Effects of Copper Source and Level on Intestinal Physiology
and Growth of Broiler Chickens1
V. J. Arias* and E. A. Koutsos*2
*Animal Science Department, One Grand Avenue, California Polytechnic State University, San Luis Obispo 93407
pared with the positive control (P < 0.05 for each). At d
30 to 31 posthatch, intestinal histology was measured. In
Experiment 1 (recycled litter), dietary TBCC, CuSO4, and
positive control decreased the number of lamina propia
lymphocytes or intraepithelial lymphocytes (IEL), or
both, compared with the negative control (P < 0.05). However, in Experiment 2 (fresh litter), TBCC and positive
control increased the number of duodenum IEL compared with the negative control (P < 0.05), and negative
control and TBCC increased the number of ileum IEL.
These data demonstrate that broiler performance and intestinal physiology can be influenced by dietary Cu
source and level as well as microbial environment (fresh
vs. recycled litter).
ABSTRACT Dietary copper sulfate (CuSO4) and tribasic copper chloride (TBCC) were examined for their effects on intestinal physiology and growth of broiler chickens. In 2 experiments (Experiments 1 and 2), day-old
broiler chicks were fed 1 of 4 diets: a basal diet with no
supplemental copper (Cu; negative control), a basal diet
+ 188 mg of Cu/kg of diet from TBCC or CuSO4, or
a basal diet + subtherapeutic antibiotics (bacitracin and
roxarsone; positive control). In Experiment 1 (recycled
litter), CuSO4 and TBCC increased carcass weight (d 45
posthatch) compared with the negative control (P < 0.05
for each). In Experiment 2 (fresh litter), negative control
and TBCC increased carcass weight (d 42 posthatch) com-
Key words: copper, antibiotic, broiler chicken, intestinal physiology, intestinal histology
2006 Poultry Science 85:999–1007
natives to AGP that can produce similar results in terms of
intestinal microflora regulation, as well as optimal growth
and performance.
The intestinal environment is a specialized system that
is highly regulated, allowing for the absorption of nutrients and the proliferation of commensal microorganisms
and also maintaining defenses against pathogenic bacteria and other antigens. These defenses include the mucosal barrier and components of the innate and adaptive
immune system (Sanderson, 2003). The mucosal barrier
provides protection through mucus production and secretion, peristalsis, and secretion of lysozymes (Sanderson
and Walker, 1999) vs. the innate immune system, which
uses pattern recognition receptors (i.e., toll-like receptors)
to recognize pathogenic and commensal bacteria (Tlaskalova-Hogenova et al., 1995). Additionally, cells of the innate and acquired immune system, such as T and B cells,
macrophages, mast cells, and M cells, are located throughout the lamina propia, intraepithelial area, and lymphoid
tissue and maintain a regulated environment that develops tolerance or induces an immune response when necessary (Shao et al., 2001; Tlaskalova-Hogenova et al.,
2004). The intestinal microflora can influence the mucosal
immune system as well as the development of the systemic immune system (Tlaskalova-Hogenova et al., 1983);
germ-free animals have reduced macrophage chemotaxis
and phagocytosis activity (Tlaskalova-Hogenova et al.,
2004). Therefore, some microflora populations are critical
INTRODUCTION
For many years, subtherapeutic antibiotics (AGP) have
been incorporated into poultry and swine diets because
of their favorable effects on growth, feed intake, and feed
efficiency (Eyssen and deSomer, 1963). It is thought that
AGP promote or allow optimal growth by regulating the
microflora in the small intestine, allowing commensal
bacteria to maintain an environment for maximum nutrient absorption and reducing pathogenic bacteria that can
produce toxins and damage the intestine (Coates et al.,
1955; Lev et al., 1957). As a result, animals fed AGP have
fewer incidences of subclinical immune challenges from
pathogenic bacteria, which would negatively affect performance (Barber et al., 1955; Coates et al., 1955; Stanley
et al., 2004). However, there are growing concerns that the
continual feeding of AGP is leading to the development of
antibiotic resistance in many pathogenic bacteria isolated
from production animals as well as from humans
(Mamber and Katz, 1985; Aarestrup, 1999; Aarestrup et
al., 2001). Therefore, there is considerable interest in alter-
2006 Poultry Science Association Inc.
Received October 5, 2005.
Accepted January 19, 2006.
1
Financial support was provided by California Agricultural Research
Initiative Grant #04-018 and by Micronutrients, Indianapolis, IN.
2
Corresponding author: [email protected]
999
1000
ARIAS AND KOUTSOS
for normal immune system development, but excessive
levels of pathogenic bacteria can reduce performance and
increase disease risks.
With the increasing public concern of bacterial resistance to antibiotics, animal production industries are
looking at AGP alternatives that have antimicrobial properties, maintain intestinal health, and allow for optimal
growth. Copper has received considerable attention due
to its antimicrobial properties that improve performance
in animals when fed over the minimum requirement (Barber et al., 1955; Smith, 1969; Jenkins et al., 1970; Miles et
al., 1998). Studies have shown that supplementation with
various Cu sources (e.g., Cu sulfate, Cu citrate, or Cu
chloride) increases growth in poultry (Smith, 1969; Pesti
and Bakalli, 1996; Miles et al., 1998) and swine (Braude,
1967; Hill et al., 2000). In particular, tribasic copper chloride (TBCC) and CuSO4 fed to pigs at 200 mg of Cu/
kg of diet improved growth rate, feed intake, and feed
efficiency (Cromwell et al., 1998). Different sources of
Cu have different bioavailability; in broiler chickens, the
bioavailability of TBCC has consistently been equal to or
greater than that of CuSO4 (Miles et al., 1998), and water
solubility also varies between TBCC (<1% soluble) and
CuSO4 (>99% solubility). Therefore, the purpose of these
trials was to examine the effects of TBCC or CuSO4 on
performance and intestinal histology of broiler chickens,
in comparison to AGP. Additionally, these sources were
examined in fresh and recycled litter environments.
MATERIALS AND METHODS
Animal Husbandry
For each of the following experiments broiler chicks
were obtained at day of hatch from a commercial hatchery
(Cobb × Cobb, Foster Farms, Fresno, CA) and housed in
a curtain-sided floor pen facility (blocked for row and
location within building). At d 0 posthatch, chicks were
randomly assigned to pens and experimental diets, which
were fed in 3 diet phases (i.e., starter, grower, and finisher
phases) until birds reached market weight (Tables 1 and
2). All chicks received ad libitum access to assigned diets
and water, and the formulated diets met or exceeded the
requirements of growing broiler chicks (NRC, 1994). The
volume of feed offered was recorded throughout the trials. All procedures were approved by the California Polytechnic State University Animal Care and Use Committee.
Experiment 1
Broiler chicks (n = 1,760) were placed on recycled litter
(rice hulls, 2 previous flocks) and randomly assigned to
1 of 4 diets (8 pens/diet; n = 55/pen; 0.85 ft2/bird × pen).
Birds were fed a basal diet (Table 1) with no supplemental
Cu (8 mg of Cu/kg of diet; negative control) or a basal diet
supplemented with 188 mg of Cu/kg of diet as CuSO4;
(209201, Sigma, St. Louis, MO), 188 mg of Cu/kg of diet
as TBCC (Micronutrients, Indianapolis, IN), or subtherapeutic antibiotics [bacitracin at 0.055 g/kg of diet
(BB130217, Alpharma Inc., Fort Lee, NJ); roxarsone at
0.025 g/kg of diet (AB510067, Alpharma Inc.; positive
control)]. At d 14 posthatch, 16 birds/pen were individually weighed and vaccinated intramuscularly (pectoralis
major) with keyhole limpet conjugated with 2,4-dinitrophenyl hemocyanin (KLH, 32412, EMD Biosciences, CA;
2 mg/kg of BW). At d 21 posthatch, blood was collected
with heparinized needles by cardiac puncture, and
plasma was isolated for further analysis. On d 30 posthatch, another 10 birds/pen were individually weighed,
then euthanized by cervical dislocation followed by tissue
collection. On d 45 posthatch, 10 birds/pen were processed using commercial processing equipment, and carcass parameters were assessed.
Dependent variables measured included primary antiKLH antibody titers (d 21 posthatch), intestinal histology
(d 30 posthatch), plasma minerals (d 21 posthatch), BW
(all birds within a pen on d 1, 14, 31, and 40 posthatch),
carcass weights (hot, eviscerated carcass; d 45 posthatch),
and breast muscle mass (pectoralis major and minor from
hot carcass; d 45 posthatch).
Experiment 2
Chicks (n = 1,033) were placed on fresh litter (rice hulls)
and randomly assigned to 1 of 4 diets (8 pens/diet; n =
33/pen; 0.85 ft2/bird × pen). Diets were similar to Experiment 1 and consisted of basal diet with no supplemental
copper (8 mg of Cu/kg of diet; negative control), or basal
diet supplemented with 188 mg of Cu/kg of diet as
CuSO4, 188 mg of Cu/kg of diet as TBCC, or subtherapeutic antibiotics (bacitracin and roxarsone as described for
Experiment 1). At d 21 posthatch, the inflammatory immune response (2 birds/pen) was stimulated by intraabdominal administration with lipopolysaccharide (LPS,
L7261, Sigma; 2 mg/kg of BW). At 24 h post-LPS (d 22
posthatch), blood was collected from LPS-vaccinated
birds as well as 2 unvaccinated birds/pen by cardiac
puncture for plasma isolation. Birds were then euthanized
by cervical dislocation, and liver and spleen weights were
recorded. On d 30 posthatch, 3 birds/pen were individually weighed, then euthanized by cervical dislocation,
and intestinal samples were isolated for further analysis.
On d 47 posthatch, 10 birds/pen were processed using
commercial processing equipment, and carcass parameters were assessed.
Dependent variables measured included liver and
spleen weight (% BW) and hepatic minerals (d 22 posthatch), intestinal histology (d 30 posthatch), BW (all birds
within a pen on d 1, 7, 21, 30, and 42 posthatch), carcass
weights (hot, eviscerated carcass; d 47 posthatch), and
breast muscle mass (pectoralis major and minor from hot
carcass; d 47 posthatch).
Analysis of Blood, Livers, and Intestines
The primary antibody response to KLH was measured
(Experiment 1) in plasma at d 21 posthatch (7 d postKLH vaccination) by ELISA assay. Briefly, 150 ␮L of DNP-
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EFFECTS OF COPPER ON BROILER INTESTINAL PHYSIOLOGY
1
Table 1. Diet composition for growing broiler chicks from d 0 posthatch to d 42 to 47 posthatch
Starter
d 0 to 14
Raw material
Corn, grain, ground
Soybean meal, 47%
Soybean oil
Calcium phosphate, dibasic
Calcium carbonate
Poultry feather meal, hydrolyzed
Cellulose
Cornstarch
NaCl
DL-Methionine
Mineral mix2
Vitamin mix3
Lysine
Threonine
Choline chloride-70%
α-Tocopherol (20,000 IU/kg)
Zinc sulfate, monohydrate
Calculated
Crude protein (%)
Methionine (%)
ME (kcal/kg)
Grower
d 14 to 35
Finisher
d 35 to 42 or 47
60.50
30.68
4.62
1.66
1.00
59.23
29.42
5.29
1.20
0.86
2.00
0.57
0.27
0.25
0.25
0.13
0.05
0.02
0.53
0.38
0.25
0.25
0.20
0.18
0.13
0.05
0.02
57.22
16.71
5.83
1.27
0.83
0.60
0.50
0.50
0.47
0.24
0.25
0.25
0.42
0.29
0.14
0.05
0.02
19.7
0.60
3,239
20.7
0.65
3,250
17.6
0.50
3,240
1
All birds had ad libitum access to feed.
Mineral mix provided (IU/kg of mix): Fe (42.18), Mn (6.6), Zn (2.8), Se (0.09).
3
Vitamin mix provided (IU/kg of mix): retinol (806,000), cholecalciferol (160,000), folate (10.3), B12 (6), biotin
(10.1), choline (1,082.6), riboflavin (1,002), vitamin K (0.33) thiamin (23.7), pantothenic acid (1,387.7), niacin
(7,544.1), pyroxidine (26.7).
2
KLH in carbonate buffer (2 ␮g/mL) was incubated in a
96-well plate for 24 h at 22°C. After incubation, the plate
was washed with PBS + 0.05% Tween 20 (P7949, Fisher,
Pittsburgh, PA) with a EL×50 Auto Strip plate washer
(Bio-Tek Instrument Inc., Winooski, VT), then washed
with Superblock buffer. Dilutions of sample plasma were
made in Superblock buffer + 0.05% Tween 20 (100 ␮L/
well; 1:16, 1:32, 1:64, and 1:128) and incubated for 30 min
at 22°C. The plate was washed again and incubated with
anti-chicken IgG-peroxidase (A9046, Sigma, 1:30,000 in
Superblock buffer) for 20 min at 22°C. The washed plate
was incubated with BM Blue POD Substrate solution
(1484281, Roche, Indianapolis, IN) for 10 min at 22°C.
Then the reaction was stopped with 1 M H2SO4 and OD
read at 450 nm within 5 min. Data presentation of KLH
antibody titers are expressed as log2 values of the highest
dilution giving a positive value.
Table 2. Analysis of dietary Cu for diets fed to growing broiler chicks1
Diet
Experiment 1
Negative control
CuSO4
TBCC
Positive control
Experiment 2
Negative control
CuSO4
TBCC
Positive control
Calculated Cu
(mg/kg of diet)
Analyzed Cu
(mg/kg of diet)
8.0
188.0
188.0
8.0
29.3
178.0
220.0
34.3
±
±
±
±
2.4
12.3
29.3
1.8
8.0
188.0
188.0
8.0
20.2
161.3
199.2
35.5
±
±
±
±
4.0
12.5
35.8
6.6
1
Birds were fed either a basal diet (8 mg of Cu/kg of diet; negative
control), a basal diet + 188 mg of Cu/kg of diet as CuSO4 or tribasic
copper chloride (TBCC), or a basal diet + subtherapeutic antibiotics
(positive control). Data represent means ± SEM.
Plasma (Experiment 1, d 21 posthatch) and livers (Experiment 2, d 22 posthatch) were examined for Cu, Zn,
P, K, S, Ca, Mg, Fe, Na, Mn, and Al by ICP analysis
(Central Analytical Labs, University of Arkansas, Fayetteville, AR).
Intestinal histology was examined in 10 birds/pen (Experiment 1; d 31 posthatch) and 3 birds/pen (Experiment
2; d 30 posthatch). Intestinal segments were taken from
the midpoint of the duodenum (duodenum), from the
midpoint of the section cut to the Meckel’s diverticulum
(jejunum), and the midpoint of the Meckel’s diverticulum
to the ileocecal junction (ileum). Duodenum, jejunum,
and ileum samples were isolated, washed with cold phosphate-buffered saline, rinsed with formalin, cut (2 cm),
and placed in 50 mL of 10% formalin for embedding
and hematoxylin and eosin staining by a commercial lab
(IDEXX, Sacramento, CA).
For every slide (per bird), 3 villi were randomly selected
to measure villus length, villus width, lamina propia
width, crypt depth, and to count lamina propia lymphocytes (LPL) and intraepithelial lymphocytes (IEL) by hematoxylin and eosin staining. Using a 5× field, villus
length was the distance between the apical region to the
base of the villus, and villus width was the distance between 1 side of the brush border membrane to the other
side of the brush border membrane (Iji et al., 2001). Using
a 10× field, lamina propia width was determined by measuring the width of the vasculature region located in the
center of the villus (Sanderson, 2003; Mestecky et al.,
2005), and crypt depth was the depth of the invaginations
at the base of each villus. The LPL were counted from in
the lamina propia area and IEL from the epithelial area
surrounding the lamina propia (Bjerregaard, 1975).
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ARIAS AND KOUTSOS
Table 3. Effect of dietary levels and sources of Cu on broiler live and carcass weights1
Experiment 1
Experiment 2
Day posthatch
Diet
d 31
(kg of LW/bird)
d 45
(kg of CW/bird)
d 42
(kg of LW/bird)
d 47
(kg of CW/bird)
0.958b
1.056a
0.983ab
0.963ab
0.01
1.879b
1.988a
1.969a
1.921ab
0.01
2.23
2.20
2.22
2.12
0.02
2.15a
2.05ab
2.16a
1.93b
0.03
Negative control
CuSO4
TBCC
Positive control
SEM
When superscripts are different within a column, broiler weight means are significantly different (P < 0.05).
Average broiler body weights were calculated at selected days over the experimental time period and
represent live weight (LW) and carcass weight (CW). Broilers (n = 1,760 for Exp. 1, n = 1,033 for Exp. 2) were
fed either a basal diet (8 mg of Cu/kg diet; negative control), a basal diet + 188 mg of Cu/kg of diet as CuSO4
or tribasic copper chloride (TBCC), or a basal diet + subtherapeutic antibiotics (positive control).
a,b
1
Statistics
Data were analyzed with ANOVA (JMP program, SAS,
Cary, NC) to examine the main effects of diet, treatment,
location within building (row), and their interactions.
Row and the interaction of row × diet were included
in the model to ensure that blocking was appropriate.
Therefore, when row and the interaction of row × diet
were not significant (P > 0.20), they were removed from
the model. When main effects were significant (P < 0.05),
differences between means were determined using Student’s t-test.
RESULTS
Experiment 1
Initial BW did not differ between groups prior to diet
assignment (P = 0.34; average = 43.1 ± 0.5 g/bird). On d 14
posthatch, chicks fed CuSO4 and TBCC had significantly
greater BW than negative controls (P < 0.05; CuSO4 average = 269 ± 5 g/bird; TBCC average = 267 ± 9 g/bird;
negative control average = 240 ± 8 g/bird), and positive
control was intermediate. On d 31 posthatch, only chicks
fed CuSO4 were heavier than negative controls (P < 0.05;
Table 3), but carcass weights at d 45 posthatch were significantly greater for chicks fed CuSO4 and TBCC as compared with those fed negative control (P < 0.05; Table 3).
At both time points, positive control birds were intermediate to negative controls and Cu-supplemented diets.
Breast muscle mass was not different due to diet (P =
0.69; average = 443 ± 9 g/bird).
Primary log2 anti-KLH antibody titers were not different due to diet (P = 0.61). At d 21 posthatch, there was
no effect of diet on plasma Cu, Zn, S, Ca, Mg, Fe, and
Na (P > 0.20 for all). However, chicks fed CuSO4 had
higher plasma P compared with those fed the negative
control (P < 0.05, data not shown), and chicks fed CuSO4
and TBCC had higher plasma K than those fed the positive control (P < 0.05, data not shown). These differences
were not observed at d 31 posthatch, and there was no
effect of diet on plasma Cu, Zn, P, K, Ca, Mg, Fe, Na, S,
and Mn (P > 0.20 for all), although chicks fed TBCC had
higher plasma Al than those fed negative control (P <
0.05; data not shown).
Villi length and width in the duodenum, jejunum, and
ileum were not affected by diet (P > 0.11 for all). In the
duodenum, there was a trend in which chicks fed TBCC
had greater LP width compared with those fed CuSO4
(P = 0.06; data not shown). In the jejunum, chicks fed
negative control had greater crypt depth compared with
CuSO4, TBCC, or positive control, and those fed CuSO4
had greater crypt depth compared with positive control
(P < 0.05, Figure 1A). Chicks fed negative control or CuSO4
had greater crypt depth in the ileum compared with those
fed TBCC and positive control (P < 0.05; Figure 1B).
Lymphocyte numbers were affected by diet in all 3
anatomical regions in the intestine. In the duodenum,
chicks fed the negative control had a greater number of
LPL compared with those fed CuSO4, TBCC, or positive
control (P < 0.05 for each, Figure 2A) and also had a
greater number of IEL than chicks fed CuSO4 or TBCC
(P < 0.05 for each; Figure 2A). In the jejunum, chicks fed
negative control had greater number of LPL compared
with those fed CuSO4 or TBCC, and those fed positive
control were greater compared with TBCC (P < 0.05 for
each; Figure 2B). In the ileum, those fed negative control
had greater LPL compared with those fed TBCC (P < 0.05
for each; Figure 2C).
Experiment 2
At d 0 posthatch, initial BW differed between groups
prior to diet assignment (P = 0.02; average = 35.7 ± 0.1 g/
bird); chicks assigned to TBCC had greater BW compared
with chicks assigned to negative control (P < 0.05; TBCC
average = 36.2 ± 0.3 g/bird; negative control average =
35.2 ± 0.3 g/bird). However, these differences were not
seen on d 7 posthatch or throughout the rest of the trial
(P > 0.10 for d 21, 31, and 42 posthatch). At market weight
(d 47 posthatch), carcasses from chicks fed negative control and TBCC were heavier than those of positive control
(P < 0.05; Table 3); however, diet did not affect breast
muscle mass (P = 0.27; average = 399.7 ± 11.5 g/bird).
EFFECTS OF COPPER ON BROILER INTESTINAL PHYSIOLOGY
1003
there was no effect of diet on duodenum LP width or
crypt depth, or ileum crypt depth (P > 0.15 for all). In the
jejunum, crypt depth was greater in chicks fed CuSO4
than those fed negative control or TBCC, and those fed
positive control had greater crypt depth than TBCC (P <
0.05; Figure 3A). The LP width increased in the jejunum
of chicks fed positive control compared with those fed
negative control or TBCC (P < 0.05; Figure 3B). In the
ileum, chicks fed CuSO4 tended to have greater LP width
compared with those fed positive control (P = 0.07).
Lymphocyte numbers were affected by diet, although
not as consistently as in Experiment 1. Although duodenum LPL were not affected by diet (P > 0.15), chicks
fed TBCC or positive control had more duodenum IEL
compared with negative control (P < 0.05; Figure 4A).
Diet did not affect the number of jejunum LPL or IEL, or
ileum LPL (P > 0.20 for each), but chicks fed negative
control or TBCC had more ileum IEL compared with
those fed positive control (P < 0.05; Figure 4B).
DISCUSSION
Figure 1. The effect of dietary Cu source and level on A) jejunum
and B) ileum crypt depth (Experiment 1). Broiler chicks (n = 320 birds;
3 measurements/tissue*bird) were placed on recycled litter and fed
either a basal diet (8 mg of Cu/kg diet; negative control), a basal diet
+ 188 mg of Cu/kg of diet as CuSO4 or tribasic copper chloride (TBCC),
or a basal diet + subtherapeutic antibiotics (positive control). a–cWhen
superscripts are different within a tissue, crypt depth means are significantly different (P < 0.05).
At d 22 posthatch (24 h post-LPS treatment), there was
no effect of diet or LPS vaccination on spleen weight (%
BW; P = 0.29 and P = 0.22, respectively). Similarly, there
was no effect of diet on liver weight (P = 0.90), although
LPS vaccination increased liver weight (% BW, 20% increase, P < 0.01). At d 22 posthatch, liver P, K, Al, Mg,
and Cu were not affected by diet or LPS (P > 0.20 for all).
The LPS stimulation increased liver Zn and decreased
liver Fe, S, Na, and Mn (P < 0.05 for all), and chicks fed
CuSO4 had higher liver Fe than those fed TBCC (P < 0.05;
86.7 vs. 60.3 ± 4.4 ␮g/g). In addition, chicks fed positive
control had greater liver Ca compared with those fed
negative control (P < 0.05; 62.9 vs. 58.8 ± 0.9 ␮g/g).
Villi length and width were not affected by diet in any
region of the small intestine (P > 0.15 for all). Additionally,
These experiments examined broiler chick responses
to different dietary Cu sources and levels. Dietary Cu
treatments did not affect plasma Cu (Experiment 1) or
liver Cu (Experiment 2) concentrations. Previous research
has shown that only when diet Cu levels are >200 mg of
Cu/kg of diet did liver or plasma Cu levels change (Miles
et al., 1998). Because these experiments did not include
levels >200 mg of Cu/kg of diet, changes in plasma and
liver Cu were not expected.
Previous research has demonstrated that dietary Cu
enhances performance in poultry and swine when fed
over the NRC minimum requirements (Hill et al., 2000;
Skrivan et al., 2000). Therefore in our experiments, it was
hypothesized that chicks fed supplemental dietary Cu
should perform similarly to chicks fed AGP and better
than chicks fed no supplemental Cu. In Experiment 1,
this hypothesis was confirmed, in that chicks fed TBCC
or CuSO4 had higher carcass weights compared with
those fed negative control but were not significantly different from positive control. These birds were grown on
recycled litter, and the effect of Cu supplementation and
AGP was likely related to the fact that bacteria and other
microorganisms are found at higher concentration in recycled litter compared with fresh litter (e.g., Coates et al.,
1955; Coates et al., 1963; Reyna et al., 1983). Increased
exposure to bacteria and other pathogens will increase
the chance of infection, and AGP have been shown to
reduce the number of pathogenic bacteria in the intestine,
therefore reducing the incidences of bacterial infections
and maximizing growth potential (McDougald and Reid,
1991; Stanley et al., 2004). Copper supplementation may
also affect intestinal microflora and has been shown to
affect the presence of bacteria in the litter (Johnson et al.,
1985). Regardless of the mechanism, when antigen load
is high and immune responses are stimulated at the level
of the intestine, an infiltration of lymphocytes occurs in
the infected area (Bienenstock, 1984; Anderson et al., 1990;
1004
ARIAS AND KOUTSOS
Figure 2. The effect of dietary Cu source and level on the number of intestinal lamina propria lymphocytes (LPL) and intraepithelial lymphocytes
(IEL) and total number of lymphocytes in the A) duodenum, B) jejunum, and C) ileum (Experiment 1) were identified by hematoxylin and eosin
staining. Broiler chicks (n = 320 birds; 3 measurements/tissue × bird) were placed on recycled litter and fed either a basal diet (8 mg of Cu/kg
of diet; negative control), a basal diet + 188 mg of Cu/kg of diet as CuSO4 or tribasic copper chloride (TBCC), or a basal diet + subtherapeutic
antibiotics (positive control). a–cWhen superscripts are different within a tissue, LPL means are significantly different (P < 0.05). x,yWhen superscripts
are different within a tissue, IEL means are significantly different (P < 0.05). *When superscripts are different within a tissue, number of total
lymphocyte means are significantly different compared with negative control (P < 0.05).
Tlaskalova-Hogenova et al., 2004). By regulating the intestinal microflora and reducing the incidences of bacteria
translocation, the number of lymphocytes associated with
the villus area should be decreased. In Experiment 1, in
all 3 tissues examined (duodenum, jejunum, and ileum),
chicks fed negative control had more LPL and IEL compared with chicks fed Cu or AGP supplemented diets.
Therefore, chicks that did not receive supplemental Cu
or AGP and were grown on recycled litter had increased
subclinical immune challenges, which correlates to observed carcass weights.
In contrast to the positive effects of AGP and Cu supplementation on performance of chicks grown on recycled
litter, chicks grown on fresh litter (Experiment 2) and fed
negative control or TBCC had greater carcass weight and
decreased IEL compared with those fed positive control.
Again, this difference may be explained in the context of
the type of microbial environment to which the bird was
exposed (Mireles et al., 2004a,b). In a “low” microbial
environment as seen with fresh litter, AGP may negatively affect commensal bacteria needed for proper digestion (Forbes and Park, 1959), resulting in decreased
growth. Interestingly, TBCC supplementation did not
have negative effects on performance in the fresh litter
environment, suggesting that Cu supplementation may
be more broadly applicable for growth promotion as com-
pared with AGP, although environmental concerns
should be noted.
In addition to differential effects of Cu sources on performance, the Cu sources (CuSO4 and TBCC) used in
these experiments affected different areas of the small
intestine. In Experiment 1, CuSO4 affected the number of
LPL and IEL and crypt depth in the duodenum, whereas
TBCC affected similar parameters in the jejunum and
ileum. The CuSO4 may have more efficacy in the proximal
intestine due to dissociation of Cu2+ from SO42−, which
can then bind to the prevalent H+ in the duodenum. The
TBCC may have more efficacy in the jejunum and ileum
due to chloride (Cl−) dissociating with Cu and binding
to either monovalent cations (e.g., K+, Na+, or P+) or participating in Na+/K+ pumps allowing Cu to associate with
the small intestine in the distal intestine. Lastly, diets
supplemented with AGP (bacitracin and roxarsone)
seemed to have effects on the whole small intestine .
Finally, the systemic inflammatory response was examined in response to an LPS challenge (Experiment 2),
which normally results in a dramatic decrease in plasma
Fe and Zn and an increase in liver Zn (Butler and Curtis,
1973; Butler et al., 1973; Klasing, 1984; Roura et al., 1992).
This response was seen in our experiment; additionally,
liver weight (% of BW) increased in response to LPS, as
previously demonstrated (Roura et al., 1992; Xie et al.,
EFFECTS OF COPPER ON BROILER INTESTINAL PHYSIOLOGY
1005
Figure 3. The effect of different Cu level and source on broiler A)
jejunum crypt depth and B) jejunum lamina propia width (Experiment
2). Broiler chicks (n = 96 birds; 3 measurements/tissue × bird) were
placed on fresh litter and fed either a basal diet (8 mg of Cu/kg of diet;
negative control), a basal diet + 188 mg of Cu/kg of diet as CuSO4
or tribasic copper chloride (TBCC), or a basal diet + subtherapeutic
antibiotics (positive control). a–cWhen superscripts are different within
a graph, crypt depth, or lamina propia width means are significantly
different, P < 0.05.
2000; Mireles et al., 2005). Together these observations
indicate that an inflammatory response was induced.
However, dietary treatments had no effect on measured
parameters of the systemic inflammatory response, although other trials have shown reduced liver weights
(with or without LPS stimulation) when poultry or swine
were fed AGP or Cu supplements (Barber et al., 1957;
Roura et al., 1992). In Experiment 2, chicks fed CuSO4
had significantly more liver Fe compared with those fed
TBCC (negative and positive controls intermediate). Because Fe accumulates in the liver during the acute phase
response (Klasing, 1984; Klasing et al., 1987), this observation suggests that Cu source may differentially modulate
the inflammatory response. Our cell culture experiments
have shown that HD11 cells (avian macrophage cell line)
incubated with plasma isolated from chicks fed TBCC
had reduced nitric oxide production after LPS stimulation
compared with cells incubated with plasma from chicks
Figure 4. The effect of different Cu level and source on broiler A)
duodenum and B) ileum intraepithelial lymphocytes (IEL; Experiment
2) were identified by hematoxylin and eosin staining. Broiler chicks (n =
96 birds; 3 measurements/tissue × bird) were placed on fresh litter and
fed either a basal diet (8 mg of Cu/kg of diet; negative control), a basal
diet + 188 mg of Cu/kg of diet as CuSO4 or tribasic copper chloride
(TBCC), or a basal diet + subtherapeutic antibiotics (positive control).
a,b
When superscripts are different within a tissue, IEL means are significantly different (P < 0.05).
1006
ARIAS AND KOUTSOS
fed negative control and CuSO4 plasma treatments (Arias
and Koutsos, 2005). Therefore, Cu source may affect systemic or local inflammation differently. Finally, Experiment 1 showed that unchallenged chicks fed CuSO4 had
higher plasma K and P vs. those fed positive control and
negative control, respectively. These changes may reflect
the disregulation of electrolyte balance due to environmental stresses or time of sampling (Borges et al., 2003,
2004). Prolonged electrolyte balance can have dramatic
effects on performance; however, these changes in minerals were not observed at d 31 posthatch (Experiment 1)
and therefore should not have been a factor affecting
growth for the rest of the trial (Nesheim et al., 1964;
Melliere and Forbes, 1966).
On the basis of these data, in a “high” microbial environment (recycled litter), Cu (TBCC or CuSO4) and AGP
supplementation improved growth and decreased the
number of intestinal lymphocytes. In contrast, in a “low”
microbial environment (fresh litter), AGP negatively affected performance, and birds fed TBCC or no supplementation had the highest BW. Differences in these effects
may be related to mechanism of action; AGP are generally
thought to regulate microflora in the intestine, whereas
Cu supplementation may affect intestinal microflora in
addition to litter microflora. Differences between Cu
sources seem to be related to effects on different regions of
the small intestine. Overall, dietary Cu supplementation
may allow for more consistent growth and performance
of birds grown on fresh or recycled litter as compared
with AGP supplementation.
ACKNOWLEDGMENTS
We would like to thank J. Beckett and D. Peterson from
California Polytechnic State University for their assistance
with manuscript preparation. We would like to thank P.
Bass, G. Guenther, L. Hylle, J. Olson, L. Shuey, and M.
Torres from California Polytechnic State University for
their assistance with animal care, sampling, and data collection.
REFERENCES
Aarestrup, F. M. 1999. Association between the consumption of
antimicrobial agents in animal husbandry and the occurrence
of resistant bacteria among food animals. Int. J. Antimicrob.
Agents 12:279–285.
Aarestrup, F. M., A. M. Seyfarth, H. D. Emborg, K. Pedersen,
R. S. Hendriksen, and F. Bager. 2001. Effect of abolishment
of the use of antimicrobial agents for growth promotion on
occurrence of antimicrobial resistance in fecal enterococci
from food animals in Denmark. Antimicrob. Agents Chemother. 45:2054–2059.
Anderson, D. B., V. J. McCracken, R. I. Aminov, J. M. Simpson,
R. I. Mackie, M. W. Verstegen, and H. R. Gaskins. 1990. Gut
microbiology and growth-promoting antibiotics in swine.
Pigs News Info. 20:115N–122N.
Arias, V. J., and E. A. Koutsos. 2005. Dietary copper source
and level lead to differences in plasma that modulate the
inflammatory response in cultured macrophages. Proc. Soc.
Exp. Biol., San Diego, CA (Abstr.)
Barber, R. S., R. Braude, and K. G. Mitchell. 1955. Antibiotic and
copper supplements for fattening pigs. Br. J. Nutr. 9:378–381.
Barber, R. S., R. Braude, K. G. Mitchell, J. A. Rook, and J. G.
Rowell. 1957. Further studies on antibiotic and copper supplements for fattening pigs. Br. J. Nutr. 11:70–79.
Bienenstock, J. 1984. The mucosal immunologic network. Ann.
Allergy 53:535–540.
Bjerregaard, P. 1975. Lymphoid cells in chicken intestinal epithelium. Cell Tissue Res. 161:485–495.
Borges, S. A., A. V. Fischer da Silva, J. Ariki, D. M. Hooge, and
K. R. Cummings. 2003. Dietary electrolyte balance for broiler
chickens exposed to thermoneutral or heat-stress environments. Poult. Sci. 82:428–435.
Borges, S. A., A. V. Fischer da Silva, A. Majorka, D. M. Hooge,
and K. R. Cummings. 2004. Physiological responses of broiler
chickens to heat stress and dietary electrolyte balance (sodium plus potassium minus chloride, milliequivalents per
kilogram). Poult. Sci. 83:1551–1558.
Braude, R. 1967. Copper as a stimulant in pig feeding (Cuprum
pro pecunia). World Rev. Anim. Prod. 3:69–81.
Butler, E. J., and M. J. Curtis. 1973. The effects of Escherichia coli
endotoxin and ACTH on the plasma zinc concentration in
the domestic fowl. Res. Vet. Sci. 15:363–367.
Butler, E. J., M. J. Curtis, and M. Watford. 1973. The effect of
Escherichia coli endotoxin on the plasma iron concentration
in the domestic fowl. Res. Vet. Sci. 15:267–269.
Coates, M. E., M. K. Davies, and S. K. Kon. 1955. The effect of
antibiotics on the intestine of the chick. Br. J. Nutr. 9:110–119.
Coates, M. E., R. Fuller, G. F. Harrison, M. Lev, and S. F. Suffolk.
1963. A comparison of the growth of chicks in the Gustafsson
germ-free apparatus and in a conventional environment,
with and without dietary supplements of penicillin. Br. J.
Nutr. 17:141–150.
Cromwell, G. L., M. D. Lindemann, H. J. Monegue, D. D. Hall,
and D. E. Orr, Jr. 1998. Tribasic copper chloride and copper
sulfate as copper sources for weanling pigs. J. Anim. Sci.
76:118–123.
Eyssen, H., and P. deSomer. 1963. The mode of action of antibiotics in stimulation growth of chicks. J. Exp. Med. 117:127–138.
Forbes, M., and J. T. Park. 1959. Growth of germ-free and conventional chicks: Effect of diet, dietary penicillin and bacterial
environment. J. Nutr. 67:69–84.
Hill, G. M., G. L. Cromwell, T. D. Crenshaw, C. R. Dove, R. C.
Ewan, D. A. Knabe, A. J. Lewis, G. W. Libal, D. C. Mahan,
G. C. Shurson, L. L. Southern, and T. L. Veum. 2000. Growth
promotion effects and plasma changes from feeding high
dietary concentrations of zinc and copper to weanling pigs
(regional study). J. Anim. Sci. 78:1010–1016.
Iji, P. A., A. Saki, and D. R. Tivey. 2001. Body and intestinal
growth of broiler chicks on a commercial starter diet. 1. Intestinal weight and mucosal development. Br. Poult. Sci.
42:505–513.
Jenkins, N. K., T. R. Morris, and D. Valamotis. 1970. The effect
of diet and copper supplementation on chick growth. Br.
Poult. Sci. 11:241–248.
Johnson, E. L., J. L. Nicholoson, and J. A. Doerr. 1985. Effect of
dietary copper on litter microbial population and broiler
performance. Br. Poult. Sci. 26:171–177.
Klasing, K. C. 1984. Effect of inflammatory agents and interleukin 1 on iron and zinc metabolism. Am. J. Physiol.
247:R901–R904.
Klasing, K. C., D. E. Laurin, R. K. Peng, and D. M. Fry. 1987.
Immunologically mediated growth depression in chicks: Influence of feed intake, corticosterone and interleukin-1. J.
Nutr. 117:1629–1637.
Lev, M., C. A. Briggs, and M. E. Coates. 1957. The gut flora of
the chick. 3. Differences in caecal flora between infected,
uninfected and penicillin-fed chicks. Br. J. Nutr. 11:364–372.
Mamber, S. W., and S. E. Katz. 1985. Effects of antimicrobial
agents fed to chickens on some gram-negative enteric bacilli.
Appl. Environ. Microbiol. 50:638–648.
McDougald, L. R., and W. M. Reid. 1991. Disease in Poultry.
7th ed. Iowa State Univ. Press, Ames.
EFFECTS OF COPPER ON BROILER INTESTINAL PHYSIOLOGY
Melliere, A. L., and R. M. Forbes. 1966. Effect of altering the
dietary cation-anion ratio on food consumption and growth
of young chicks. J. Nutr. 90:310–314.
Mestecky, J., M. E. Lamm, W. Strober, J. Bienenstock, J. R.
McGhee, and L. Mayer. 2005. Mucosal Immunology. 3rd ed.
Elsevier Acad. Press, Burlington, MA.
Miles, R. D., S. F. O’Keefe, P. R. Henry, C. B. Ammerman, and
X. G. Luo. 1998. The effect of dietary supplementation with
copper sulfate or tribasic copper chloride on broiler performance, relative copper bioavailability, and dietary prooxidant activity. Poult. Sci. 77:416–425.
Mireles, A. J., S. M. Kim, and K. C. Klasing. 2005. An acute
inflammatory response alters bone homeostasis, body composition, and the humoral immune response of broiler chickens. Poult. Sci. 84:553–560.
Mireles, A. J., D. Sutton, E. Koutsos, and R. Spiller. and S. Kim.
2004a. Effect of growth promoters (antibiotics + roxarsone +
copper sulfate) on broiler composition during stess: Possible
mechanisms of growth promotion. Poult. Sci. 83(Suppl.
1):397.(Abstr.)
Mireles, A. J., D. Sutton, E. Koutsos, R. Spiller, and S. Kim.
2004b. Effect of growth promoters (antibiotics + roxarsone +
copper sulfate) on broiler performance during stress: Possible
mechanisms of growth promotion. Poult. Sci. 83(Suppl.
1):396.(Abstr.)
National Research Council. 1994. Nutrient Requirements of
Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC.
Nesheim, M. C., R. M. Leach, Jr., T. R. Zeigler, and J. A. Serafin.
1964. Interrelationships between dietary levels of sodium,
chlorine and potassium. J. Nutr. 84:361–366.
Pesti, G. M., and R. I. Bakalli. 1996. Studies on the feeding
of cupric sulfate pentahydrate and cupric citrate to broiler
chickens. Poult. Sci. 75:1086–1091.
Reyna, P. S., L. R. McDougald, and G. F. Mathis. 1983. Survival
of coccidia in poultry litter and reservoirs of infection. Avian
Dis. 27:464–473.
Roura, E., J. Homedes, and K. C. Klasing. 1992. Prevention
of immunologic stress contributes to the growth-permitting
ability of dietary antibiotics in chicks. J. Nutr. 122:2383–2390.
1007
Sanderson, I. R. 2003. The innate immune system of the gastrointestinal tract. Mol. Immunol. 40:393–394.
Sanderson, I. R., and W. A. Walker. 1999. Handbook of Mucosal
Immunology. 2nd ed. Academic Press, New York, NY.
Shao, L., D. Serrano, and L. Mayer. 2001. The role of epithelial
cells in immune regulation in the gut. Semin. Immunol.
13:163–176.
Skrivan, M., V. Skrivanova, M. Marounek, E. Tumova, and J.
Wolf. 2000. Influence of dietary fat source and copper supplementation on broiler performance, fatty acid profile of meat
and depot fat, and on cholesterol content in meat. Br. Poult.
Sci. 41:608–614.
Smith, M. S. 1969. Responses of chicks to dietary supplements
of copper sulphate. Br. Poult. Sci. 10:97–108.
Stanley, V. G., C. Gray, M. Daley, W. F. Krueger, and A. E.
Sefton. 2004. An alternative to antibiotic-based drugs in feed
for enhancing performance of broilers grown on Eimeria spp.infected litter. Poult. Sci. 83:39–44.
Tlaskalova-Hogenova, H., M. A. Farre-Castany, R. Stepankova,
H. Kozakova, L. Tuckova, D. P. Funda, R. Barot, B. Cukrowska, J. Sinkora, L. Mandel. 1995. The gut as a lymphoepithelial
organ: The role of intestinal epithelial cells in mucosal immunity. Folia Microbiol. (Praha) 40:385–391.
Tlaskalova-Hogenova, H., R. Stepankova, T. Hudcovic, L. Tuckova, B. Cukrowska, R. Lodinova-Zadnikova, H. Kozakova,
P. Rossmann, J. Bartova, D. Sokol, D. P. Funda, D. Borovska,
Z. Rehakova, J. Sinkora, J. Hofman, P. Drastich, and A. Kokesova. 2004. Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune
diseases. Immunol. Lett. 93:97–108.
Tlaskalova-Hogenova, H., J. Sterzl, R. Stepankova, V. Dlabac,
V. Veticka, P. Rossmann, L. Mandel, and J. Rejnek. 1983.
Development of immunological capacity under germfree and
conventional conditions. Ann. N. Y. Acad. Sci. 409:96–113.
Xie, H., N. C. Rath, G. R. Huff, W. E. Huff, and J. M. Balog.
2000. Effects of Salmonella typhimurium lipopolysaccharide
on broiler chickens. Poult. Sci. 79:33–40.