Aquaculture 310 (2010) 173177

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Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Dietary copper requirement reevaluation for juvenile grouper, Epinephelus

malabaricus, with an organic copper source
Yu-Hung Lin a, Chia-Chi Shih a, Megan Kent a, Shi-Yen Shiau a,b,
a
b

Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Road, Keelung, 202, Taiwan
Department of Food and Nutrition, Providence University, 200 Chung-Chi Road, Shalu, Taichung County 433, Taiwan

a r t i c l e

i n f o

Article history:
Received 13 August 2010
Received in revised form 5 October 2010
Accepted 6 October 2010
Keywords:
Grouper
Copper
Requirement
Organic form

a b s t r a c t
To reevaluate the dietary copper (Cu) requirements of grouper, Epinephelus malabaricus, with an organic Cu
source, Cu peptide was added to a basal diet at 0, 1, 2, 3, 4, 8 and 12 mg Cu/kg, providing actual dietary
values of 0.19, 1.81, 2.55, 3.43, 4.37, 8.99 and 12.81 mg Cu/kg for a total of 7 experimental diets. Each diet
was fed to triplicate groups of sh (initial body weight: 21.60 0.08 g) in a recirculating seawater rearing
system for 8 wks. Weight gain (WG) was highest (P b 0.05) in sh fed the diet with 2.55 mg Cu/kg, followed
by sh fed the diet with 3.43 mg Cu/kg, and lowest in sh fed diets with 0.19, 8.99 and 12.81 mg Cu/kg.
Hepatic copperzinc superoxide dismutase (CuZn SOD) activity was highest in sh fed the diet with
2.55 mg Cu/kg, followed by sh fed diets with 3.43, 8.99, 1.81 and 12.81 mg Cu/kg (in order of decreasing
activity), and lowest in sh fed the diet with 0.19 mg Cu/kg. Hepatic thiobarbituric acid-reactive substance
(TBARS) value was lowest in sh fed diets with 2.55 and 3.43 mg Cu/kg, followed by sh fed diets with 0.19
and 4.37 mg Cu/kg, and highest in sh fed the diet with 12.81 mg Cu/kg. Analysis by broken-line regression
of WG, hepatic CuZn SOD activity and TBARS value, and by linear regression of whole-body Cu retention of
the sh indicated that the adequate dietary Cu requirement for grouper determined by Cu-peptide is about
23 mg Cu/kg.
2010 Elsevier B.V. All rights reserved.

1. Introduction
Trace minerals are an essential component of sh nutrition which
lags in inquiry when compared to research devoted to other nutrients.
Although sh can absorb minerals across the gill membrane and
intestinal mucosa, diet is considered to be the major source of
minerals for sh (Watanabe et al., 1997). Trace minerals are largely
present in their inorganic chemical forms; few minerals, namely
selenium (Se), copper (Cu), iron (Fe), zinc (Zn), and manganese (Mn)
also have an organic chemical form.
Copper functions in hematopoiesis and as a central structural
component in numerous Cu-dependent enzymes including lysyl
oxidase, cytochrome c oxidase (CCO), ferroxidase, tyrosinase and
superoxide dismutase (SOD) (O'Dell, 1976). Dietary Cu requirements
have been quantied in common carp (Ogino and Yang, 1980), rainbow
trout (Julshamn et al., 1988), hybrid tilapia (Shiau and Ning, 2003),
channel catsh (Murai et al., 1981; Gatlin and Wilson, 1986), Atlantic
salmon (Lorentzen et al., 1998) and grouper (Lin et al., 2008), ranging
from 1.5 to 6 mg Cu/kg. It should be noted that the dietary Cu sources
used in these studies were all inorganic forms. Chick (Guo et al., 2001;
Mondal et al., 2007) and lamb (Eckert et al., 1999; Senthilkumar et al.,

Corresponding author. Fax: +886 2 2462 1684.

E-mail address: syshiau@pu.edu.tw (S.-Y. Shiau).
0044-8486/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2010.10.004

2009) have been reported to utilize organic Cu better than inorganic Cu,
which was evident from tissue storage or plasma ceruloplasmin activity.
Pork (Coffey et al., 1994) and cattle (Kegley and Spears, 1994; Ward
et al., 1996; Du et al., 1996), however, were reported as having similar
utilization of both forms of Cu. There is a lack of research with regard to
the utilization of organic Cu in sh.
The purpose of the present study was to estimate the dietary Cu
requirements for grouper using an organic copper source. The
utilization of organic and inorganic Cu by grouper was also compared.
2. Materials and methods
2.1. Diet preparation
Diet formulation and proximate composition analysis (AOAC,
1995) are shown in Table 1. Casein (Sigma Chemical Co., St. Louis,
MO), sh oil (Semi-rened sh oil, Oleaginosa Victoria S.A., Peru) and
corn oil and starch (Tai-Tang Industrial, Taiwan; Sigma Chemical)
were used as dietary protein, lipid and carbohydrate sources, respectively. All diets were formulated to be isoenergetic at 360 kcal/
100 g diet as recommended by Shiau and Lan (1996). An attractant
that had a similar chemical composition to squid mantle tissue
(Mackie and Mitchell, 1985) was added at 6% to all diets to increase
palatability and diet acceptance. The basal diet was supplemented
with copper peptide (Cu peptide, Alltech Inc., Lexington Kentucky,

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Y.-H. Lin et al. / Aquaculture 310 (2010) 173177

Table 1
Formulation and proximate composition of the basal dieta.
%
Ingredient
Casein
Fish oil
Corn oil
Corn starch
Vitamin mixtureb
Mineral mixturec
Attractantd
Carboxylmethylcellulose
Alpha-cellulose

51
4.5
4.5
16.7
2
4
6
3
8.3

Proximate composition
Moisture
Ash
Crude protein
Ether extract

9.29
3.40
48.11
8.47

a
The basal diet was supplemented with copper peptide at 0, 1, 2, 3, 4, 8 and 12 mg
Cu/kg diet.
b
Vitamin mixture (g/kg mixture): thiamin hydrochloride, 2.5; riboavin, 10;
calcium pantothenate, 25; nicotinic acid, 37.5; pyridoxine hydrochloride, 2.5; folic
acid, 0.75; inositol, 100; L-ascorbyl-2-monophosphate Mg, 5; choline chloride, 250;
menadione, 2; alpha-tocopheryl acetate, 5; retinyl acetate, 1; cholecalciferol, 0.0025;
biotin, 0.25; vitamin B12, 0.05. All ingredients were diluted with alpha-cellulose to 1 g.
c
Mineral mixture (g/kg mixture): calcium lactate, 327; K2PO4, 239.8; CaHPO4 2H2O,
135.8; MgSO4 7H2O, 132; Na2HPO4 2H2O, 87.2; NaCl, 43.5; ferric citrate, 29.7; ZnSO4
7H2O, 3; CoCl2 6H2O, 1; MnSO4 H2O, 0.8; KI, 0.15; AlCl3 6H2O, 0.15; selenomethionine,
0.02. All ingredients were diluted with alpha-cellulose to 1 g.
d
As mg/100 g diet: L-aspartic acid, 18; L-threonine, 44; L-serine, 33; L-glutamic
acid, 53; L-valine, 36; L-methionine, 36; L-isoleucine, 29; L-leucine, 55; L-tyrosine, 22;
L-phenylalanine, 29; L-lysine-HCl, 29; L-histidine-HCl, 15; L-proline, 1456; L-alanine,
273; L-arginine, 228; taurine, 337; glycine, 892; betain-HCl, 910; trimethylamine-HCl,
91; trimethylamine n-oxide HCl, 1138; hypoxanthine, 47; inosine, 25; adenosine-5'monophosphate, 40; L-(+)-lactic acid, 91; alpha-cellulose, 80 (Mackie and Mitchell,
1985).

USA) at 0, 1, 2, 3, 4, 8 and 12 mg Cu/kg diet. Dietary Cu concentrations

were determined by ame atomic absorption photometry (Z-5000,
Hitachi Co., Tokyo, Japan) after wet ashing (AOAC, 1995) to be 0.19
(0), 1.81 (1), 2.55 (2), 3.43 (3), 4.37 (4), 8.99 (8) and 12.81 (12) mg
Cu/kg diet, respectively. All ingredients of the experimental diet were
mixed, and then mixed with enough cold water to form stiff dough.
This was then passed through a mincer with die (1.2 mm), and the
resulting strands were dried using an electrical fan at 20 C. After
drying, the diets were broken up and sieved into pellets (1.2 mm in
diameter) and stored at 20 C until used.
2.2. Experimental procedure
E. malabaricus juveniles were obtained from a local hatchery
(Pingtung, Taiwan). Upon arrival, they were acclimated to laboratory
conditions for 4 weeks in a 1000 L plastic tank and fed a commercial
diet (Uni-President Enterprise Corp., Tainan, Taiwan). The proximate
composition and Cu concentration of the commercial diet were as
follows: moisture, 11.7%; crude protein, 43.3%; lipid, 8.8%; ash, 9.3%;
Cu, 7.5 mg Cu/kg diet. At the beginning of the experiment, 10 sh
(mean initial weight: 21.60 0.08 g) were stocked in each aquarium
(0.305 0.610 0.555 m) designed to contain 60 L. Each experimental
diet was fed to sh in three randomly chosen aquaria. Each aquarium
was part of a semi-closed recirculating system with a common
reservoir of seawater at 29 to 32 salinity. Water temperature of the
rearing system was maintained at 29 1 C. The water was circulated
at 2 L/min through two separate biological lters to remove waste
products. Half of the make up water in the system was exchanged
daily. Cu concentration in rearing water was monitored regularly and
averaged 1.25 0.12 g Cu/L.
Fish were fed 3% of their body weight per day. The diet divided
into two equal meals fed at 0830 and 1630 h. Grouper nished their

ration within 12 min after feeding. Fish were weighed once every
2 weeks and the daily ration adjusted accordingly. Excess feed and
fecal material was siphoned daily. A photoperiod of 12 h light (0800
to 2000 h), 12 h dark was used. Any dead sh were removed and not
replaced during the experiment. Fish were fed the test diets for an
8-week period.
At the end of the feeding trial, percentage of body weight gain (WG)
in each aquarium [100 (nal body weight initial body weight)/initial
body weight], feed efciency (FE) [(nal body weight initial body
weight)/feed intake], and survival [100 (nal sh number/initial sh
number)] were calculated.
After the nal weight was noted, two sh were randomly sampled
from each aquarium and pooled for whole body Cu concentration
analysis (AOAC, 1995). Six other sh were randomly selected per
aquarium and livers were removed and pooled for determining Cu
concentration, CuZn superoxide dismutase (SOD) activity, and
thiobarbituric acid reaction substance (TBARS) values. Hepatic Cu
concentrations were analyzed by the same methods as feed analysis
(AOAC, 1995). The CuZn SOD activity was assessed using a commercial kit (Merck Co., Germany). The TBARS values were analyzed
according to the method of Uchiyama and Mihara (1978). All analyses
were conducted in triplicate.
2.3. Statistical analysis
Each experimental diet was fed to three groups of sh according
to a completely randomized design. Results were analyzed by oneway analysis of variance (ANOVA) using the SAS/PC statistical software (SAS Inst. Inc., Cary, NC), and signicance was set at P b 0.05.
Multiple comparisons among means were made with Duncan's new
multiple range test. Dietary Cu requirements of grouper were
estimated by the broken-line regression method based on weight
gain, hepatic CuZn SOD activity and TBARS value and by linear
regression analysis of the dietary Cu levels vs. whole body Cu retention values.
3. Results
Weight gain (WG) was highest (P b 0.05) in sh fed the diet with
2.55 mg Cu/kg, followed by sh fed the diet with 3.43 mg Cu/kg, and
lowest in sh fed diets with 0.19, 8.99 and 12.81 mg Cu/kg (Table 2).
Feed efciency was higher in sh fed the diet with 2.55 mg Cu/kg than
that in sh fed diets with 0.19, 1.81 and 8.99 mg Cu/kg. Survival was
not affected by the dietary treatments.
Hepatic Cu concentration was highest in sh fed the diet with
12.81 mg Cu/kg diet, intermediate in sh fed the diet with 8.99 mg Cu/
kg diet, then 4.37, 3.43, 2.55 and 1.81 mg Cu/kg, and lowest in sh fed
the diet with 0.19 mg Cu/kg. Differences between each of the three
groups were statistically signicant (P b 0.05) (Table 3). The hepatic
Table 2
Weight gain, feed efciency (FE) and survival of grouper fed different diets for
8 weeks.1
Analyzed dietary
Cu concentration

Weight gain

mg/kg diet

0.19
1.81
2.55
3.43
4.37
8.99
12.81

183 4.95ab
204 10.23bc
247 21.03d
212 19.10c
204 0.13bc
179 7.49ab
179 17.35a

FE

Survival
%

0.48 0.03a
0.60 0.04ab
0.75 0.09c
0.67 0.09bc
0.64 0.01bc
0.49 0.04a
0.49 0.12a

90.00 14.14
100.00 0.00
100.00 0.00
96.67 0.00
95.00 7.07
93.33 5.77
90.00 10.00

Different superscripts in the column indicate signicant (P b 0.05) difference between

different dietary treatments.
1
Values are means SD from three groups of sh (n = 3) with the remaining 10 sh
per group.

Y.-H. Lin et al. / Aquaculture 310 (2010) 173177

175

Table 3
Hepatic Cu concentration, thiobarbituric acid reactive substances (TBARS) value and
CuZn superoxide dismutase (CuZn SOD) activity of grouper fed different diets for
8 weeks.1
Analyzed dietary
Cu concentration

Cu

TBARS

CuZn SOD

mg/kg diet

g/g tissue

nmol MDA/g tissue

units/g tissue

0.19
1.81
2.55
3.43
4.37
8.99
12.81

5.75 0.32a
6.65 0.15b
7.17 0.38bc
7.55 0.37c
8.57 0.38d
12.78 0.21e
15.62 0.52f

46.02 3.07bc
36.82 7.99ab
29.66 4.46a
30.34 5.82a
45.00 4.09bc
51.14 7.16 cd
60.34 4.69d

16.94 1.18a
56.09 4.48b
97.57 7.32f
84.25 2.12e
66.37 4.15 cd
71.42 4.54d
60.10 3.17bc

Different superscripts in the column indicate signicant (P b 0.05) difference between

different dietary treatments.
1
Values are means SD from three groups of sh (n = 3) with 6 sh per group.

thiobarbituric acid-reactive substance (TBARS) value was lowest in

sh fed diets with 2.55 and 3.43 mg Cu/kg, followed by sh fed diets
with 0.19 and 4.37 mg Cu/kg, and highest in sh fed the diet with
12.81 mg Cu/kg. Hepatic copperzinc superoxide dismutase (CuZn
SOD) activity was highest in sh fed the diet with 2.55 mg Cu/kg,
followed by sh fed the diet with 3.43 mg Cu/kg, then in sh fed diets
with 8.99 mg Cu/kg, 1.81 and 12.81 mg Cu/kg, and lowest in sh fed
the diet with 0.19 mg Cu/kg.
Analysis by broken-line regression of WG, hepatic CuZn SOD
activity, and TBARS value of the sh indicated that the adequate
requirements of dietary copper were 2.19, 2.43 and 2.38 mg Cu/kg,
respectively (Fig. 1).
Whole-body Cu content in sh generally increased as the dietary
Cu supplementation level increased (Y = 9.16X + 73.48, R2 = 0.97,
Table 4). When these data were used to calculate the whole-body Cu
retention for each group of sh, linear regression analysis of the
dietary Cu level vs. whole-body Cu retention values (Fig. 2) indicated
a Cu requirement of 3.34 mg/kg diet (Y = 32.24X + 107.84,
R2 = 0.97) using the organic copper source.
4. Discussion
In the present study, dietary Cu requirements determined with
organic Cu (Cu peptide) for grouper were 23 mg Cu/kg diet. This
value is considerably lower than the value (46 mg Cu/kg diet)
obtained previously (Lin et al., 2008) for the same species fed inorganic Cu (CuSO4). Fish size, basal diet composition, rearing system,
water temperature, salinity, and water Cu concentration were
identical in the two studies. Highest WG of sh obtained (247%,
Table 2) compares favorably with those of previous studies in our
laboratory (Lin and Shiau, 2003, 2007; Lin et al., 2008), in which 250
300% WG was observed in similar sized sh fed a nutritionally
adequate diet for 8 wks. Generally, sh growth under controlled
laboratory condition would be slower compared to commercial
husbandry. Whether requirement for Cu would be higher due to
faster growth is interesting. Since Cu requirement is presented on the
basis of feed intake, e.g. mg Cu/kg diet. Therefore, faster growth sh
under commercial rearing condition could get their required Cu by
ingesting more feed.
To use broken-line model or linear regression model (continuous
response) to quantify nutrient requirement for sh is an open issue.
Zeitoun et al. (1976) have suggested the use of polynomial regression
as a means of approximating the relationship between weight gain
and essential nutrient intake. As indicated by Zeitoun et al., the value
corresponding to maximal gain estimated by a quadratic regression is
dened as the maximum concentration of dietary nutrient that
produces optimal growth, and beyond which growth is depressed. In
the present study, when the polynomial (second) regression model

Fig. 1. Broken-line analysis of weight gain, hepatic copperzinc superoxide dismutase

(CuZn SOD) activity, and thiobarbituric acid reaction substance (TBARS) values on
analyzed dietary copper concentration indicate that the optimal Cu levels for juvenile
grouper are 2.19, 2.43 and 2.38 mg Cu/kg diet, respectively. The 95% condence interval
around the prediction are 2.41 N N 1.97 (weight gain); 2.55 N N 2.31 (CuZn SOD); and
2.68 N N 2.08 (TBARS). Each point represents the value of three aquaria within a
treatment, with 10 sh per aquarium for weight gain and 6 sh per aquarium for
hepatic CuZn SOD activity and TBARS value, respectively.

(Zeitoun et al., 1976) was employed to express the relationship

between sh growth and copper levels, the regression line:
Y=

0:56X + 4:82X + 200:59;R = 0:36

was obtained and a growth peak was reached when dietary Cu was
4.23 mg/kg diet. It should be noted that the sh growth declined
Table 4
Whole-body copper retention of grouper fed diets containing various levels of copper
for 8 weeks.1
Analyzed
dietary Cu
concentration

Initial
whole-body
Cu content

Total
Cu fed

Final
whole-body
Cu content

Finalinitial
whole-body
Cu content

Whole-body
Cu retention2

mg/kg diet

0.19
1.81
2.55
3.43
4.37
8.99
12.81

11.53
11.53
11.53
11.53
11.53
11.53
11.53

6.98
67.29
107.57
124.09
157.22
314.33
437.15

75.33
139.73
170.91
137.42
159.68
128.92
140.80

63.80
128.20
159.38
125.89
148.15
117.39
129.27

56.81
60.91
51.81
1.80
9.07
196.93
307.87

Different superscripts in the column indicate signicant (P b 0.05) difference between

different dietary treatments.
1
Values are means SD from three groups of sh (n = 3) with 2 sh per group.
2
(Final initial whole-body Cu content) total Cu fed.

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Y.-H. Lin et al. / Aquaculture 310 (2010) 173177

Fig. 2. The relationship between dietary copper concentration and whole body copper
retention in grouper. Each point represents the mean of three groups of sh (n = 3) with 2
sh per group. Requirement derived with the linear regression method is 3.34 mg Cu/kg
diet (Y = 32.24X+ 107.84, R2 = 0.97).

when the supplement Cu levels were higher than 3.43 mg/kg diet. It is
also noted that weight gain in the 2.55 mg/kg group was signicantly
higher than in the groups with supplementation 3.43 mg/kg. It is
very doubtful that the requirement should be higher than 2.55 mg/kg.
Moreover, low correlation coefcient value (R2 = 0.36) indicates that
data does not t the model well. Thus, the broken-line model appears
to better t the data for sh growth and suggest that dietary Cu at
2.19 mg/kg diet is adequate to meet the requirement for growth of
grouper.
Cowey (1976) suggested that besides growth, other physiological
parameters such as tissue enzyme activity may also be appropriate to
quantify nutrient requirement in sh. In the present study, hepatic
CuZn SOD activity and TBARS value were also used to estimate the Cu
requirement in grouper (Fig. 1). These results agreed well with that
obtained from growth performance of the sh.
The lower Cu requirement for grouper found in this study
may be attributed to the organic Cu source (Cu peptide). Organic
minerals which are commonly used in cultured animal feeds are
chelated with organic compounds, such as carbohydrates, amino
acids and hydrolyzed protein (peptides). If an element is chelated
by a compound that can either release it in its ionic form at the site
of absorption or readily be absorbed as an intact chelate, this
chelation may greatly enhance the absorption of the element by
preventing its conversion to insoluble chemical compounds in the
intestine or by preventing its strong adsorption of insoluble colloids
(Apines et al., 2003). In addition, adding chelated trace minerals to
the diet may minimize inhibitory interaction with other minerals
and dietary factors (e.g., phytate and ber) compared with inorganic sources (Peter and Mahan, 2008). Another potential benet of
stable chelated organic minerals for aquatic species is reduced
solubility in water. Researchers have reported that several organic
Cu chelates were less soluble than the inorganic form of Cu, CuSO4,
when tested at similar concentrations in deionized water (Guo
et al., 2001).
Although there may be numerous advantages to using organic Cu
sources in aquaculture, organic Cu studies in sh nutrition thus far
have only been done on one sh species. Apines et al. (2003) and
Apines-Amar et al. (2004) indicated that trace elements chelated with
amino acids, which include Cu, Zn, and Mn, seem to be more available
for mineral deposition than those from inorganic compounds in
rainbow trout. It should be noted that in the two rainbow trout studies
the authors were able to ignore the interaction of Cu with other trace
elements, i.e. Zn and Mn. This is because organic minerals are

generally considered less sensitive to the inhibitory action of other

compounds due to the different absorption pathway (Ashmead,
1992). In the Apines-Amar et al. studies, dietary organic Cu utilization
was unclear. Our present study provided primary information on
organic Cu utilization in sh.
Previous studies have shown that the amount of dietary inorganic
Cu required to reduce growth performance varies between sh
species. For example, 8 mg Cu/kg (2 adequate) and 16 mg Cu/kg (3
adequate) inorganic Cu were reported to reduce growth of tilapia
(Shiau and Ning, 2003) and channel catsh (Gatlin and Wilson, 1986),
respectively. In grouper studies, however, growth was depressed in
sh fed diets with 8.97 mg Cu/kg (~2 adequate) inorganic Cu
(CuSO4) (Lin et al., 2008) and 8.99 mg Cu/kg (~4 adequate)
organic Cu (Cu peptide) (Table 2). These differential levels required
for growth reduction suggest that grouper have a lower toxicity
threshold for inorganic Cu than organic Cu.
The liver is the major Cu storage organ in sh (Miller et al., 1993;
Lorentzen et al., 1998; Shiau and Ning, 2003). In the present study,
hepatic Cu concentration increased with increasing dietary Cu
supplementation levels (Y = 0.82X + 5.18, R2 = 0.99, Table 3). A
similar trend was observed in our previous study (Y = 3.62X + 7.08,
R2 = 0.86, Lin et al., 2008). By way of assessment through the slope
ratio method (Fox et al., 1981), this previous study also suggested that
hepatic Cu accumulation rate was higher in sh fed diets with
inorganic Cu than in sh fed diets with organic Cu. This phenomenon
was also reported in pig (Coffey et al., 1994), cow (Du et al., 1996) and
ewe (Eckert et al., 1999). High levels of Cu accumulation in the liver
might cause toxic oxidative stress for sh (Lin and Shiau, 2007). This
lower accumulation rate of Cu in organic Cu-fed sh also provides
additional evidence for why organic Cu had a higher toxicity threshold
for grouper than inorganic Cu in this study.
The different metabolic pathways of organic and inorganic Cu,
however, might inuence the Cu storage site. For instance, muscle and
liver were the major storage tissues in sh fed diets with organic and
inorganic Se, respectively (Wang and Lovell, 1997). Other tissues such
as bone, scale, kidney or muscle, were not assessed in the present
study. Further study with regard to this is needed.
Although Cu is central to many enzymatic reactions, oxidative
stress caused by hepatic Cu accumulation or deciency must also be
considered in order to determine optimal dietary levels of Cu. The
family of antioxidant enzymes, called the superoxide dismutase
(SOD), function to remove damaging reactive oxygen species (ROS)
from the cellular environment by catalyzing the dismutation of two
superoxide radicals to hydrogen peroxide and oxygen (Fattman et al.,
2003). Depressed hepatic CuZn SOD activity in grouper was observed
when the dietary Cu was insufcient ( 1.81 mg Cu/kg diet, Table 3)
and in excess (12.81 mg Cu/kg diet, Table 3). TBARS analysis is one of
the most popular and commonly used indicators of oxidative stress,
which works by detecting lipid oxidation (Rosmini et al., 1996). The
hepatic TBARS values showed an inverse trend with the performance
of hepatic CuZn SOD activity (Table 3). The high hepatic TBARS
values in sh fed the Cu-decient diet might be due to low activity of
CuZn SOD. Moreover, free Cu has been reported to induce the
oxidation and destruction of SOD in vitro (Cecconi et al., 2002). It is
suggested, then, that excess Cu stored in the liver caused high
oxidative stress and destroyed the enzyme activity. This further
claries why the lower Cu accumulation rate in organic Cu may be
favorable for sh tness.
The higher cost of most mineral chelates relative to inorganic
sources has generally limited their use in aquaculture to date. The
prices of feed-grade CuSO4 and Cu-peptide are about 2 and 13 US
dollars per kilogram, respectively (Nice Garden Industrial Co., Ltd.,
Tainan, Taiwan; Uni-President Enterprise Corp., Tainan, Taiwan). If
only considering cost, a half-supplementation level of organic Cu,
which, according to this study, should produce growth equivalent to a
whole supplementation of inorganic Cu, in sh diets may not be a

Y.-H. Lin et al. / Aquaculture 310 (2010) 173177

good candidate as a dietary source. Nevertheless, Cu solubility and its

excretion from cultured animals due to high ingestion continue to
draw environmental concern and hence lend importance to organic
mineral research.
In conclusion, this study of sh utilization of organic Cu was the
rst of its kind, revealing through growth, physiological response, and
Cu retention data that grouper can better utilize organic Cu (Cu
peptide) than inorganic Cu (CuSO4). Although the cost of using
organic minerals is high even relative to the lower adequate dietary
Cu requirement for organic-form Cu found in this study, the lower
environmental impact due to higher utilization rate and lower
solubility support the need for further research.
Acknowledgements
This work was supported by a grant from the National Science
Council of the Republic of China, grant number NSC 98-2321-B-126001-MY3.
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