Aquaculture Nutrition
2017 23; 171–180
..........................................................................................
1 1 2 1
1 2
School of Fisheries, Aquaculture and Aquatic Sciences, Auburn University, Auburn, AL, USA;
and Nutritional Engineering, Wuhan Polytechnic University, Wuhan, China
Two trials were conducted to evaluate the performance of
Pacific white shrimp Litopenaeus vannamei offered diets con-
taining various copper (Cu) levels from Cu hydroxychloride
(Cu2(OH)3Cl) containing 58.81% copper in the clear water
recirculating system. In both trials, the basal diet (360 g kg 1
protein, 80 g kg 1 lipid) containing approximately
10 mg Cu kg 1 was primarily comprised of fishmeal, soy-
bean meal, corn protein concentrate and whole wheat. In trial
1, test diets were produced supplementing the basal diet with
5, 10, 20, 40 and 60 mg Cu kg 1 from Cu hydroxychloride.
Four replicate groups of 15 shrimp per tank (initial weight
0.28 g) were offered diets in slight excess over 8 weeks. In
trial 2, the basal diet was supplemented with 30, 90, 150, 210
and 270 mg Cu kg 1 from Cu hydroxychloride. Seven repli-
cate groups of 15 shrimp per tank (initial weight 0.22 g) were
offered feed in slight excess over 7 weeks. At the end of the
two growth trials, no significant differences were observed in
final biomass, final mean weight, percentage weight gain, feed
conversion ratio (FCR) and survival. In trial 1, the Cu con-
centrations of the carapace, hepatopancreas and whole
shrimp linearly increased with increasing dietary Cu supple-
ments. In trial 2, polynomial regression of Cu concentrations
of the carapace, hepatopancreas and whole shrimp against
analysed dietary Cu content indicated that a plateau was
reached at 215 mg analysed Cu kg 1. Results of this study
indicate that there was no negative effect of high levels of Cu
supplement with regard to growth and survival. Tissue levels
generally increased up to around 200 mg Cu kg 1 diet and
then decreased, possibly indicating a shift in physiology.
KEY WORDS:bioavailability, copper hydroxychloride, growth
performance, Litopenaeus vannamei, tissue response
..............................................................................................
ª 2015 John Wiley & Sons Ltd
doi: 10.1111/anu.12378
School of Animal Science
Received 1 July 2015; accepted 25 August 2015
Correspondence: D.A. Davis, School of Fisheries, Aquaculture, and
Aquatic Sciences, 203 Swingle Hall, Auburn University, Auburn, AL
36849-5419, USA. E-mail: davisda@auburn.edu
The essentiality of copper (Cu) for several species of
shrimp and fish is well documented (O’Dell 1976; Depledge
1989; Watanabe et al. 1997; NRC 2011). Cu is involved in
many important metabolic processes such as an allosteric
factor for numerous Cu-dependent enzyme systems (O’Dell
1976; Rubino & Franz 2012), electron carrier (Hay 1984),
induction to oxidative stress (Al-Subiai et al. 2011) and
transcriptional response (Varotto et al. 2013). In malacos-
traca crustacean, Cu plays a particularly vital role in oxy-
gen transport by haemocyanin (Terwilliger 1998). Studies
have demonstrated that Cu is also essential for moulting
and reproduction in shrimp (Rao & Anjaneyulu 2008; Rao
et al. 2008).
Potential Cu sources for dietary Cu supplementation in
the commercial feed industry should be assessed for effi-
cacy and bioavailability. Cu sulphate is the most common
Cu source to include in animal and aquatic feeds.
Another Cu source is Cu hydroxychloride (Cu2(OH)3Cl),
often referred to as tribasic copper chloride (TBCC),
dicopper chloride trihydroxide or copper trihydroxyl chlo-
ride. Cu hydroxychloride possesses several valuable char-
acteristics such as a lower solubility in water and very
low chemical reactivity, thus making it an attractive Cu
source in animal and aquatic feeds (Cromwell et al. 1998).
Several investigators have demonstrated Cu hydroxychlo-
ride as an effective copper source in pigs (Cromwell et al.
1998), broilers (Luo et al. 2005), lambs (Cheng et al. 2008),
crucian carp Carassius auratus gibelio (Shao et al. 2010),
blunt snout bream Megalobrama amblycephala (Shao et al.
2012) and Pacific white shrimp Litopenaeus vannamei
(Zhou et al. 2014). With the exception of the study from
Zhou et al. (2014), no information on dietary Cu supple-
mentation from Cu hydroxychloride in shrimp feed is
available. Zhou et al. (2014) demonstrated that Cu hydrox-
ychloride is an effective dietary Cu supplement in shrimp
cultured in systems with or without primary productivity.
However, the tested range of Cu levels were limited to 21
to 45 mg Cu kg 1 in order to confer to the reported Cu
requirement levels of 16-32 mg kg 1 (NRC 2011), and
there is no available data assessing high supplementation
levels from Cu hydroxychloride in shrimp feed. The
objective of this study was to confirm the efficacy of
Cu hydroxychloride in practical diets across a range of
Cu levels (at low in trial 1 or both low and high in
trial 2) which included those that may be considered
therapeutic.
In both trials, Cu hydroxychloride (Aquashield C,
Micronutrients, Div. of Heritage Technologies, LCC, Indi-
anapolis, IN, USA) was supplied to a basal diet mainly
composed of fishmeal, soybean meal, corn protein concen-
trate and whole wheat containing approximately
10 mg Cu kg 1 (Table 1 and 2). In trial 1, test diets were
produced by supplementing the basal diet with 5, 10, 20,
40 and 60 mg Cu kg 1 from Cu hydroxychloride. In trial

Table 1 Composition of six practical diets formulated with increasing levels of dietary copper 0, 5, 10, 20, 40 and 60 mg kg 1
from Cu
hydroxychloride. The diets were formulated to be isonitrogenous at 360 g kg 1 protein and 80 g kg 1 lipid

Ingredients (g kg 1, as-is) B10 H15 H20 H30 H50 H70

Fishmeal1 45.0 45.0 45.0 45.0 45.0 45.0

Soybean meal2 495.5 495.5 495.5 495.5 495.5 495.5
Corn protein concentrate3 56.5 56.5 56.5 56.5 56.5 56.5
Corn starch4 32.0 32.0 32.0 32.0 32.0 32.0
Whole wheat5 250.1 250.1 250.1 250.1 250.1 250.1
Fish oil1 58.4 58.4 58.4 58.4 58.4 58.4
Mineral premix Cu-free6 5.0 5.0 5.0 5.0 5.0 5.0
Vitamin premix7 18.0 18.0 18.0 18.0 18.0 18.0
Choline chloride5 2.0 2.0 2.0 2.0 2.0 2.0
Stay C8 1.0 1.0 1.0 1.0 1.0 1.0
CaP dibasic9 25.0 25.0 25.0 25.0 25.0 25.0
Lecithin10 10.0 10.0 10.0 10.0 10.0 10.0
Cholesterol11 0.5 0.5 0.5 0.5 0.5 0.5
Cellulose12 1.0 0.992 0.983 0.966 0.932 0.898
Cu hydroxychloride13 – 0.009 0.017 0.034 0.068 0.102
Proximate composition (g kg 1, as-is)
Crude protein 356.0 340.0 339.0 361.0 354.0 357.0
Moisture 65.7 78.9 73.3 78.0 76.2 68.6
Lipid 91.7 86.9 86.2 85.3 86.1 89.2
Ash 70.2 69.8 71.9 67.4 70.0 68.2
Cu (mg kg 1) 10.0 15.0 19.0 29.0 48.0 66.0
1
Omega Protein Inc., Reedville, VA, USA.
2
Faithway Feed Co., Guntersville, AL, USA.
3
Empyreal 75 Cargill Corn Milling, Cargill, Inc., Blair, NE, USA.
4
Grain Processing Corporation, Muscatine, IA, USA.
5
MP Biochemicals Inc., Solon, OH, USA.
6
Trace mineral premix Cu free (g kg 1): cobalt chloride, 0.04; ferrous sulphate, 40.0; magnesium sulphate heptahydrate, 283.98; manga-
nous sulphate monohydrate, 6.50; potassium iodide, 0.67; sodium selenite, 0.10; zinc sulphate heptahydrate, 131.93; filler, 534.28.
7
Vitamin premix (g kg 1): thiamine HCL, 4.95; riboflavin, 3.83; pyridoxine HCL, 4.00; Ca pantothenate, 10.00; nicotinic acid, 10.00; bio-
tin, 0.50; folic acid, 4.00; cyanocobalamin, 0.05; inositol, 25.00; vitamin A acetate (500,000 IU/g), 0.32; vitamin D3 (1,000,000 IU/g), 0.05;
vitamin E acetate (250 IU/g), 80.00; menadione, 0.50; alpha-cellulose, 756.81.
8
Stay C, (L-ascorbyl-2-polyphosphate 35%), Roche Vitamins Inc., Parsippany, NJ, USA.
9
Fisher Scientific, Fair Lawn, NJ, USA.
10
Solae Company, St. Louis, MO, USA.
11
USB Biochemicals, Cleveland, OH, USA.
12
Sigma Chemical Co., St. Louis, MO, USA.
13
Micronutrients, Div. of Heritage Technologies, LCC, Indianapolis, IN, USA.
..............................................................................................

Aquaculture Nutrition, 23; 171–180 ª 2015 John Wiley & Sons Ltd
 2, the similar basal diet was supplemented with 30, 90, 150,
210 and 270 mg Cu kg 1 from Cu hydroxychloride. The
formulation (360 g kg 1 protein, 80 g kg 1 lipid) and anal-
ysed composition of the diets in trials 1 and 2 are shown in
Tables 1 and 2, respectively (analysed by the Experiment
Station Chemical Laboratories, University of Missouri,
Columbia, MO, USA).
Experimental diets in two trials were produced at the
Aquatic Animal Nutrition Laboratory in the School of
Fisheries, Aquaculture and Aquatic Sciences, Auburn
University (Auburn, AL, USA), using standard protocol
for shrimp feed. The procedure for preparing diets
was described by Zhou et al. (2014). Briefly, all dry
ingredients were weighed and thoroughly mixed before
adding the fish oil and boiling water. The diets were
then pelleted utilizing a food mixer with 3-mm die, and
the resulting pellets were dried to less than 10% mois-
ture. Subsequently, the diets were stored at 20 °C until
used.
Shrimp postlarvae (PL) L. vannamei were purchased
from Shrimp Improvement Systems (Islamorada, FL,
USA) and nursed in an indoor recirculating system (six
6000-L nursery tanks). PL were fed four times daily with
commercial PL feeds (Zeigler Bros., Inc., Gardners, PA,
USA; 500 g kg 1 protein and 150 g kg 1 lipid) and then
switching to crumbled commercial shrimp feed (Rangen
Inc., Buhl, ID, USA; 400 g kg 1 protein and 80 g kg 1
lipid) using an equivalent procedures as described by
Sookying & Davis (2012). Two trials were conducted at the
Alabama Department of Conservation and Natural
Resources Marine Resource Division, Claude Peteet Mari-
culture Center (Gulf Shores, AL, USA).

Table 2 Composition of six diets formulated with graded levels of dietary copper 0, 30, 90, 150, 210 and 270 mg kg 1 from Cu hydroxy-
chloride containing 588.1 g kg 1 copper. The diets were formulated to be isonitrogenous at 360 g kg 1 protein and 80 g kg 1 lipid

Ingredients (g kg 1, as-is) B10 H40 H100 H160 H220 H280

1
Fishmeal 65.0 65.0 65.0 65.0 65.0 65.0
Soybean meal2 500.0 500.0 500.0 500.0 500.0 500.0
Corn protein concentrate3 63.5 63.5 63.5 63.5 63.5 63.5
Corn starch4 25.4 25.4 25.4 25.4 25.4 25.4
Whole wheat4 220.0 220.0 220.0 220.0 220.0 220.0
Fish oil1 55.6 55.6 55.6 55.6 55.6 55.6
Mineral premix Cu-free5 5.0 5.0 5.0 5.0 5.0 5.0
Vitamin premix6 18.0 18.0 18.0 18.0 18.0 18.0
Choline chloride4 2.0 2.0 2.0 2.0 2.0 2.0
Stay C7 1.0 1.0 1.0 1.0 1.0 1.0
CaP dibasic8 25.0 25.0 25.0 25.0 25.0 25.0
Lecithin9 10.0 10.0 10.0 10.0 10.0 10.0
Cholesterol10 0.5 0.5 0.5 0.5 0.5 0.5
Cellulose11 9.0 8.949 8.847 8.745 8.643 8.541
Cu hydroxychloride12 – 0.051 0.153 0.255 0.357 0.459
Proximate composition (g kg 1, as-is)
Crude protein 357.9 349.0 364.5 361.6 363.2 362.5
Lipid 81.5 72.5 78.7 70.5 63.6 69.6
Moisture 88.5 118.8 78.7 87.0 84.9 75.3
Ash 70.6 69.5 71.6 71.9 71.6 72.2
Cu (mg kg 1) 11.1 39.3 94.6 165.6 214.6 257.1
1
Omega Protein Inc., Reedville, VA, USA.
2
De-hulled solvent extracted soybean meal, Bunge, AL, USA.
3
Empyreal 75 Cargill Corn Milling, Cargill, Inc., Blair, NE, USA.
4
MP Biochemicals Inc., Solon, OH, USA.
5
Trace mineral premix Cu free (g kg 1): cobalt chloride, 0.04; ferrous sulphate, 40.0; magnesium sulphate heptahydrate, 283.98; manga-
nous sulphate monohydrate, 6.50; potassium iodide, 0.67; sodium selenite, 0.10; zinc sulphate heptahydrate, 131.93; filler, 534.28.
6
Vitamin premix (g kg 1): thiamine HCL, 4.95; riboflavin, 3.83; pyridoxine HCL, 4.00; Ca pantothenate, 10.00; nicotinic acid, 10.00; bio-
tin, 0.50; folic acid, 4.00; cyanocobalamin, 0.05; inositol, 25.00; vitamin A acetate (500,000 IU/g), 0.32; vitamin D3 (1,000,000 IU/g), 0.05;
vitamin E acetate (250 IU/g), 80.00; menadione, 0.50; alpha-cellulose, 756.81.
7
Stay C, (L-ascorbyl-2-polyphosphate 35%), Roche Vitamins Inc., Parsippany, NJ, USA.
8
Fisher Scientific, Fair Lawn, NJ, USA.
9
Solae Company, St. Louis, MO, USA.
10
USB Biochemicals, Cleveland, OH, USA.
11
Sigma Chemical Co., St. Louis, MO, USA.
12
Micronutrients, Div. of Heritage Technologies, LCC, Indianapolis, IN, USA.
..............................................................................................

Aquaculture Nutrition, 23; 171–180 ª 2015 John Wiley & Sons Ltd
 using the test kits (3352-01, 3354-01; LaMotte, Chester-
town, MD, USA) once per week. Water samples were col-
At the end of the nursery phase, juvenile shrimp (mean ini-
lected from the system reservoir once weekly prior to water
tial weight 0.28 g trial 1; 0.22 g trial 2) were sorted by
exchange and kept at 20 °C for subsequent Cu analyses.
hand to the uniform size. In trial 1, the shrimp were
The procedures of Cu analyses in water samples were the
stocked at 15 shrimp per tank with four replications per
same as described by Zhou et al. (2014). Briefly, water sam-
treatment in the recirculating systems containing 24 square
ples were analysed using the Hach Porphyrin Method
tanks (162 L volume, 0.60 9 0.60 9 0.45 m) for an 8-week
(Hach Chemical Company, Loveland, CO, USA) and eval-
period. In trial 2, the shrimp were stocked at 15 shrimp per
uated using a standard spectrophotometer (Aquamate 9423
tank with seven replications per treatment in the recirculat-
AQA 2000E; Thermo Scientific, Cambridge, UK).
ing systems containing 42 square tanks (162 L volume,
0.60 9 0.60 9 0.45 m) for a 7-week period. In both trials,
the indoor recirculating culture system composed of culture
tanks, a sump with biological filter, sand filter, a circulation
At the end of two trials, haemolymph from all shrimps in
pump and supplemental aeration. Each tank was covered
each tank were collected using 250-lL capillary tubes to
with netting to prevent shrimp loss due to jump and was
pierce the abdominal segments 1, 2 or 3. Both the shrimp and
provided with continuous aeration by two air stones. Water
haemolymph samples were then immediately frozen at
was exchanged twice weekly at approximately 40% of the
20 °C for subsequent Cu analyses. Frozen shrimp were
whole system at each exchange. Water was pumped from
rinsed with deionized water; the carapace and hepatopancreas
the intracostal waterway canal between Bon Secour Bay
from 6 to 8 shrimp per tank were dissected and oven-dried
and Perdido Bay or the Gulf of Mexico.
(90 °C) overnight to a constant weight. Whole shrimp (3–4
In both trials, test diets were offered four times per day
per tank) were rinsed and oven-dried overnight. Carapace
at 0800–0830, 1100–1130, 1500–1530 and 1830–1900. Feed
and whole-shrimp dried samples were ground to a power.
inputs were predetermined assuming the shrimp would
Samples were wet-ashed and determined for Cu concentration
double their weight each week up to one gram. Thereafter,
using the atomic absorption spectrophotometer (Model AA-
the weekly basis used a feed conversion ratio (FCR) of 2.0
6630, Shimadzu, Japan) as described by Zhou et al. (2014).
in trial 1 and 1.8 in trial 2. Shrimp were counted weekly
and feed inputs adjusted based on observed survival. At
the end of each trial, shrimp were counted and group-
weighed. Final biomass, final mean weight, percentage Statistical analyses were performed using SAS 9.4 (SAS
weight gain, FCR, survival and thermal-unit growth coeffi- Institute, Cary, NC, USA). Data were analysed using one-
cient (TGC) were determined. The formula of TGC is way analysis of variance (ANOVA) to determine whether
described by Iwama & Tautz (1981) as follows: significant differences existed among treatment means. The
TGC = 100 9 ( FBW1/3 IBW1/3)/∑DT. Student–Newman–Keuls multiple comparison tests were used
Where IBW is initial body weight (g/shrimp), FBW is to identify significant differences among treatments means.
final mean weight (g shrimp 1), D is the number of days, Linear, second- or third-order polynomial regressions were
and T is average daily water temperature. performed to investigate the relationship between tissues Cu
concentrations and analysed dietary Cu. To identify the most
appropriate regression model, we evaluated P-value of the
model components, R2 value, adjust R2 value and the sum of
In both trials, water temperature, dissolved oxygen (DO), squares for error (SSE) with different regression models. All
salinity and pH were measured twice daily (from two tanks statistical tests were considered significant at P < 0.05.
in trial 1 and in three tanks in trial 2) at 0730-0800 h and
1500-1530 h using a YSI Professional Plus meter (Yellow
Spring Instrument Co., Yellow Spring, OH, USA). Water
samples were collected twice weekly to measure total
ammonia nitrogen (TAN) using an Orion ammonia elec-
trode probe (Thermo Fisher Scientific Inc., Waltham, MA, In trial 1, water quality parameters (mean  standard devi-
USA). The nitrite and nitrate concentrations were measured ation) were maintained for temperature at 27.40  1.48 °C,
..............................................................................................

Aquaculture Nutrition, 23; 171–180 ª 2015 John Wiley & Sons Ltd
 DO at 6.19  0.31 mg L 1, salinity at 19.00  0.60 g L 1
and pH at 7.63  0.10. Total ammonia nitrogen (TAN)
was 0.04  0.02 mg L 1. In trial 2, water quality parame-
ters were as follows: temperature at 27.42  1.42, DO
at 6.02  0.29, salinity at 27.28  1.23 g L 1, pH
at 7.81  0.24, TAN 0.12  0.13 mg L 1; nitrite at
0.75  0.31 mg L 1 and nitrate at 9.43  3.53 mg L 1.
The Cu concentrations of water samples during the culture
period were below 2.0 lg L 1 in trial 1 and
15.08  7.86 lg L 1 in trial 2. Water quality conditions in
two trials were maintained appropriately for the normal
growth and survival in L. vannamei.
No significant differences with regard to the dietary Cu
levels on the shrimp’s final biomass, final mean weight,
FCR and survival were observed in either of two trials. In
trial 1, final mean weight of shrimp ranged from 8.47 to
9.08 g, final biomass was between 111.1 and 127.2 g, per-
centage weight gain ranged from 2903% to 3128%, FCR
varied from 1.10 to 1.20, TGC was between 0.0885 and
0.0916, and survival ranged between 86.7 and 100.0%
(Table 3). In trial 2, final mean weight of shrimp ranged
from 5.06 to 5.43 g, final biomass was between 64.0 and
69.1 g, percentage weight gain ranged from 2199% to
2388%, FCR varied from 1.32 to 1.41, TGC was between
0.0829 and 0.0852, and survival ranged between 81.9 and
89.5% (Table 3).
Dietary copper levels affected tissues Cu concentrations in
both trials (Table 4). In trial 1, the carapace Cu concentra-
tions of shrimps fed diets H50 or H70 were significantly
higher than those of shrimp reared on H20 diet. The cara-
pace, hepatopancreas and shrimp Cu contents increased
linearly with increasing analysed dietary Cu up to 66 mg
analysed Cu kg 1 (Fig 1a,b,d). The Cu concentration in
the hepatopancreas and whole shrimp reared on diets H50
and H70 was significantly higher than that of shrimp
reared on other diets. The haemolymph Cu concentrations
of shrimp maintained on H30, H50 and H70 diets were sig-
nificantly higher than other treatments (Table 4). Relation-
ship between haemolymph Cu content and analysed dietary
Cu (10–66 mg analysed Cu kg 1) supported that this was a
second-order polynomial relationship (Fig. 1c).
In trial 2, the carapace Cu concentrations of shrimp fed
on diets H220 were significantly higher than those of
shrimp reared on B10, H40, H100 and H280. The hep-
atopancreas and whole-shrimp Cu contents were signifi-
cantly increased with graded Cu levels from 40 to 220 mg
total target Cu kg 1 (Table 4). The third-order polynomial
regression analysis was applied to relationship between

Table 3 Effect of dietary copper sources and levels on performance of Litopenaeus vannamei (initial weight 0.28 g, 8 weeks in trial 1; initial
weight 0.22 g, 7 weeks in trial 2)

Trial Diet Analysed Cu (mg kg 1) Final biomass (g) Final weight (g) Weight gain (%)1 FCR2 TGC Survival (%)

Trial 1 B10 10.0 123.0 8.47 2903 1.18 0.0885 96.7

n=4 H15 15.0 127.2 8.48 2906 1.20 0.0886 100.0
H20 19.0 120.2 8.70 2960 1.15 0.0896 91.7
H30 29.0 111.1 8.54 2955 1.17 0.0890 86.7
H50 48.0 124.7 8.77 3003 1.14 0.0901 95.0
H70 66.0 121.4 9.08 3128 1.10 0.0916 90.0
PSE3 2.6 0.11 49 0.01 0.0014 1.7
P-value 0.5694 0.6104 0.7879 0.6524 0.6693 0.3130
Trial 2 B10 11.1 64.0 5.09 2204 1.70 0.0831 83.8
n=7 H40 39.3 69.1 5.33 2352 1.65 0.0852 86.7
H100 94.6 68.0 5.06 2199 1.74 0.0829 89.5
H160 165.6 67.6 5.43 2388 1.60 0.0861 82.9
H220 214.6 65.4 5.32 2315 1.62 0.0851 81.9
H280 257.1 65.8 5.38 2323 1.66 0.0852 81.9
PSE3 3.2 0.18 79 0.06 0.0014 3.3
P-value 0.8736 0.4429 0.4429 0.5041 0.5254 0.5032

Values are means of four or seven replicates. Means within columns with the same letter are not significantly different (P > 0.05) based
on the analysis of variance followed by the Student–Newman–Keuls multiple range test.
1
Weight gain (%) = (Final weight – Initial weight)/Initial weight 9 100.
2
Feed conversion ratio (FCR) = Feed intake/(Final weight Initial weight).
3
Pooled standard error.
..............................................................................................

Aquaculture Nutrition, 23; 171–180 ª 2015 John Wiley & Sons Ltd
Table 4 Effects of dietary copper sources and levels on copper concentrations of carapace, hepatopancreas and haemolymph in Litopenaeus
vannamei

Analysed Cu Carapace Hepatopancreas Haemolymph Whole shrimp

Trial Diet (mg kg 1) (lg g 1) (lg g 1) (lg g 1) (lg g 1)

Trial B10 10 15.5ab 12.3b 92.7b 25.8c

1 H15 15 14.8ab 12.7b 98.2b 26.3c
n=4 H20 19 13.2b 12.7b 95.6b 28.7bc
H30 29 16.8ab 19.3b 112.5a 33.8b
H50 48 19.4a 29.5a 123.8a 42.7a
H70 66 19.9a 33.0a 123.0a 46.3a
PSE1 1.5 3.2 4.5 1.9
P-value 0.0261 0.0009 <0.0001 <0.0001
Trial B10 11 36.1b 63.3d 86.9 82.5d
2 H40 39 36.9b 87.3d 130.9 96.2d
n=7 H100 95 36.0b 209.6c 118.1 112.6c
H160 166 44.9ab 328.7b 135.6 132.5b
H220 215 52.9a 545.3a 121.2 152.7a
H280 257 39.0b 338.1b 130.9 136.3b
PSE1 3.8 33.8 16.1 5.1
P-value 0.0215 <0.0001 0.3199 <0.0001

Values are means of four or seven replicates. Means within columns with the same letter are not significantly different (P > 0.05) based
on the analysis of variance followed by the Student–Newman–Keuls multiple range test.
1
Pooled standard error.

carapace, hepatopancreas Cu contents and analysed dietary
Cu (11–257 mg analysed Cu kg 1) in Fig. 2a,b. The sec-
ond-order polynomial was supported to the relationship
between shrimp Cu contents and analysed dietary Cu
(Fig. 2c). No significant differences in the haemolymph
copper concentrations of shrimp were observed.
The results reported here confirmed our hypothesis that the
application of Cu hydroxychloride as a Cu source in
shrimp practical feeds is appropriate. Our growth data
indicate that final biomass, final mean weight and percent-
age weight gain in groups with Cu supplementation were
numerically higher than the control group (Table 3). This
is in agreement with previous studies in aquatic species for
crucial carp (Shao et al. 2010), blunt snout bream (Shao
et al. 2012) and Pacific white shrimp (Zhou et al. 2014).
Many studies indicated that a limited response between
growth performance and dietary Cu supplementation was
observed in abalone (Wang et al. 2009), channel catfish
(Gatlin & Wilson 1986), rainbow trout (Knox et al. 1982,
1984) and Pacific white shrimp (Zhou et al. 2014). In other
studies, some investigators reported that the final mean
weight or percentage weight gain increased with dietary Cu
supplementation in L. vannamei (Davis et al. 1993; Bharad-
waj et al. 2014). For example, Davis et al. (1993) found
significantly higher percentage weight gain in shrimp fed
between 2 and 130 mg total Cu kg 1 from Cu sulphate as
described by a second-order polynomial regression.
Bharadwaj et al. (2014) found that the final mean weight
and growth rate of shrimp fed diet with 286 mg analysed
Cu kg 1 were significantly higher than those of shrimp fed
diet with 93 mg analysed Cu kg 1 from Cu sulphate.
Moreover, the differences of percentage weight gain and
FCR were observed between these two trials. The different
results may be caused by several factors including the
nutrition and composition of diets (semipurified vs practi-
cal), the degree to which tissues were replaced (i.e. percent-
age weight gain), different rearing conditions/period,
leaching of mineral from the diets prior to consumption
and availability of test diets with a low concentration of
targeted mineral (NRC 2011).
With the exception of osmoregulation and oxygen trans-
port, biochemically the function of Cu in aquatic animals
is similar to that in terrestrial animals (NRC 2011). Many
studies have demonstrated that numerous minerals includ-
ing Cu can be absorbed from the water in shrimp and fish
(Djangmah & Grove 1970; Davis et al. 1993; Davis &
Gatlin 1996; NRC 2011). Hence, mineral levels in the water
should be noted when conducting mineral studies (Davis &
Gatlin 1996). In the current study, the water Cu content
was below 2 lg L 1 in trial 1 and 15.08  7.86 lg L 1 in
trial 2. To minimize the water Cu accumulation in the
..............................................................................................

Aquaculture Nutrition, 23; 171–180 ª 2015 John Wiley & Sons Ltd
 (a) (b)
30 45
40
25

Hepatopancreas Cu (µg g–1)

35
Carapace Cu (µg g–1)

20 30
25
15
20
10 15
10 R² = 0.4413
5 R² = 0.3842
P = 0.0012 5 P = 0.0004
0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Analyzed dietary Cu (mg kg–1) Analyzed dietary Cu (mg kg–1)
(c) (d)
60
140

120 50
Hemolymph Cu (µg g–1)

Shrimp Cu (µg g–1)

100 40

80
30
60
20
40
R² = 0.7132 10 R ² = 0.8402
20 P < 0.0001 P < 0.0001
0 0
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
Analyzed dietary Cu (mg kg–1) Analyzed dietary Cu (mg kg–1)

Figure 1 (a) In trial 1, relationship between carapace Cu contents and analysed dietary Cu of L. vannamei. The regression line is described
by carapace Cu = 0.1097 dietary Cu + 13.16 (R² = 0.3842; P = 0.0012). (b) In trial 1, relationship between hepatopancreas Cu contents
and analysed dietary Cu of L. vannamei. The regression line is described by hepatopancreas Cu = 0.3370 dietary Cu + 11.16 (R2 = 0.4413,
P = 0.0004). (c) In trial 1, relationship between haemolymph Cu contents and analysed dietary Cu of L. vannamei. The regression line is
described by haemolymph Cu = -0.0127 dietary Cu2 + 1.5728 dietary Cu + 76.07 (R2 = 0.7132, P < 0.0001). (d) In trial 1, relationship
between shrimp Cu contents and analysed dietary Cu of L. vannamei. The regression line is described by shrimp Cu = 0.3981 dietary Cu +
21.50 (R2 = 0.8402, P < 0.0001).

system, 40% of the whole system water was exchanged confirms the carapace is a good indicator of Cu intake
twice weekly, resulting in approximately 80% of the rearing and storage.
water exchanged each week. In the Gulf of Mexico waters, Of the tissues analysed, Cu concentrations were the high-
total Cu contents ranged from 0.10 to 12.3 lg L 1 (Slowey est in the hepatopancreas (Table 4). This finding is consis-
& Hood 1971). Hence, most of our values were within the tent with the fact that the crustacean hepatopancreas plays a
range of reported levels for nearshore waters. Thus, the vital role in nutrient absorption and regulation (Loizzi &
impact of potential water Cu contribution to shrimp should Peterson 1971; Johnson 1980; Verri et al. 2001). Davis et al.
be negligible and within natural limits seen for coastal (1993) and Bharadwaj et al. (2014) also suggested the hep-
waters. atopancreas acts as a site for Cu storage and/or excretion.
In the current study, the selected tissues (carapace and For example, Bharadwaj et al. (2014) reported the Cu con-
hepatopancreas) and haemolymph of crustacean were centration of hepatopancreas ranging from 327 to
assessed (Table 4). Several investigators found these tis- 1487 lg kg 1, when shrimp fed diets containing 64-430 mg
sues are good indicators of nutrient status with a signifi- analysed Cu kg 1 from Cu sulphate and 30-96 mg analysed
cant portion of body burden of Cu (Icely & Nott 1980; Cu kg 1 from chelated Cu, respectively. In the current
Baker 1986; Depledge 1989; Davis et al. 1993; Davis & study, the Cu concentration of hepatopancreas increased
Gatlin 1996). Our results in trial 1 for the Cu concentra- with increasing dietary Cu up to 215 mg analysed Cu kg 1.
tions of carapace are similar to previous studies with L. The increasing trend is consistent with the results reported
vannamei (Davis et al. 1993; Zhou et al. 2014). This by Davis et al. (1993). In trial 2, the Cu concentration of
..............................................................................................

Aquaculture Nutrition, 23; 171–180 ª 2015 John Wiley & Sons Ltd
 (a) 80 trend with the Cu concentration of carapace was observed in
70 trial 2 (Fig. 2a). The increase and then decrease in Cu accu-
mulation at high levels could simply be a residual or varia-
Carapace Cu (µg g–1)

60
50
tion caused by small sample size variation in analysing
samples or moult cycle (Keteles & Fleeger 2001; Bharadwaj
40
et al. 2014). However, the rise and then decrease in tissue
30
deposition may also be due to regulation or oversaturation
20
R² = 0.2161 of absorption and/or transport sites.
10 P = 0.0309
Haemolymph is an important store of Cu as approxi-
0
0 50 100 150 200 250 300 mately 40% of body burden of Cu is stored in the haemo-
Analyzed dietary Cu (mg kg–1) lymph and the response has been associated with Cu status
(Depledge 1989; Davis et al. 1993; Sun et al. 2013). In the
(b) 900 present study, the Cu contents of haemolymph ranged from
800 92.7 to 135.6 lg kg 1 when shrimp fed diets containing 10-
Hepatopancreas Cu (µg g–1)

700 257 mg analysed Cu kg 1 for both trials. This is similar to

600 the results reported by Zhou et al. (2014), who found that
500 the Cu contents of haemolymph from Cu hydroxychloride
400 in L. vannamei ranged from 82.0 to 149.3 lg kg 1 when
300 shrimp fed diets containing 21–45 mg analysed Cu kg 1.
200 This may indicate that shrimp modulate Cu concentration
100 R² = 0.7532
P < 0.0001 within this range to release and transport Cu to other tis-
0
0 50 100 150 200 250 300 sues. Depressions noted in other studies may be due to
Analyzed dietary Cu (mg kg–1)
lower levels of Cu in the basal diet or possibly higher levels
of tissue replacement in these studies.
(c) 180 It is important to note that the Cu levels of the carapace,
160 hepatopancreas and whole shrimp fed the basal diet (B10)
140 in trial 2 were higher than those fed basal diet (B10) in trial
Shrimp Cu (µg g–1)

120
100
80
60
40
20
0
0 50 100 150 200
Analyzed dietary Cu (mg
Figure 2 (a) In trial 2, relationship between carapace Cu contents
and analysed dietary Cu of L. vannamei. The regression line is
described by carapace Cu = 0.0000096 dietary Cu3 + 0.0036 diet-
ary Cu2 - 0.2932 dietary Cu + 40.56 (R2 = 0.2161, P = 0.0309). (b)
In trial 2, relationship between hepatopancreas Cu contents and
analysed dietary Cu of L. vannamei. The regression line is described
by hepatopancreas Cu = 0.00016 dietary Cu3 + 0.0561 dietary Cu2
3.1305 dietary Cu + 98.21 (R2 = 0.7532, P < 0.0001). (c) In trial 2,
relationship between shrimp Cu contents and analysed dietary Cu
of L. vannamei. The regression line is described by y = 0.0011
dietary Cu2 + 0.5416 dietary Cu + 75.54 (R² = 0.7360; P = 0.0001).
hepatopancreas reached a peak at 215 mg analysed Cu kg 1
diet than the hepatopancreas Cu content decreased at diet-
ary levels of 257 mg analysed Cu kg 1 (Fig. 2b). A similar
1. The diet formulation of the basal diet in both trials was
almost the same and contained 10 mg analysed Cu kg 1 in
trial 1 and 11 mg analysed Cu kg 1 in trial 2. The elevated
R² = 0.7360 levels in trial 2 are likely due to variations in shrimp stocks,
P = 0.0001
moult cycle and sample analyses and preparation. Zhou
et al. (2014) reported variations in carapace, haemolymph
250 300 and hepatopancreas Cu concentrations in shrimp fed the
kg–1) same diets in different rearing systems (clear water system
vs green water system). This may indicate the Cu concentra-
tion not only varied in different tissues, but also varies due
to rearing conditions, shrimp strains, size and moult stage.
In addition to animal health, Cu supplementation may
influence intestinal microflora and has affected the presence
of bacteria in the litter of broilers (Johnson et al. 1985;
Arias & Koutsos 2006). Scaletti et al. (2003) reported that
Cu supplementation appeared to reduce the severity of an
Escherichia coli Mastitis in heifers. Arias & Koutsos (2006)
found the Cu sources (Cu sulphate and Cu hydroxychlo-
ride) influenced different regions of small intestine in broil-
ers. Additionally, C ß elik et al. (2005) suggest Cu
hydroxychloride might be used for decreasing the negative
effects of aflatoxin in broilers. In a study of blunt snout
..............................................................................................

Aquaculture Nutrition, 23; 171–180 ª 2015 John Wiley & Sons Ltd
 bream, Shao et al. (2012) reported that the number of bac-
teria species in fish fed diets with 6–150 mg Cu kg 1 was
higher than that in fish fed with 0–3 mg Cu kg 1 from Cu
hydroxychloride. They explained the intestinal bacterial
community was affected by dietary Cu supplementation.
Therefore, the effect of Cu supplementation on intestinal
microflora in shrimp should be investigated in future
studies.
In conclusion, our data demonstrated that Cu hydroxy-
chloride is an appropriate Cu source in shrimp diets. Anal-
ysis of selected tissue indicated that each tissue responded
differently as dietary Cu increased. There was very little
response and no discernible trends to Cu levels on the hae-
molymph. However, the Cu concentration of carapace,
hepatopancreas and shrimp increased numerically with
graded Cu level up to 215 mg analysed Cu kg 1. Our data
suggest high levels (257 mg analysed Cu kg 1) do not
appear to be detrimental to the shrimp. The effects of diet-
ary high copper on food safety and environment should be
considered in further work.
The authors would like to express our gratitude and
appreciation to those who have taken the time to criti-
cally review this manuscript as well as those who helped
support this research at the Claude Peteet Mariculture
Center and at Auburn University. We thank Rebecca L.
Cook, Van To, Chuck Roe and Igor Simone Tiagua Vice-
nte to help maintain the daily management in the trials.
This publication was supported by Hatch Funding Pro-
gram of Alabama Agriculture Experiment Station and
Micronutrients, Div. of Heritage Technologies, LCC, Indi-
anapolis, IN, USA. Mention of a trademark or propri-
etary product does not constitute an endorsement of the
product by Auburn University and does not imply its
approval to the exclusion of other products that may also
be suitable.
Al-Subiai, S.N., Moody, A.J., Mustafa, S.A. & Jha, A.N. (2011) A
multiple biomarker approach to investigate the effects of copper
on the marine bivalve mollusc, Mytilus edulis. Ecotoxicol. Envi-
ron. Saf., 74, 1913–1920.
Arias, V. & Koutsos, E. (2006) Effects of copper source and level
on intestinal physiology and growth of broiler chickens. Poult.
Sci., 85, 999–1007.
Baker, D.H. (1986) Problems and pitfalls in animal experiments
designed to establish dietary requirements for essential nutrients.
J. Nutr., 116, 2339–2349.
..............................................................................................
Aquaculture Nutrition, 23; 171–180 ª 2015 John Wiley & Sons Ltd
Bharadwaj, A.S., Patnaik, S., Browdy, C.L. & Lawrence, A.L.
(2014) Comparative evaluation of an inorganic and a commer-
cial chelated copper source in Pacific white shrimp Litopenaeus
vannamei (Boone) fed diets containing phytic acid. Aquaculture,
422, 63–68.
ß elik, S., Erdogan, Z., Erdogan, S. & Bal, R. (2005) Efficacy of
C
tribasic copper chloride (TBCC) to reduce the harmful
effects of aflatoxin in broilers. Turk. J. Vet. Anim. Sci., 29,
909–916.
Cheng, J., Fan, C., Zhang, W., Zhu, X., Yan, X., Wang, R. &
Jia, Z. (2008) Effects of dietary copper source and level on per-
formance, carcass characteristics and lipid metabolism in lambs.
Asian-Aust. J. Anim. Sci., 21, 685–691.
Cromwell, G., Lindemann, M., Monegue, H., Hall, D. & Orr, D.
(1998) Tribasic copper chloride and copper sulfate as copper
sources for weanling pigs. J. Anim. Sci., 76, 118–123.
Davis, D.A. & Gatlin, D.M. III (1996) Dietary mineral require-
ments of fish and marine crustaceans. Rev. Fish. Sci., 4, 75–99.
Davis, D.A., Lawrence, A.L. & Gatlin, D.M. III (1993) Dietary
copper requirement of Penaeus vannamei. Nippon Suisan
Gakkaishi, 59, 117–122.
Depledge, M. (1989) Re-evaluation of metabolic requirements for
copper and zinc in decapod crustaceans. Mar. Environ. Res., 27,
115–126.
Djangmah, J. & Grove, D. (1970) Blood and hepatopancreas cop-
per in Crangon vulgaris (Fabricus). Comp. Biochem. Physiol., 32,
733–745.
Gatlin, D.M. III & Wilson, R.P. (1986) Dietary copper require-
ment of fingerling channel catfish. Aquaculture, 54, 277–285.
Hay, R.W. (1984) Bio-inorganic chemistry. Halsted Press, Chich-
ester, U.K.
Icely, J. & Nott, J. (1980) Accumulation of copper within the
“hepatopancreatic” caeca of Corophium volutator (Crustacea:
Amphipoda). Mar. Biol., 57, 193–199.
Iwama, G.K. & Tautz, A.F. (1981) A simple growth-model for sal-
monids in hatcheries. Can. J. Fish Aquat. Sci., 38, 649–656.
Johnson, P.T. (1980) Histology of the blue crab, Callinectes sapi-
dus: A model for the Decapoda. Praeger, New York.
Johnson, E.L., Nicholson, J.L. & Doerr, J.A. (1985) Effect of diet-
ary copper on litter microbial population and broiler perfor-
mance. Br. Poult. Sci., 26, 171–177.
Keteles, K.A. & Fleeger, J.W. (2001) The contribution of ecdysis
to the fate of copper, zinc and cadmium in grass shrimp,
Palaemonetes pugio Holthius. Mar. Pollut. Bull., 42, 1397–
1402.
Knox, D., Cowey, C.B. & Adron, J.W. (1982) Effects of dietary
copper and copper: Zinc ratio on rainbow trout Salmo gairdneri.
Aquaculture, 27, 111–119.
Knox, D., Cowey, C.B. & Adron, J.W. (1984) Effects of dietary
zinc intake upon copper metabolism in rainbow trout (Salmo
gairdneri). Aquaculture, 40, 199–207.
Loizzi, R.F. & Peterson, D.R. (1971) Lipolytic Sites in crayfish
Hepatopancreas and correlation with fine structure. Comp. Bio-
chem. Physiol. B Biochem. Mol. Biol., 39, 231–236.
Luo, X., Ji, F., Lin, Y., Steward, F., Lu, L., Liu, B. & Yu, S.
(2005) Effects of dietary supplementation with copper sulfate or
tribasic copper chloride on broiler performance, relative copper
bioavailability, and oxidation stability of vitamin E in feed.
Poult. Sci., 84, 888–893.
NRC (2011) Nutrient Requirements of Fish and Shrimp. National
Academic Press, Washington D.C.
O’Dell, B.L. (1976) Biochemistry of copper. Med. Clin. N. Am.,
60, 687–703.
Rao, M.S. & Anjaneyulu, N. (2008) Effect of copper sulfate on
molt and reproduction in shrimp Litopenaeus vannamei. Int.
J. Biol. Chem., 2, 35–41.
Rao, M.S., Rajitha, B., Pavitra, E. & Anjaneyulu, N. (2008)
Changes of copper and protein profiles in hepatopancreas and
hemolymph tissues during different molt stages of white shrimp,
Litopenaeus vammmei (Boone, 1931). Biotechnology, 7, 153–156.
Rubino, J.T. & Franz, K.J. (2012) Coordination chemistry of cop-
per proteins: how nature handles a toxic cargo for essential func-
tion. J. Inorg. Biochem., 107, 129–143.
Scaletti, R.W., Trammell, D.S., Smith, B.A. & Harmon, R.J.
(2003) Role of dietary copper in enhancing resistance to Escheri-
chia coli mastitis. J. Dairy Sci., 86, 1240–1249.
Shao, X., Liu, W., Xu, W., Lu, K., Xia, W. & Jiang, Y. (2010) Ef-
fects of dietary copper sources and levels on performance, copper
status, plasma antioxidant activities and relative copper bioavail-
ability in Carassius auratus gibelio. Aquaculture, 308, 60–65.
Shao, X.-P., Liu, W.-B., Lu, K.-L., Xu, W.-N., Zhang, W.-W.,
Wang, Y. & Zhu, J. (2012) Effects of tribasic copper chloride on
growth, copper status, antioxidant activities, immune responses
and intestinal microflora of blunt snout bream (Megalobrama
amblycephala) fed practical diets. Aquaculture, 338, 154–159.
Slowey, J.F. & Hood, D.W. (1971) Copper, manganese and zinc
concentrations in Gulf of Mexico waters. Geochim. Cosmochim.
Acta, 35, 121–138.
Sookying, D. & Davis, D.A. (2012) Use of soy protein concentrate
in practical diets for Pacific white shrimp (Litopenaeus vannamei)
reared under field conditions. Aquac. Int., 20, 357–371.
Sun, S., Qin, J., Yu, N., Ge, X., Jiang, H. & Chen, L. (2013)
Effect of dietary copper on the growth performance, non-specific
immunity and resistance to Aeromonas hydrophila of juvenile
Chinese mitten crab, Eriocheir sinensis. Fish Shellfish Immunol.,
34, 1195–1201.
Terwilliger, N.B. (1998) Functional adaptations of oxygen-
transport proteins. J. Exp. Biol., 201, 1085–1098.
Varotto, L., Domeneghetti, S., Rosani, U., Manfrin, C., Cajar-
aville, M.P., Raccanelli, S., Pallavicini, A. & Venier, P. (2013)
DNA damage and transcriptional changes in the gills of mytilus
galloprovincialis exposed to nanomolar doses of combined metal
salts (Cd, Cu, Hg). PLoS ONE, 8, e54602.
Verri, T., Mandal, A., Zilli, L., Bossa, D., Mandal, P.K.,
Ingrosso, L., Zonno, V., Vilella, S., Ahearn, G.A. & Storelli,
C. (2001) D-glucose transport in decapod crustacean hep-
atopancreas. Comp. Biochem. Physiol. A Mol. Integr. Physiol.,
130, 585–606.
Wang, W., Mai, K., Zhang, W., Ai, Q., Yao, C., Li, H. & Liufu,
Z. (2009) Effects of dietary copper on survival, growth and
immune response of juvenile abalone, Haliotis discus hannai Ino.
Aquaculture, 297, 122–127.
Watanabe, T., Kiron, V. & Satoh, S. (1997) Trace minerals in fish
nutrition. Aquaculture, 151, 185–207.
Zhou, Y., Davis, D.A. & Rhodes, M.A. (2014) Comparative
evaluation of copper sulfate and tribasic copper chloride on
growth performance and tissue response in Pacific white
shrimp Litopenaeus vannamei fed practical diets. Aquaculture,
434, 411–417.
..............................................................................................
Aquaculture Nutrition, 23; 171–180 ª 2015 John Wiley & Sons Ltd