The essentiality of copper for poultry and livestock is well documented (Davis and Mertz, 1987). The high level copper from CuSO4 has been added into swine diets as growth promoter generally, but it is still controversial for broiler diets. This dispute is enhanced by the shortcomings of supplementation with high level of CuSO4, including its damages to nutrients by strengthening their oxidation (Miles et al., 1998; Hooge et al., 2000; Luo et al., 2005). Tribasic Copper Chloride has low hygroscopicity, very low chemical reactivity, less active in catalyzing the destruction of certain vitamins and other organic compounds when, concentrated in base mixes or when included in supplements and diets (Cromwell et al., 1998; Miles et al., 1998; Luo et al., 2005). And this copper source is as available and as safe as that in copper sulfate (Ammerman et al., 1995; Miles et al., 1998; Luo et al., 2005). However, paying more attention to the potential adverse effects of high level copper on the characteristics of animal products and ecological environment, we need think highly of the efficiency and safety of copper sources when adding them in manufacture.
The following study was conducted to investigate the effects of copper from TBCC and CuSO4 on the carcass characteristics, tissular nutrients deposition and oxidation in broilers and to compare the prooxidation and product quality safety of the different copper sources for broilers.
MATERIALS AND METHODS
Experimental design and dietary treatments: A completely randomized design involving a 2x5 factorial arrangement of treatments was used in this study. Five supplemental levels including 0, 50, 150, 250 and 350 mg/kg copper from the two copper sources, TBCC and feed-grade Copper Sulfate (CuSO4.5H2O), were added into a basal diet. Because, treatments with no additions of both copper sources shared the same basal diet, there were a total of 9 treatments in this experiment. The basal corn-soybean meal diet (Table 1) was formulated to meet the requirements of starting and grower chicken (Ministry of Agriculture of the People's Republic, 2004), containing copper 10.18 or 9.52 mg/kg. All diets were made into pellets.
Birds and feeding: A total of 1890, 1 day old, Cobb 48 commercial male
chicks were fed to 40 days in 63 floor pens of 30 birds each. Broilers were
randomly allotted by weight to 1 of 9 treatments for 7 replicate pens. The broilers
were assigned to floor pens (0.15 m2 per bird) in a randomized complete
block design, which had been vaccinated against Mareks disease and Infectious
Bursal Disease. New wood shavings at a depth of approximately 8 cm were used
as litter material and each pen contained one hanging heat lamp, one tube feeder
and one Plasson waterer. Light was provided 24 h to encourage feed intake. Feeds
and water were available ad libitum.
||Composition and nutrient levels of basal diet
|*Provided per kilogram of diet: Vitamine A 13500
IU, VD3 3000 IU, VE 30 IU, VK3 6 mg, VB2
9 mg, VB6 6 mg, VB12 0.63 ug, Pantothenic acid 18
mg, Niacin 60 mg, Biotin 0.15 mg, Folic acid 1.5 mg, Choline (50%) 750 mg;
Fe (FeSO4.7H2O) 100 mg, Zn (ZnSO4. 7H2O)
100 mg, Mn (MnSO4.7H2O) 120 mg, I (KI) 0.7 mg, Se
(Na2SeO3) 0.3 mg, Sodium Bicarbonate 100 mg, Salinomycin 60 mg
Carcass traits test and sample collections: On day 40, after weighing the birds per pen, 2 birds from each replicate of treatments (close to mean body weight) were picked out. They were starved for 12 h (indeed drinking water was supplied ad libitum) and were sacrificed as per standard procedure for evaluation of carcass characteristics including the yield of evisceration weight, half-evisceration yield, breast yield and thigh yield. The various carcass traits recorded, were then expressed in terms of percentage of live weight. Liver samples, heart samples and pectoral muscles were collected and frozen individually in heat sealed plastic bags for vitamin E, MDA, copper and crude fat analysis.
Laboratory analysis: After homogenizing 0.2 g of liver tissue in 1.8 mL (wt/vol) of dehydrated alcohol (0.9%, wt/vol) and then extracting it with heptane, vitamin E levels of liver and heart were determined by a vitamin E detection kit (JianCheng Bioengineering Institute, Nanjing, China.) according to instructions provided by the manufacturer. The optical densities were read in a spectrophotometer (Shanghai Meipuda instrument co. Ltd., China; UV-1100) at 533 nm.
After homogenizing 0.2 g of liver tissue in 1.8 mL (wt/vol) of cold saline
(0.9%, wt/vol), levels of MDA in liver, heart and breast were determined by
a MDA detection kit (Nanjing JianCheng Bioengineering Institute, Nanjing, China)
following the colorimetric method provided by the manufacturer. The optical
densities were read in above spectrophotometer at 532 nm. Each sample was analyzed
in duplicate and the results were averaged.
Liver and breast samples were predigested in 5 mL HNO3 for 4 h on a hotplate, then all samples were dry ashed at 550°C for 12 h and solubilized in 4 mL 10% HNO3, then metered volume to 10 mL. Copper concentrations were determined by a flame atomic absorption spectrophotometry (Analytik Jena AG, Germany; novAA 400) with the methods descripted by Zhang (2004). Validations of the mineral analysis were conducted using bovine liver powder as a standard reference. Standards were matched for acid and macroelement concentrations as needed and standard reference materials were included with samples.
After dried all liver or muscle samples at 65°C, 2 g of sample were weighed accurately into the thimble. Then, samples in thimble were dried in an oven at 105°C for 5 h and cooled subsequently in desiccators for 30 min. Constant weights of the samples were gain. Then the sample was placed directly into the distillation apparatus over a water bath for a 6 hours extraction. And then, constant dried weight of the sample without fat was gain. And the fat content in the samples can be concluded.
Statistical analysis: One-way ANOVA with post hoc multiple comparisons test (Duncan test) was performed to determine significant differences among treatment groups (p<0.05). Data were also analyzed for potential effects of copper source, copper levels and interactions between copper source and copper level using the general linear model procedure of SPSS 11.0 software (SPSS Inc., Chicago, IL). Data were expressed as means±SEM. A value of p<0.05 (or p<0.01) was considered statistically significant and denoted by different letter superscripts.
Body weight and carcass characteristics: There was no significant difference on initial body weight. Copper source, added copper level or an interaction between copper source and level had no significant effects on body weight of 40 days. Chicks consuming 150 mg/kg copper as CuSO4 had a higher BW than those of chicks fed 350 mg/kg copper as TBCC (p<0.05).
Copper source affected evisceration yield (p<0.01) and chicks fed TBCC had a higher evisceration yield. Added copper level and an interaction between copper source and level did not affect (p<0.05) evisceration yield. Copper source, added copper level or an interaction between copper source and level had no significant effects on half-evisceration yield, breast yield and thigh yield. Chicks fed 150 mg/kg copper had the highest half-evisceration yield and breast yield (p<0.05).
||Effects of dietary copper sources and levels on body weight
carcass characteristics of broilers*
|*n = 14; Within a column, values with different small letter
superscripts mean significant difference (p<0.05), differ rent capital
letter superscripts mean significant difference (p<0.01)
||Effects of dietary copper sources and levels on concentration
of VE or MDA in tissues of broilers*
|*n = 14; on fresh basis, by analysis; Within a column, values
with different small letter superscripts mean significant difference (p<0.05),
different capital letter superscripts mean significant difference (p<0.01)
Tissue VE and MDA content: Added copper level affected vitamin E contents in livers and hearts of broilers (p<0.05). Liver vitamin E content decreased with added copper level increasing and liver vitamin E contents of broilers fed 350 mg/kg copper were less than the control group sharply (p<0.01). The heart vitamin E increased and subsequently decreased following added copper level. Heart vitamin E contents of broilers fed 150 mg/kg copper were higher than the control group significantly (p<0.01). Copper source and an interaction between copper source and level had no significant effects on liver and heart vitamin E content of broilers.
Added copper level affected liver MDA significantly (p<0.05). Copper source,
added copper level and an interaction between copper source and level had no
significant effects on heart and breast MDA content of broilers.
||Effects of dietary copper sources and levels on tissular contents
of copper and fat in broilers*
|*n = 14; on air-dried basis, by analysis; General linear model
of log10 transformed copper concentration; Within a column, values with
different small letter superscripts mean significant difference (p<0.05),
differ rent capital letter superscripts mean significant difference (p<0.01)
Tissue fat and copper concentrations: Copper source, added copper level and an interaction between copper source and level had no significant effects on the content of crude fat in liver. But the content of crude fat in livers increased with the copper level increasing. The contents of crude fat in livers of broilers fed 250 mg/kg copper were higher than the control group and the birds fed 50 mg/kg copper (p<0.05). Copper source and an interaction between copper source and level did not affect the content of crude fat in breast muscle. Added copper level affected the content of crude fat in breast (p<0.05). The content of crude fat in breast increased with added copper level increasing. The content of crude fat in breast muscle of broilers fed 350 mg/kg copper were higher than the control and the birds fed 50 mg/kg copper (p<0.01).
Copper source, added copper level, or an interaction between copper source and level affected (p<0.01) liver copper significantly (Table 4). Liver copper concentrations in chicks were increased by supplementation with copper (p<0.01). Chicks consuming diets with 350 mg/kg copper from CuSO4 had the highest liver copper concentration and the control group had the lowest one. When added level of copper exceeded 150 mg/kg, liver copper of broilers fed with TBCC was less than that of the broilers fed CuSO4. Added copper level affected copper concentrations of breast muscle in chicks (p<0.01). Copper concentrations of breast muscle in chicks were increased by supplementation with copper too (p<0.01). Copper source and an interaction between copper source and level had no significant effects on copper concentrations of breast muscle.
It was reported by Arias and Koutsos (2006), that carcass weight (45 days posthatch) of chicks fed diet with copper 188 mg/kg from CuSO4 or carcass weight (47 days posthatch) of chicks fed diet with copper 188 mg/kg from TBCC were heavier than those of chicks fed diet without supplemental copper (p<0.05); while, these diets did not affect breast muscle mass. In the present study, results indicated that diets with high levels of copper from the two sources had no improvement on the bird bodyweight of d40 and 350 mg/kg copper in diets even reduced the bodyweight. But chicks fed 150 mg/kg copper as CuSO4 had a bigger evisceration yield, a bigger half-evisceration yield and a greatest breast yield. And half-evisceration yield, breast yield and thigh yield of the broilers fed with 150 mg/kg copper as TBCC were non-significantly more than those of the control group.
Otherwise, copper sources in this study affected the evisceration yield of broilers significantly (p<0.01). The evisceration yield of broilers fed CuSO4 decreased while, those of broilers fed TBCC increased. Chicks fed 50 mg/kg copper as TBCC had the greatest evisceration yield, which was more than those of birds consuming 250 and 350 mg/kg copper as CuSO4 significantly (p<0.05). The difference between the half-evisceration yield and the evisceration yield represented the percentage of heart, liver, proventriculus, gizzard, abdominal fat, head, neck and shank (Yang et al., 2003). This part reduced 11% while, the group fed with 50 mg/kg copper as TBCC compared with the control group. A smaller percentage of this part implicates a better feed convert rate.
In the present study, the crude fat in liver and breast increased when broilers fed with high level copper. Liver crude fat of broilers fed with 250 mg/kg copper as CuSO4 increased significantly comparing with the control (p<0.05). Crude fat in muscle of broilers consuming 350 mg/kg copper as TBCC increased notably (p<0.05). These increments of crude fat content might be due to promoting fat absorption by dietary supplementation with copper (Luo et al., 1996). However, Chowdhury et al. (2004) reported that dietary Cu-Met although accumulated more fat in liver at high levels of supplementation (150 and 200 g/kg), but had no effect on excreta fat as well as digestibility of fat.
Tribasic copper chloride possesses several chemical characteristics that make it less oxidation as a copper source in production, such as small particle size, excellent flow and low water solubility (Ammerman et al., 1995; Miles et al., 1998; Luo et al., 2005). Miles et al. (1998) reported that copper source and copper concentration affected peroxide values of lipid extracted from stored diets significantly. TBCC promoted less oxidation than coarse CuSO4 at 300 mg/kg added copper, as the modal particle size of TBCC was lower (67 mm) than CuSO4 (455 mm). In current study, with the increasing of added copper level, the liver MDA of broilers fed CuSO4 decreased, while, the liver MDA of broilers fed TBCC changed as an initial increasing and a subsequent decreasing. And supplementation with either CuSO4 or TBCC into diets did not affected MDA contents in heart and muscle.
Hooge et al. (2000) reported that vitamin E is greater in TBCC diets than in copper sulfate diets at the same copper levels. It was also reported by Luo et al. (2005) that the vitamin E contents in liver and plasma of broilers access to TBCC were higher than those of birds fed copper sulfate (p<0.01). In the present study, dietary supplementation with copper influenced the VE contents in liver and heart of broilers (p<0.05). While, liver VE of the broilers decreased with the copper level increasing, the heart VE content changed as an initial notable increasing and a subsequent little decreasing. The above findings implicate it is a complex effect of dietary supplementation with high copper on fat and VE, which dietary copper may get involved in absorption, deposition and oxidation of lipids.
Liver copper concentration increases linearly with the increasing of added
copper level. It was reported by Miles et al. (1998) that liver copper
increased significantly when, dietary copper level exceeded 300 mg/kg. And in
the study of Bakalli et al. (1995), muscle copper (35 and 42 days) increased
lightly (14.5%, p>0.05) when, supplemented 250 mg/kg copper as CuSO4
into birds diets, but the copper content in the breast muscle was lower. Results
obtained in the present study were in agreement with the above studies. Chicks
consuming added copper diets had the higher liver copper and muscle copper contents.
Liver copper increased significantly (p<0.01) when, broilers fed diets added
copper from either sources. Chicks fed 350 mg/kg copper as CuSO4 or
TBCC had 15.9 and 8.4 times liver copper of the control, respectively. Liver
copper of chicks fed with 150~350 mg/kg copper as CuSO4 was higher
than those of TBCC. Meanwhile, chicks consuming copper as CuSO4 or
TBCC had 1.3~2.9 or 1.8~2.5 times muscle copper of the control, respectively.
Results of this trail (data not shown) showed that high level copper promoted the performance in the starter not but the grower and resulted in chronic copper poisoning with a depressing performance in the grower period. Thus, it need a withdrawal time when we added copper into the broiler diets. Liver copper of birds fed TBCC was similar to those of birds fed CuSO4 when, copper played a role as growth promotion factor (50~150 mg/kg copper, data not shown). Liver copper of chicks consuming 250 mg/kg copper as TBCC or CuSO4 were 2.8 or 9.1 times of the control group. Chicks supplemented with 350 mg/kg copper as CuSO4 had a higher liver copper than those fed TBCC (p<0.01). It implicated that chicks were more sensitive to copper as CuSO4 than TBCC when copper levels >250 mg/kg. Thus, there was a higher toxic dose of copper as TBCC than CuSO4. This is in agreement with the study of Miles et al. (1998), who reported that the Cu sulfate had a greater percentage of larger particles than TBCC and TBCC had a better uniformity of particle size. Luo et al. (2005) reported also that TBCC was a safer product and more available to broilers than copper sulfate.
Tolerance limit of copper in meat is 10 mg/kg (Ministry of Health of the Peoples Republic, 1994). In the present study, when muscle copper levels were converted into values on fresh basis, they were all <5 mg/kg and in the safety margin. While, added level of copper >50 mg/kg from either copper sources, levels of liver copper in birds are exceed the tolerance limit, even though liver copper of chicks consuming TBCC was lower than those fed CuSO4.
Conclusion: In conclusion, due to its similar growth promotion effect with a less prooxidant activity to damage nutrients, a better carcass characteristics and a higher toxic dose in broilers, TBCC can be used as a safe dietary copper source.
This work was supported by Heritage Dongjiang Micro- Nutrients Additive (Shenzhen) Company Limited and the specific research supporting program for academic sustentation research team in Sichuan Agricultural University. The corresponding author is Zhang Ke-ying who wishes to thank Dr. Chao Liang and Ms. Jun-qin Gao for their help.