6 mmol/L) for unexplained reasons. The plasma was
separated from cells within 1 h of collection, and
shipped to the diagnostic laboratory on ice packs the
same day. Glucose concentrations reported here are
low compared to those in 2-year-old cultured Atlantic
sturgeon (3.8 mmol/L  0.1 SD).30 Mean glucose con-
centrations reported elsewhere from cultured and wild
fish ranged from 2.5 mmol/L in Chinese sturgeon (A
sinensis)21 to 9.5 mmol/L in female Stellate sturgeon.19
Urea concentrations reported here for Atlantic sturgeon
are similar to mean values in Stellate sturgeon (5.1–
5.9 mmol/L)19, but are substantially higher than in
hybrid sturgeon (0.8 mmol/L  0.004 SE)17, Amur
sturgeon (A schrenckii) (0.8 mmol/L  0.4 SD)21, and
Chinese sturgeon (0.5 mmol/L  0.2 SD).21 Urea con-
centrations reported here and by others19 are also high
compared to values in teleosts, which typically have
< 1.8 mmol/L in freshwater species and < 3.6 mmol/L
in marine species.42 Plasma phosphate concentrations
and factors that regulate their variations are not well
studied in fish. The RI for phosphate in Atlantic stur-
geon in this study are higher than in Shortnose stur-
geon (1.6–2.6 mmol/L)18, but were within the
phosphate RI in hybrid sturgeon (1.7–5.8 mmol/L).17
In other sturgeon species, mean phosphate concentra-
tions ranged from 2.1 to 4.3 mmol/L.15,19
Sodium and chloride are the predominant mono-
valent electrolytes in fish, and are primarily affected by
393
Atlantic sturgeon hematology and plasma chemistry
disturbances in osmoregulation.41 Concentrations of
sodium and chloride in freshwater teleosts are approxi-
mately 150 and 130 mmol/L, respectively, and are
generally higher in marine teleosts (175–200 mmol/L
sodium, and 165–175 mmol/L chloride).41,42 In stur-
geon, sodium concentrations range from 132 to
154 mmol/L, while chloride concentrations range
from 107 to 125 mmol/L.15,17–19,30,43 Plasma concen-
trations of sodium and chloride were significantly
higher in fish maintained in freshwater than in fish
from both brackish water hatcheries in this study. The
RI for osmolality of Atlantic sturgeon in this study were
on the high end of the osmolality RI reported for cul-
tured Shortnose sturgeon (232–289 mmol/kg).18
Higher mean osmolality values have been reported in
Atlantic sturgeon (272 mmol/kg  6 SD)30 compared
to Shortnose sturgeon (255 mmol/kg  3 SD).30 Potas-
sium concentrations measured in Atlantic sturgeon in
this study are similar to values reported for other Aci-
penserid sturgeon17–19,30, but are low compared to
potassium concentrations Shovelnose sturgeon (mean
potassium: 4.8 and 5.7 mmol/L).43 Potassium concen-
trations are typically 2–4 mmol/L in freshwater fish and
5–10 mmol/L in marine fish.41 A number of stress fac-
tors such as handling, exertion, confinement, and expo-
sure to toxicants can affect plasma electrolyte
concentration by disrupting gill function.41
Concentrations of calcium and other divalent ions
are predominantly regulated by the kidneys in fish,
and are typically less variable than monovalent ion
levels.41 In general, calcium concentrations are
2–3 mmol/L in freshwater fish and 3–5 mmol/L in
marine fish.41 The RI for calcium concentration in
Atlantic sturgeon in this study are somewhat low
compared to freshwater fish, while the RI for calcium
concentration in hybrid sturgeon was markedly wide
(1.9–7.2 mmol/L).17 In other sturgeon species, cal-
cium concentrations are comparable to concentrations
in other freshwater fish.15,16,18,19,43 A fraction of
plasma calcium is protein bound, and therefore cal-
cium concentrations are typically sensitive to changes
in plasma volume or protein concentration.41
Osmoregulatory mechanisms in fish such as the
anadromous Atlantic sturgeon can influence a num-
ber of plasma chemistry analytes in response to chang-
ing salinities. Even species considered to be primarily
freshwater, such as Lake sturgeon (A fulvescens), show
some osmoregulatory ability.44 Short-term response
to increased salinity may involve hemoconcentration
(eg, increased osmolarity, and electrolyte and plasma
protein concentrations) as a result of transient
increase in gill permeability and subsequent osmotic
water loss. However, compensatory mechanisms such
394 Vet Clin Pathol 43/3 (2014) 387–396 ©2014 American Society for Veterinary Clinical Pathology and European Society for Veterinary Clinical Pathology
Matsche et al.
as increased ingestion of environmental water, activa-
tion of Na+–K+-ATPase, changes in size or number of
chloride cells in the gills, and increased excretion of
divalent ions from the kidneys may stabilize plasma
volume, and electrolyte and protein concentrations
within days.39,45–47 The sibling fish in this study were
maintained for years in either fresh or brackish water,
and therefore it would be expected that any differ-
ences in plasma biochemistry would reflect long-term
acclimation mechanisms. However, plasma osmolal-
ity, and sodium and chloride concentrations were
higher in freshwater fish (Lamar hatchery) than in
siblings maintained in brackish water (Chalk point). It
is therefore more likely that differences in plasma bio-
chemistry variables among the hatcheries in this study
resulted from husbandry-related differences among
the hatcheries such as differences in feed, growth rates
of fish, stocking densities, types of confinement enclo-
sures, temperature, or other factors.
Enzyme activities tend to be highly variable in stur-
geon and are poorly understood. In this study, the RI
for AST activity were within the RI for Shortnose
sturgeon (90–311 U/L)18 and hybrid sturgeon (17–
406 U/L).17 Mean AST activities from 18–549 U/L have
been reported for populations of Amur, Chinese, Per-
sian, and Stellate sturgeon.16,19–21 Activities for CK in
sturgeon are highly variable; reported values include RI
determined for Atlantic sturgeon in this study 149–219,
10–81, and 424–840 U/L; 389–5,559 U/L (RI for hybrid
sturgeon)17; 184–1,935 U/L (range in mean activities,
Persian sturgeon)16; and 5,959–7,233 U/L (range in
mean values, Stellate sturgeon).20 The RI for LDH
activity in Atlantic sturgeon in this study are generally
on the upper end of the RI for hybrid sturgeon (389–
4,040 U/L)17 and Amur sturgeon (827–3,624 U/L).21
In comparison, Chinese sturgeon had relatively low
LDH activities (633–908 U/L).21 Interpreting enzyme
activities in fish is complicated as they may vary by
sampling and analytic techniques, species, age, diet,
temperature, sex, maturity, habitat, and in response to
a number of stressors.41
Conclusions
Significant differences in blood values among hatcher-
ies were common in this study. We therefore recom-
mend that sturgeon propagation or research programs
determine separate hematologic and biochemical RI
for each population.10 Sex, maturity, and type of con-
finement (eg, pond vs tank) should be considered as
subcategories where feasible. While a sample size of
120 individuals for each subcategory is recommended,
Matsche et al. Atlantic sturgeon hematology and plasma chemistry

robust techniques provide the means of obtaining RI 9. Hrubec TC, Smith SA, Robertson JL. Age-related
when fewer individuals are available. changes in hematology and plasma chemistry values of
hybrid striped bass (Morone chrysops 9 Morone saxatilis).
Vet Clin Pathol. 2001;30:8–15.
Acknowledgments
10. Hrubec TC, Smith SA, Robertson JL, et al. Blood bio-
This project was funded by Maryland Department of Natural chemical reference intervals for sunshine bass (Morone
Resources (MD DNR). We thank the numerous staff mem- chrysops 9 Morone saxatilis) in three culture systems. Am
bers from the following facilities or programs that partici- J of Vet Res. 1996;57:624–627.
pated in animal husbandry and data collection: University of
11. Ram Bhaskar B, Srinivasa Rao K. Influence of environ-
Maryland, Aquatic Restoration and Ecology Laboratory,
mental variables on haematology, and compendium of
Horn Point; Lamar Fish Health Center, US Fish and Wildlife
normal haematological ranges of milkfish, Chanos cha-
Service; GenOn Energy Chalk Point Aquaculture Facility;
nos (Forskal), in brackishwater culture. Aquaculture.
Fish and Wildlife Health Program (MD DNR) and Hatcheries
1989;2:123–136.
and Finfish Restoration Program (MD DNR). We thank Dr.
Craig Harms for technical assistance and manuscript review. 12. Hrubec TC, Robertson JL, Smith SA. Effects of ammonia
Disclosure: The authors have indicated that they have and nitrate concentration on hematologic and serum
no affiliations or financial involvement with any organiza- biochemical profiles of hybrid striped bass (Morone
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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Table S1. Analytic test methods used to measure
plasma chemistry variables in blood of 3 populations of
captive Atlantic sturgeon.