Aquaculture 454 (2016) 118–124

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Dietary copper requirement of juvenile Russian sturgeon

Acipenser gueldenstaedtii
Hewei Wang a, Erchao Li a, Haoyong Zhu a, Zhenyu Du a, Jianguang Qin b, Liqiao Chen a,⁎
a
Laboratory of Aquaculture Nutrition and Environmental Health, School of Life Sciences, Shanghai 200241, PR China
b
School of Biological Sciences, Flinders University, Adelaide, SA 5001, Australia

a r t i c l e i n f o a b s t r a c t

Article history: To quantify the dietary copper (Cu) requirement of Russian sturgeon Acipenser gueldenstaedtii, copper sulfate
Received 11 September 2015 (CuSO4·5H2O) was added into the basal semi-purified diet at seven levels 0, 1, 2, 4, 6, 8 and 16 mg Cu kg−1
Received in revised form 16 December 2015 diet yielding the actual dietary values of 0.3, 1.5, 2.4, 4.4, 6.2, 8.3 and 16.1 mg Cu kg−1 diet, respectively. Each
Accepted 17 December 2015
diet was fed to sturgeon (11.84 ± 0.07 g) in triplicate in a flow-through system for 8 weeks. The Cu concentration
Available online 19 December 2015
in the rearing water was 0.8–1.2 μg L−1 during the trial period. The weight gain rate (WGR) and feed efficiency
Keywords:
were significantly higher in fish fed 2.4 to 8.3 mg Cu kg−1 than those fed 0.3, 1.5 or 16.1 mg Cu kg−1. The highest
Russian sturgeon WGR was found in fish fed 6.2 mg Cu kg−1. The whole-body Cu concentrations were highest in fish fed 6.2 to
Copper 16.1 mg Cu kg−1. The liver Cu concentration was highest in fish fed 16.1 mg Cu kg−1 diet and lowest in fish
Requirement fed diets Cu levels range from 0.3 to 4.4 mg Cu kg−1. Hepatic copper–zinc superoxide dismutase (Cu–Zn SOD)
Growth activities, total antioxidant capacity and serum ceruloplasmin activity were significantly higher in fish fed 6.2
Antioxidant ability and 8.3 mg Cu kg−1 diet than those in other treatments. The lowest hepatic malondialdehyde values were ob-
served in fish fed 6.2 and 8.3 mg Cu kg−1. No significant differences were in survival, hepatosomatic index,
viscerosomatic index and condition factor among all the treatments. There were also no significant differences
in whole body moisture, crude protein, crude lipid and ash between all the treatments. Analysis of broken-line
regression based on the WGR, whole-body Cu concentration, hepatic Cu–Zn SOD and serum ceruloplasmin
activity of the fish fed different diets indicates that the minimum Cu concentration in the semi-purified diet for
juvenile Russian sturgeon (12–70 g) is at 7–8 mg kg−1.

Statement of relevance

The dietary copper (Cu) requirement of juvenile Russian sturgeon was investigated. Dietary Cu deficiency and
over-loading significantly reduced growth, feed utilization and antioxidant capacity. The results suggest that
the appropriate dietary Cu content is considerable in Russian sturgeon farming. Minimum dietary Cu level for
this species is recommend as 7–8 mg Cu kg−1 in this study. These information will be useful in developing a
practical diet for Russian sturgeon culture.
© 2015 Elsevier B.V. All rights reserved.

1. Introduction lipids, proteins, and DNA and lead to neurodegenerative disorders

Copper (Cu) is an essential trace element and a redox-active transi-
tion metal related to oxidative damage in fish (Gropper et al., 2005; Lall,
2002). Cu is a cofactor required for structural and catalytic properties of
superoxide dismutase, ceruloplasmin, cytochrome oxidase, lysyl oxi-
dase, dopamine hydroxylase and tyrosinase (Watanabe et al., 1997).
These enzymes are involved in a series of biological processes for oxida-
tion–reduction reaction, iron utilization, cellular energy production,
collagen synthesis and brain neurotransmitters (Lall, 2002). On the
other hand, a high Cu concentration may cause oxidative damage to
⁎ Corresponding author.
E-mail address: lqchen@bio.ecnu.edu.cn (L. Chen).
(Gaetke and Chow, 2003).
Fish can obtain Cu from both diet and water, but the former provides
most Cu consumed by fish (Kamunde et al., 2002). Until now, minimum
dietary Cu requirement has been determined in rainbow trout
Oncorhynchus mykiss, common carp Cyprinus carpio (Ogino and Yang,
1980), channel catfish Ictalurus punctatus (Gatlin and Wilson, 1986),
Atlantic salmon Salmo salar (Berntssen et al., 1999), hybrid tilapia
(Shiau and Ning, 2003), grouper Epinephelus malabaricus (Lin et al.,
2008), yellow catfish Pelteobagrus fulvidraco (Tan et al., 2011), and
large yellow croaker Larimichthys croceus (Cao et al., 2014). It has been
shown that Cu deficiency decreases the activity of ceruloplasmin and
copper–zinc superoxide dismutase (Cu–Zn SOD) in grouper (Lin et al.,
2008). In contrast, excessive Cu in diet can be toxic and overloaded Cu

http://dx.doi.org/10.1016/j.aquaculture.2015.12.018
0044-8486/© 2015 Elsevier B.V. All rights reserved.
 H. Wang et al. / Aquaculture 454 (2016) 118–124 119

can also cause intestinal apoptosis and slow growth in Atlantic salmon
(Lundebye et al., 1999). Therefore, identification of minimum dietary
Cu requirement is important for aquacultured species during grow-out.
There are 27 extant species of sturgeons and paddlefishes (other
Acipenseriformes) on the earth, all of them face extinction. 15 of the
27 species are artificial cultured to their valuable meat and caviar
(Memis et al., 2006; Pikitch et al., 2005). Eleven species and hybrids
are being cultured since 1998 in China. Since 2000, China has been the
world's largest producer of sturgeon (Wei et al., 2011). The rapid in-
crease of sturgeon production in aquaculture occurred at the end of
the last century, which coincides with the massive decline of wild
caught stocks. The modern aquaculture production started only during
and after the early nineteen-eighties. (Bronzi et al., 2011). The produc-
tion of the cultured sturgeon in 2013 has reached 75,000 t in the
world (FAO, Food and Agriculture Organization of the United Nations,
2015) and was more than 64,000 t in China (China Fishery Statistical
Yearbook, 2014). Russian sturgeon accounted for 10% of the annual
sturgeon production in China from 2010 to 2012 (Shen et al., 2014).
Under culture conditions, the nutritional requirements on protein
(Stuart and Hung, 1989), lipid (Şener et al., 2005), carbohydrate
(Hung et al., 1989), and trace elements (Wen et al., 2008; Xu et al.,
2011) have been studied in various sturgeon species. However, our
knowledge on dietary Cu requirement in sturgeon is very limited.
Until now, only the dietary Cu requirement for juvenile beluga Huso
huso has been determined (Mohseni et al., 2014). The objective of this
study was to determine the dietary Cu requirement of juvenile
Russian sturgeon Acipenser gueldenstaedtii (Bronzi et al., 2011).
2. Materials and methods
2.1. Experimental diets
Basal diet formulation and composition analysis (AOAC, 1995) are
shown in Table 1. Casein and gelatin were used as the dietary protein
sources and fish oil and soybean oil were used as the main dietary
lipid source. Corn starch was used as the carbohydrate source. The
Table 1
Formulation and proximate composition of the basal diet (% dry matter).
Ingredient
Casein, vitamin-freea
Gelatinb
Corn starchc
Fish oild
Soybean oile
Choline chloride
Taurine
Monocalcium phosphate
Vitamin premixf
Mineral premix, copper-freeg
Cellulose
Proximate composition
Crude protein
Crude lipid
Moisture
Ash
a
Casein, vitamin-free: crude protein 92% (Sigma-Aldrich Trading Co.,
Ltd., Shanghai, China).
b
Gelatin: Sangon Biotech, Shanghai, China.
c
Corn starch: Sinopharm Chemical Reagent Co., Ltd., Shanghai, China.
d
Fish oil: Xiamen Xinsha Pharmaceutical Co., Ltd., Xiamen, China.
e
Soybean oil: Kerry Oils & Grains Industrial Co., Ltd., Shanghai, China.
f
Vitamin premix (mg kg−1 diet): thiamin hydrochloride, 50; riboflavin,
200; pyridoxine hydrochloride, 50; vitamin B12, 0.1; nicotinic acid, 200; cal-
cium pantothenate, 100; folic acid, 20; biotin, 5; inositol, 800; ascorbic acid,
1000; menadione sodium bisulfite, 10; retinol acetate, 15; cholecalciferol,
10; alpha-tocopherol, 200; cellulose, 7340.
g
Mineral premix, copper-free (mg kg−1 diet): ZnSO4·7H2O, 100;
FeSO4·7H2O, 700; MnSO4·H2O, 80; CoCl2·6H2O, 20; KI, 8; Na2SeO3, 3;
MgSO4·7H2O, 4000; NaCl, 1000; KCl, 1000; AlCl3·6H2O, 15; cellulose, 3074.
basal diet was supplemented with CuSO4·5H2O (Sinopharm Chemical
Reagent Co., Ltd., Shanghai, China) at 0, 1, 2, 4, 6, 8 and 16 mg kg−1
diet, respectively, resulting in final dietary Cu concentrations of 0.3,
1.5, 2.4, 4.4, 6.2, 8.3 and 16.1 mg kg−1, respectively. Dietary Cu concen-
tration was analyzed using an inductively coupled plasma-atomic emis-
sion spectrophotometer (ICP-OES Optima 5300DV; PerkinElmer
Corporation) at the Shanghai Academy of Public Measurement, China.
Diets were processed into 2.5 mm diameter strips, air dried, ground
and sieved to appropriate size and stored at −20 °C before feeding.
2.2. Experimental procedure
Russian sturgeon juveniles were obtained from a farm in Quzhou,
China. Before starting the experiment, fish were acclimated to the labo-
ratory condition for two weeks in circular fiberglass tanks (diameter:
2 m, height: 0.51 m) in a flow-through system with continuous
aeration, and fed with the basal diet during acclimation.
Prior to the feeding trial, fish were fasted for 24 h. Juvenile sturgeon
(11.84 ± 0.07 g) were randomly assigned to 21 aquariums
(40 × 45 × 100 cm) in a flow-through system at a density of 20 fish
per aquarium. Each diet was fed to fish in three randomly chosen
aquaria. The diet was divided into two equal meals fed at 08:30 and
16:30 h by hand for 8 weeks. Fish were daily fed 3% of their body weight.
Fish were weighed once every 2 weeks and the daily ration was adjust-
ed according to weight gain. Feed intake was completed within 2–3 min
after delivery, thus leaching of Cu into water was negligible. Dissolved
Cu in water was collected 10 min before and after feeding and was
0.8–1.2 μg L−1 throughout the experiment. Excess feed and feces were
siphoned daily. During the experimental period, the water temperature
was 19.0–21.4 °C, dissolved O2 7 mg L−1, pH 7.2 and ammonia
0.1 mg L−1. A photoperiod of 12 h light (08:00 to 20:00 h) and 12 h
dark was used during the study.
2.3. Sample collection and analysis
At the termination of the experiment, fish were fasted for 24 h and
anesthetized with MS222 at 150 mg L−1 before handling. Then total
number and weight of fish in each aquarium were counted and weight-
ed to calculate the body weight gain rate and survival rate. Subsequent-
ly, three fish per aquarium were randomly selected to collect blood
%
sample from the caudal vessels. Serum was separated by centrifugation
40 of blood at 1500 × g for 10 min at 4 °C using a centrifuge (5804R,
10
Eppendorf, Hamburg, Germany). And then, the serum samples were
30
5 frozen in liquid nitrogen and then stored at −80 °C for ceruloplasmin
5 activity determination. After blood collecting, the liver was rapidly re-
0.5 moved from the same fish and frozen in liquid nitrogen and then stored
0.5 at −80 °C for determining liver Cu concentration, Cu–Zn SOD activity,
3
1
total antioxidant capacity (T-AOC) and malondialdehyde (MDA).
1 Viscerosomatic index and hepatosomatic index were calculated after
4 blood and liver were collected. Another six fish per aquarium were ran-
domly selected, pooled and then stored in −20 °C for determining the
43.1
whole-body composition and Cu concentration.
9.8
10.2 Growth performance of the juvenile Russian sturgeon was evaluated
3.9 using weight gain rate (WGR), feed efficiency (FE), survival rate (SR),
condition factor (CF), hepatosomatic index (HSI) and viscerosomatic
index (VSI) and calculated as in the following:
Weight gain rate ðWGR; %Þ
¼ 100  ½ðfinal body weight−initial body weightÞ=initial body weight:
Feed efficiency ðFEÞ
¼ ðfinal body weight−initial body weightÞ=feed intake:
Survival rate ðSR; %Þ
¼ 100  ðfinal amount of fishÞ=ðinitial amount of fishÞ:
120 H. Wang et al. / Aquaculture 454 (2016) 118–124
Hepatosomatic index ðHSI; %Þ ¼ ðhepatic weight=body weightÞ  100:
Viscerosomatic index ðVSI; %Þ ¼ ðviscera weight=body weightÞ  100:
 
3
Condition factor ðCFÞ ¼ body weight=body length  100:
All of the enzyme activities and MDA concentration were measured
by the commercial kits (Nanjing Jiancheng Bioengineering Institute,
Nanjing, China). Serum ceruloplasmin activity was measured using
o-dianisidine dihydrochloride as a substrate (Schosinsky et al., 1974).
The analysis of Cu–Zn SOD activity was based on SOD-mediated
inhibition of nitrite formation from hydroxyammonium in the presence
of O−2 generators (xanthine and xanthine oxidase) (Elstner and Heupel,
1976). The total antioxidant capacity (T-AOC) was measured by the
colorimetric technique as described byMiller et al. (1993). MDA concen-
tration was measured by the thiobarbituric acid-reacting substance
(TBARS) assay (Ohkawa et al., 1979).
Contents of the moisture, crude protein, crude lipid and ash in the
whole fish were analyzed using the standard procedures (AOAC,
1995). Crude protein (N × 6.25) was determined by the Kjeldahl meth-
od after an acid digestion using an auto Kjeldahl System (Kjeltec 8000;
Foss Tecator, Sweden). Crude lipid was determined by the ether extrac-
tion method using the Soxhlet system (2055 Soxhlet Avanti; Foss
Tecator, Hoganas Sweden). Moisture was determined by oven drying
at 105 °C to a constant weight and ash was quantified using a muffle fur-
nace until it ignited to a constant weight.
Cu concentration was measured according to the modified method
by Gomez et al. (2007) and Gonzálvez et al. (2010) using the inductively
coupled plasma-atomic emission spectrophotometer (ICP-OES Optima
5300DV; PerkinElmer Corporation) at the Shanghai Academy of Public
Measurement, China. The sample preparation and analysis procedure
details are as follows.
Ground sample 0.3–0.4 g weighed into a Pyrex glass digestion tube
was added with 10 mL of concentrated nitric acid and perchloric acid
superior grade (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China)
solution with 9:1 ratio. The sample was digested by heating the mixture
until dryness. The residue was dissolved in ultrapure water
(18.2 MΩ cm−1) to a volume of 10 mL before filtering it through a
0.45 μm membrane filter (Tianjing Jinteng Instrument Factory, Tianjin,
China).
Stock standard solution of Cu (100 μg mL−1) from National Research
Center for Certified Reference Material (NRCCRMS, China) was used as
stock solutions for calibration. The calibration standards were prepared
by diluting the stock standard solution in 0.5% nitric acid. The calibration
range was from 0.005 to 1 mg L−1 and it was modified according to the
expected Cu concentration range of the samples.
Analytical wavelength of Cu was 327.39 nm. Quantitative determi-
nation of Cu concentration was calculated using calibration standards
to create calibration curves of intensity versus concentration. The limit
of quantitation (LOQ) of Cu was 0.015 mg L−1 which was below the ex-
pected minimum Cu concentration of the sample digestion solution.
2.4. Statistical analysis
The results were presented as means ± SE of three replicates. Data
were analyzed by one-way analysis of variance (ANOVA) using the
SPSS 21.0 statistical software. When overall differences were significant
(P b 0.05), Tukey's test was used to compare the mean values between
individual treatments. Dietary Cu requirement of Russian sturgeon was
estimated based on WGR, Cu–Zn SOD activity, MDA value and whole-
body Cu concentration by the broken-line regression method
(Robbins, 1986).
3. Results
3.1. Growth performance
The growth and feed utilization data are presented in Table 2. Final
body weight (FBW),WGR and FE in fish fed diets with 0.3, 1.5 and
16.1 mg Cu kg− 1 were significantly lower (P b 0.05) than those in
other treatments. The significantly highest (P b 0.05) FBW and WGR
values were observed in the 6.2 mg kg− 1 treatment. Fish survival
(88.3–98.3%) was not significantly affected by dietary Cu levels
(P N 0.05).
As shown in Table 3, HSI, VSI and CF showed no significant differ-
ences among all the treatments (P N 0.05). Their values were in the
range of 2.60% to 3.28%, 10.52% to 11.36% and 0.85 to 0.91, respectively.
3.2. Whole body compositions
The results of the whole body compositions are shown in Table 4. No
significant differences in the contents of moisture, crude protein, crude
lipid and ash were observed among all the treatments (P N 0.05). These
contents were in the range of 78.5% to 79.0%, 13.0% to 13.3%, 4.45% to
4.65% and 2.81% to 2.97%, respectively.
3.3. Whole body and liver copper concentration
The results of the whole body and liver Cu concentration are shown
in Table 5. Whole-body Cu concentrations in fish fed diets with 6.2, 8.3
and 16.1 mg Cu kg−1 were significantly higher (P b 0.05) than those in
other treatments. Fish fed diets with 0.3 and 1.5 mg Cu kg−1 had the
lowest whole-body Cu concentration (P b 0.05). Hepatic Cu concentra-
tion in fish fed diet with 16.1 mg Cu kg−1 was significantly higher
(P b 0.05) than those fed diets range from 0.3 to 6.2 mg Cu kg− 1
(P b 0.05). Fish fed diets Cu levels range from 0.3 to 4.4 mg Cu kg− 1
had the lowest hepatic Cu concentration (P b 0.05).
3.4. Liver antioxidant parameters and serum ceruloplasmin activity
The liver antioxidant parameters and serum ceruloplasmin activity
are listed in Table 6. Activities of hepatic Cu–Zn SOD and T-AOC in-
creased when dietary Cu contents were increased from 0.3 to
8.3 mg kg−1, and then declined in fish fed 16.1 mg kg−1. The significant-
ly highest (P b 0.05) hepatic Cu–Zn SOD activity was found in fish fed
diets with 6.2 and 8.3 mg Cu kg−1. Fish fed diets with 4.4, 6.2 and
8.3 mg Cu kg−1 had highest hepatic T-AOC (P b 0.05). Hepatic MDA in
fish fed diets with 6.2 and 8.3 mg Cu kg−1 was significantly lower
(P b 0.05) than those fed diets with other Cu contents. Serum cerulo-
plasmin activity in fish fed diets with 6.2 and 8.3 mg Cu kg−1 was signif-
icantly higher (P b 0.05) than those in other treatments.
3.5. Dietary copper requirement analyses
Analysis by broken-line regression on WGR, whole-body Cu concen-
tration, hepatic Cu–Zn SOD and serum ceruloplasmin activity indicated
that the minimum requirement of dietary Cu was 6.1 (Fig. 1A), 7.4
(Fig. 1B), 6.7 (Fig. 1C), and 6.5 mg kg−1 (Fig. 1D), respectively.
4. Discussion
In the present study, growth performance was best in fish fed
6.2 mg Cu kg− 1 and poorest in fish fed diet with 0.3, 1.5 and
16.1 mg Cu kg−1. These results demonstrate that a certain concentra-
tion of dietary Cu is essential for juvenile Russian sturgeon. The weight
gain rate (438.1%–510.5%) in this study is in agreement with Cui's data
(431.0%–681.9%) (Cui, 2014). The feed efficiency (1.01–1.02) is a little
bit higher than other reported data which Russian sturgeon were fed
with practical diets such as Şener's (0.69–0.77) (Şener et al., 2005)
 H. Wang et al. / Aquaculture 454 (2016) 118–124 121

Table 2
Growth and feed utilization of juvenile Russian sturgeon fed the experimental diets with different levels of copper for 8 weeks.1

Dietary Cu levels (mg/kg) IBW2 (g) FBW3 (g) WGR4 (%) FE5 (%) Survival (%)

0.3 11.81 ± 0.07 63.6 ± 1.27a 438.1 ± 12.51a 1.01 ± 0.01a 90.0 ± 5.00
1.5 11.85 ± 0.11 64.1 ± 1.40a 440.5 ± 8.22a 1.03 ± 0.01a 88.3 ± 10.41
2.4 11.83 ± 0.03 68.4 ± 1.02b 478.2 ± 7.09b 1.11 ± 0.02b 96.7 ± 2.89
4.4 11.87 ± 0.11 69.2 ± 1.26b,c 482.5 ± 5.42b 1.12 ± 0.02b 96.7 ± 5.77
6.2 11.84 ± 0.04 72.3 ± 1.06c 510.5 ± 6.81c 1.11 ± 0.01b 98.3 ± 2.87
8.3 11.89 ± 0.07 70.4 ± 1.94b,c 492.3 ± 14.16b,c 1.10 ± 0.01b 96.7 ± 2.89
16.1 11.82 ± 0.08 64.5 ± 1.37a 445.5 ± 9.14a 1. 04 ± 0.03a 95.0 ± 5.00

One-way ANOVA
P value 0.867 b0.001 b0.001 b0.001 0.278

Means in the same column with different superscript letters are significantly different (P b 0.05).
1
Values are presented as the mean ± SE (n = 3) of three replicates per treatment.
2
IBW: initial body weight.
3
FBW: final body weight.
4
WGR: weight gain rate.
5
FE: feed efficiency.

and Cui's (0.80–0.91) (Cui, 2014). The reduction of feed efficiency was differences of dietary Cu content in diet, test duration, species or rearing
seen in fish fed both Cu-deficient and Cu excessive diets in this study, conditions between those trials, which makes the comparison of these
in agreement with other aquatic animals such as grouper (Lin et al., indexes between studies difficult. In the present study, the possible rea-
2010), beluga (Mohseni et al., 2014) and yellow catfish (Tan et al., son was that the Cu content in diets was not high enough to affect body
2011). The low feed efficiency may partly contribute to the poor growth indices and whole body compositions.
performance. As estimated by weight gain, the minimum dietary Cu Animals absorb dietary Cu in the intestine and store the Cu in the
content in juvenile Russian sturgeon is 6.1 mg kg−1. This value is similar body tissues but release Cu when intake is insufficient (Harris, 1991;
to those reported with other fish species, such as 4.78 mg kg−1 for grass Kamunde et al., 2001) and with the Cu level changes to affect the
carp Ctenopharyngodon idella (Tang et al., 2013), 5 mg kg−1 for channel growth performance of animal (Cowey, 1992). In this study, the
catfish (Murai et al., 1981) and 5.36 mg kg−1 for grouper (Lin et al., whole-body Cu concentration increased when dietary Cu content in-
2008). However, it is higher than the minimum value for rainbow creased from 0.3 to 4.4 mg kg−1 and then reached the platform when
trout (3 mg kg− 1) (Ogino and Yang, 1980), yellow catfish dietary Cu content increased from 6.2 to 16.1 mg kg−1. This suggests
(3.13 mg kg− 1) (Tan et al., 2011) and large yellow croaker that Russian sturgeons have the ability to keep a balance between Cu
(3.41 mg kg− 1) (Cao et al., 2014), but is lower than that for beluga deficiency and surplus via balancing deposition and excretion. Even
(10.3 mg kg− 1), cobia Rachycentron canadum (11.5 mg kg−1) (Qiao, so, the growth performance was poor in fish fed excessive Cu at
2007) and spotted steed Hemibarbus maculatus Bleeker 16.1 mg kg−1. The results caution that excessive dietary Cu is adverse
(11.5 mg kg−1) (Lu, C.X., 2009). The discrepancy of minimum Cu re- to its growth. Similar results were found in soft-shelled turtles
quirement is related to fish species, age, feed, rearing environment Pelodiscus sinensis (Wu and Huang, 2008), abalone Haliotis discus hannai
and the method of sample analysis (Clearwater et al., 2002; Wapnir, Ino (Wang et al., 2009), grouper (Lin et al., 2010) and large yellow
1998). croaker (Cao et al., 2014). Liver is the central organ for the copper ho-
In the present study, there were no significant differences in meostasis and represents the highest tissue Cu concentration (Heidary
hepatosomatic index (HSI), viscerosomatic index (VSI), condition factor et al., 2012; Luza and Speisky, 1996). The liver Cu concentration has
(CF), whole-body moisture, crude protein, crude lipid and ash between been shown to reflect the dietary Cu intake in Atlantic salmon
all treatments which is similar to results on the impact of Cu on large (Lorentzen et al., 1998) and grouper (Lin et al., 2010). In the present
yellow croaker (Cao et al., 2014). But in previous studies, the over- study, the liver Cu concentration has the similar trends with the
loading of dietary Cu shows significant change of these indexes in Nile whole-body Cu concentrations which values agree well with the growth
tilapia (Shaw and Handy, 2006),yellow catfish (Tan et al., 2011) and be- variable. It has been reported that the concentration of whole-body
luga (Mohseni et al., 2014). The controversial results may be due to the mineral elements was an excellent indicator to assess mineral nutrition
status (Cao et al., 2014; Liang et al., 2012). Based on the whole-body Cu
concentration, the minimum dietary Cu content in juvenile Russian
sturgeon was estimated to be 7.4 mg kg−1. This value is consistent
Table 3 with the result in a previous study on large yellow croaker (Cao et al.,
The body indices of juvenile Russian sturgeon fed the experimental diets with different 2014), which is a little higher than the Cu requirement based on the
levels of copper for 8 weeks.1 change of weight gain rate (6.1 mg kg−1).
Dietary Cu levels (mg/kg) HSI2 VSI3 CF4 Cu intake, distribution, utilization and excretion must be bound to
0.3 3.16 ± 0.88 11.19 ± 1.52 0.89 ± 0.07
some carrier proteins rather than “free” ions throughout these process-
1.5 2.60 ± 0.60 10.52 ± 1.11 0.86 ± 0.07 es (O'Halloran and Culotta, 2000; Rae et al., 1999). Superoxide dismut-
2.4 2.81 ± 0.55 10.79 ± 1.73 0.85 ± 0.05 ase (SOD) is one of the important Cu-containing enzymes identified as
4.4 2.97 ± 0.76 10.62 ± 1.11 0.91 ± 0.07 an essential component in the oxidative stress defense mechanism in
6.2 3.00 ± 0.63 11.36 ± 1.90 0.88 ± 0.07
organisms (Fridovich, 1995; Linder et al., 1998; Vulpe and Packman,
8.3 2.92 ± 0.74 10.54 ± 1.80 0.87 ± 0.09
16.1 3.28 ± 0.79 11.23 ± 1.56 0.89 ± 0.08 1995). Although the specific enzymatic activity of SOD is defined as
the ratio of the enzyme activity to the immunoreactive protein, it is
One-way ANOVA
also a sensitive indicator to assess Cu nutrition status (Louro et al.,
P value 0.191 0.573 0.344
2001; Milne, 1994). The activity of SOD is usually used as an indicator
1
Values are presented as the mean ± SE (n = 3) of one determination per fish, six fish of Cu nutrition status in animals due to the difficult and expensive of
per aquarium and three aquariums per treatment.
2
HSI: hepatosomatic index.
specific antibody production for immunoreactive protein (Lin et al.,
3
VSI: viscerosomatic index. 2008; Tang et al., 2013). In order to fully understand the effect of dietary
4
CF: condition factor. Cu on antioxidant capacity of Russian sturgeon another two antioxidant
122 H. Wang et al. / Aquaculture 454 (2016) 118–124

Table 4
The whole-body compositions (% live weight) of juvenile Russian sturgeon fed the experimental diets with different levels of copper for 8 weeks.1

Dietary Cu levels (mg kg−1) Moisture (%) Crude protein (%) Crude lipid (%) Ash (%)

0.3 78.5 ± 0.25 13.0 ± 0.09 4.48 ± 0.08 2.92 ± 0.12

1.5 78.9 ± 0.65 13.1 ± 0.09 4.49 ± 0.14 2.95 ± 0.09
2.4 78.70 ± 0.52 13.0 ± 0.27 4.65 ± 0.13 2.91 ± 0.07
4.4 78.9 ± 0.56 13.1 ± 0.3 4.56 ± 0.14 2.97 ± 0.12
6.2 78.6 ± 0.26 13.2 ± 0.08 4.60 ± 0.15 2.85 ± 0.07
8.3 79.0 ± 0.64 13.3 ± 0.13 4.54 ± 0.19 2.81 ± 0.09
16.1 78.8 ± 0.75 13.1 ± 0.25 4.45 ± 0.14 2.93 ± 0.04

One-way ANOVA
P value 0.909 0.578 0.594 0.346
1
Values are presented as the mean ± SE (n = 3) of three determinations of pooled samples of 6 fish per aquarium and three aquariums per treatment.

indexes, malondialdehyde (MDA) value and total antioxidant capacity
(T-AOC), were also used in this study (Ghiselli et al., 2000; Janero,
1990). The lowest Cu–Zn SOD activity was found in fish fed diets with
0.3, 1.5 and 16.1 mg Cu kg−1, suggesting that both excessive and defi-
cient dietary Cu content can decrease the hepatic Cu–Zn SOD activity.
The lower activity of Cu–Zn SOD under dietary Cu deficiency is possibly
due to a reduction in Cu–Zn SOD proprotein and insufficient cofactor
concentration to saturate Cu–Zn SOD proprotein (Prohaska and
Brokate, 2001). Free Cu ions have been reported to induce the formation
of reactive oxygen species (ROS) and damage Cu–Zn SOD activity
(Gaetke and Chow, 2003; Schuessel et al., 2005), which may explain
the decreased Cu–Zn SOD activity in fish fed high Cu content diets.
Based on the hepatic Cu–Zn SOD activity, the minimum dietary Cu con-
tent in juvenile Russian sturgeon was estimated to be 6.7 mg kg− 1.
Compared to the Cu–Zn SOD activity, T-AOC showed the same change
trend but the MDA value showed the opposite trend. Significantly
lower T-AOC and higher MDA values were found in fish fed diets with
0.3, 1.5 and 16.1 mg Cu kg−1. The low T-AOC may be related to the de-
creased Cu–Zn SOD activity and fish could not produce other antioxi-
dants to compensate the reduction of T-AOC. Elevated MDA is possibly
a result of poor anti-oxidative capability.
Ceruloplasmin is a Cu carrier protein and Cu-containing enzyme to
deliver Cu from liver to cell of other tissues, which is a good indicator
for the Cu nutrition status in human and fish (Lin et al., 2008; Milne,
1998; Tang et al., 2013). Ceruloplasmin itself is an enzyme possessing
ferroxidase activity (Osaki et al., 1966) and oxidase activity (Goldstein
et al., 1979). In this study, the lowest plasma ceruloplasmin activities
were seen in fish fed Cu deficient diets (0.3 and 1.5 mg Cu kg− 1),
which is in line with the report on hybrid tilapia (Lin et al., 2008;
Shiau and Ning, 2003; Tang et al., 2013). Thus, dietary Cu deficiency
may have negative effect on ceruloplasmin activity. Previous research
has shown that Cu does not affect the rate of synthesis or secretion of
apoceruloplasmin (Gitlin et al., 1992). Therefore, the depressed cerulo-
plasmin activity in fish fed Cu deficient diets may be caused by
apoceruloplasmin failure to incorporate Cu during biosynthesis (Gitlin
et al., 1992). Lower ceruloplasmin activity was observed in fish fed ex-
cessive Cu at 16.1 mg kg−1. Similar, increased free Cu ions concentration
is associated with depressed ceruloplasmin activity in human (Harris,
1993). Therefore, high free Cu ions may account for the lower cerulo-
plasmin activity of fish. Based on the serum ceruloplasmin activity, the
minimum dietary Cu content in juvenile Russian sturgeon was estimat-
ed to be 6.5 mg kg−1.
From the above, the minimum dietary Cu level in Russian sturgeon is
7–8 mg kg−1. This Cu level was determined with semi-purified diets
which will give a theoretical minimum Cu requirements of juvenile
Russian sturgeon weighted 12–70 g. When Cu is supplemented to the
practical diet, the supplemented Cu content should be adjusted accord-
ing to the source of Cu, type of feed ingredients and the size of the fish. It
is reported that the bioavailability of dietary chelated mineral elements
are higher than the inorganic forms such as zinc (Zn) methionine for
channel catfish (Paripatananont and Lovell, 1995), amino acid-chelate
Zn and Cu for rainbow trout (Apines-Amar et al., 2004) and cupric cit-
rate for pig (Armstrong et al., 2004). Besides, more mineral elements
should be supplemented to the practical diet than the purified diet
due to the endogenous inhibitors such as phytic acid and tricalcium
phosphate in the feedstuffs will impair the bioavailability of mineral
elements (Apines-Amar et al., 2004; Gatlin and Wilson, 1984).
5. Conclusion
In conclusion, both dietary Cu deficiency and over-loading reduced
sturgeon growth, feed utilization, antioxidant capacity and ceruloplas-
min activity. Meanwhile, its whole-body and liver Cu concentrations

Table 6
Table 5 Activity of the Cu–Zn superoxide dismutase (Cu–Zn SOD), total antioxidant capacity
The whole-body and liver copper concentration (mg kg−1 dry matter) of juvenile Russian (T-AOC) and malondialdehyde (MDA) value in liver and serum ceruloplasmin activ-
sturgeon fed the experimental diets with different levels of copper for 8 weeks.1 ity of juvenile Russian sturgeon fed the experimental diets with different levels of
copper for 8 weeks.1
Dietary Cu levels Whole-body Cu concentration Liver Cu concentration
(mg kg−1) (mg kg−1)1 (mg kg−1)2 Dietary Cu levels Cu–Zn SOD T-AOC MDA Ceruloplasmin
(mg kg−1) (U/mg prot.) (U/mg prot.) (nmol/mg prot.) activity(U/L)
0.3 1.9 ± 0.09a 10.8 ± 1.12a
1.5 2.2 ± 0.12a 12.1 ± 1.41a 0.3 146.2 ± 2.87a 5.8 ± 0.85a 43.3 ± 5.91b 13.7 ± 1.42a
2.4 2.4 ± 0.05a,b 14.4 ± 2.37a 1.5 148.0 ± 4.46a 6.2 ± 1.00a,b 45.0 ± 4.73b 14.7 ± 1.03a
4.4 2.8 ± 0.12b 22.1 ± 1.71b 2.4 159.3 ± 3.46b 7.5 ± 0.96b,c 38.9 ± 4.92a,b 18.6 ± 1.07b
6.2 3.9 ± 0.26c 37.8 ± 0.62c 4.4 161.3 ± 4.38b 8.1 ± 0.87c 37.2 ± 3.15a,b 19.1 ± 1.70b
8.3 4.2 ± 0.19c 41.0 ± 2.25c,d 6.2 176.8 ± 4.41c 8.4 ± 0.52c 31.4 ± 3.23a 22.8 ± 1.78c
16.1 4.4 ± 0.26c 44.4 ± 1.91d 8.3 174.4 ± 3.38c 8.3 ± 0.60c 32.7 ± 2.86a 22.5 ± 1.97c
16.1 148.9 ± 4.57a 5.0 ± 0.54a 57.7 ± 5.81c 17.7 ± 0.61b
One-way ANOVA
P value b0.001 b0.001 One-way ANOVA
P value b0.001 b0.001 b0.001 b0.001
Means in the same column with different superscript letters are significantly
different (P b 0.05). Means in the same column with different superscript letters are significantly
1
Values are presented as the mean ± SE (n = 3) of three determinations of pooled different (P b 0.05).
1
samples of 6 fish per aquarium for whole-body and 3 fish per aquarium for liver, and three Values are presented as the mean ± SE (n = 3) of one determination per fish, three
aquariums per treatment. fish per aquarium and three aquariums per treatment.
 H. Wang et al. / Aquaculture 454 (2016) 118–124
Fig. 1. Broken-line analyses of weight gain rate (A), whole body Cu concentration (B),
hepatic Cu–Zn SOD activity (C) and serum ceruloplasmin activity (D) on analyzed
dietary copper concentration indicate that the minimum dietary copper content for
Russian sturgeon juveniles was at 6.1, 7.4, 6.7 and 6.5 mg kg−1, respectively.
varied with dietary Cu contents. Thus, a certain concentration of dietary
Cu is essential for juvenile Russian sturgeon. Broken-line regression
analysis on WGR, whole-body Cu concentration, hepatic Cu–Zn SOD
activity and serum ceruloplasmin activity indicated that the minimum
Cu level in semi-purified diet for 12–70 g Russian sturgeon was
7–8 mg kg−1. What we want to remind is that the supplemented Cu
content should be adjusted according to the source of Cu, type of feed
ingredients and the size of fish.
Acknowledgments
This research was supported by grants from the National ‘Twelfth
Five-Year’ Plan for Science & Technology Support (2012BAD25B03),
the National Basic Research Program (973 Program, No.
2014CB138803), the Special Fund for Agro-scientific Research in the
Public Interest (No. 201203065), the National Natural Science Founda-
tion of China (No. 31572629) and partly by the E-Institutes of Shanghai
123
Municipal Education Commission (No. E03009).The authors thank
Haoyong Zhu, Qi Li, Tingting Zhu and other members of the laboratory
for their kind assistance.
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