Introduction

The essentiality of copper (Cu) for poultry and livestock is well documented [1]. Knowledge of the effect of supplemental Cu sources on performance of animals is critical in the selection of a source for use in poultry and livestock production.

Micronutrients Tribasic Copper Chloride (TBCC, Cu2(OH)3Cl) is a new Cu source, which is a more concentrated form of Cu than Cu sulfate pentahydrate (CuSO4·5H2O) (57% vs. 25% Cu). It is also believed that TBCC is a less reactive and destructive form of Cu relative to Cu sulfate because it has lower hygroscopicity and solubility in water. Therefore, TBCC may be a potential Cu source with higher bioavailability and less oxidant-promoting activity than Cu sulfate.

Some studies indicated that TBCC improved performance of poultry, pig, and cattle more efficiently as compared to Cu sulfate [2–6]. It was also demonstrated that TBCC could improve the stability of animal feeds by reducing oxidation reactions and reducing the loss of vitamin in feeds [2, 7–9]. In addition, Luo et al. [10] reported that TBCC is safer and more effective than Cu sulfate, and it is chemically less active than Cu sulfate in promoting the oxidation of vitamin E in feed. However, the additional levels (150, 300, and 450 mg/kg) of Cu as TBCC used in their study were much higher than levels normally used in the commercial poultry industry.

It was documented that Cu as Cu sulfate at low additional levels (≤150 mg/kg) in diets is of anti-microbial effect for broilers [11, 12]. Broilers fed in floor pens are easily susceptible to bacterial diseases. It is speculated that Cu as TBCC supplemented to diets at low levels might be more effective in inhibiting the bacterial diseases and promoting the growth performance of the broilers fed in floor pens than Cu as Cu sulfate. In addition, TBCC may improve the stability of organic compounds like phytase in broilers’ feeds more effectively than Cu sulfate. However, no experiment on the effect of TBCC on the performance of broilers fed in floor pens and phytase stability in their feeds has been done so far. The objectives of the following study were to compare the efficacy of Cu as feed grade TBCC with that in the sulfate form in improving growth of broilers fed in floor pens and to determine if Cu source influenced the stability of dietary vitamin E and phytase in stored feed.

Materials and Methods

Experimental Design and Treatments

A completely randomized design involving a 2 × 4 factorial arrangement of treatments was used in this study. Two supplemental Cu sources were TBCC and feed-grade Cu sulfate (CuSO4·5H2O), respectively. The Cu sources were added to the basal diet at 0, 100, 150, or 200 mg/kg Cu. The diets were fed for 21 days. Because two Cu sources shared the same basal diet supplemented with Cu at the requirement level, there were a total of seven treatments in this experiment.

Birds and Diets

A total of 840, 1-day-old, Arbor Acres commercial male chicks were used in the 21-day experiment. Broilers were randomly allotted by body weight to one of seven treatments for six replicate floor pens of 20 birds each. Broilers were housed on the mixed litter (2/3 used litter at the bottom plus 1/3 new litter on the top). Birds were maintained on a 24-h constant-light schedule. Feed and tap water were available ad libitum.

The corn–soybean meal basal diet (Table 1) was in mash form, and formulated to meet the requirements of starting chicks [13]. The Cu products were added to the basal diet according to the experimental treatments. Dietary actually analyzed Cu concentrations are shown in Table 2. The analyzed content of vitamin E in the premix was 64 IU/kg, and the content of supplemental vitamin E in feeds was 16 IU/kg.

Table 1 Composition of the Basal Diet for Broilers (As-fed Basis)
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Table 2 Added and Analyzed Copper Concentrations (mg/kg) of Diets for Broilers
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In addition, 30 kg of the basal diet were removed at the time of mixing and supplemented with phytase (BASF, Mount Olive, NJ) at 1,000 PU/kg. This mixture was then divided into three equal parts. One part was used as the control treatment, and the other two were supplemented with 200 mg/kg Cu as either TBCC or Cu sulfate in order to investigate the effect of Cu from two sources on phytase stability in feeds. Therefore, there were a total of three treatments in the phytase stability test.

Sample Collections and Preparations

Feed samples were taken from all the treatments of the animal feeding trial and submitted for Cu analysis prior to the initiation of the trial to confirm Cu contents in the diets. Ten replicate feed samples were taken from the diets of the treatments supplemented with 200 mg/kg Cu from two sources, respectively, for Cu analysis to compare the distribution of Cu in feed mixing between the two sources.

In addition, ten replicate feed samples were taken from the control and the treatments with 200 mg/kg added Cu from two sources in the animal feeding trial for vitamin E analysis, and another ten replicate samples were also taken from the diet of the control treatment and the treatments with 200 mg/kg added Cu from two sources in the phytase stability test for phytase activity analysis. On days 10, 21, 31, and 41 of feed storage at room temperature (18 ± 5°C), ten replicate feed samples were taken from the control treatment and the treatments with 200 mg/kg added Cu from two sources in the animal feeding trial, respectively, for vitamin E analysis, and ten replicate samples were taken from the diets of all three treatments in the phytase stability test, respectively, for phytase activity analysis.

At the end of the animal feeding trial, chicks were weighed by each pen following a 12-h fast, and three chicks were chosen from every pen according to the pen average body weight. Blood samples were taken from each of the three birds via cardiac puncture, which were then centrifuged to harvest plasma samples for plasma vitamin E analysis. After the three chicks were killed by cervical dislocation, liver samples were collected for vitamin E and Cu analyses. All plasma and liver samples were stored at −50°C until analyses. Three samples of plasma or livers of the birds from each pen were pooled in equal ratios into one sample before analysis.

Sample Analysis

Copper concentrations in feeds, livers, and two sources were measured by inductively coupled plasma emission spectroscopy (IRIS Intrepid II, TE, Madison, WI, USA) according to a method described by Luo et al. [10]. Copper contents in feed samples supplemented with 200 mg/kg Cu from two sources were analyzed, and variation coefficients of analyzed values were calculated to compare the distribution of Cu in feed mixing between the two sources. Validations of the mineral analysis were conducted using bovine liver powder (National Institute of Standards and Technology, Beijing, China) as a standard reference. The contents of vitamin E in feed (ten replicate assays) and plasma (triplicate assays) were determined as described by Luo et al. [10]. Liver vitamin E (triplicate assays) was determined as described for plasma vitamin E by homogenizing 0.4 g of liver tissue in 3.6 mL (wt/vol) of cold saline (0.9%, wt/vol) and then extracting it with heptane. The activity of phytase in feeds was determined by the method of Engelen et al. [14].

Statistical Analysis

Data were analyzed by least squares analysis of variance using the General Linear Models (GLM) procedure of the SAS Institute [15]. The replicate pen served as the experimental unit. For the data of vitamin E contents and phytase activities in feeds, the model included the main effect of treatment and time, and their interaction. For other data, the model included the main effect of Cu source and supplemental Cu level, and their interaction. Liver Cu concentrations exhibited variance heterogeneity, and were subjected to log10 transformation prior to analysis.

Multiple linear regression equation was calculated by least squares using the GLM procedure of SAS. In this study, the supplemental Cu level intervals were so small that the log10 transformed liver Cu concentrations had no linear relations with Cu intake; thus, relative bioavailability value of Cu as TBCC was not able to be determined by slope ratio comparison from multiple linear regressions. Linear regression equations of vitamin E contents or phytase activities in feed against time were calculated by least squares using the GLM procedure of SAS. In all cases, P < 0.05 was considered to be statistically significant.

Results

Copper Contents of Sources and Uniformity of Copper Mixing in Feeds

Analyzed Cu concentrations were 25.4% and 56.7% for CuSO4·5H2O and TBCC, respectively. When the added Cu level was 200 mg/kg, the variation coefficients of the Cu concentrations in the feeds supplemented with either Cu sulfate or TBCC were 2.94 and 2.05, respectively.

Growth Performance of Broilers

Copper source, added Cu level, or an interaction between Cu source and level did not affect (P > 0.05) average daily feed intake (ADFI) and feed per gain (F/G), but affected (P < 0.05) average daily gain (ADG) (Table 3). Chicks fed 200 mg/kg Cu as TBCC had a higher (P < 0.05) ADG than those consuming other diets, and no differences in ADG were detected (P > 0.05) among birds fed other diets.

Table 3 Effects of Dietary Copper Source and Level on Growth Performance of Broilers Fed 21 d (n = 6)
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Copper Concentrations in Liver

Liver Cu was not affected (P > 0.05) by Cu source, added Cu level, and their interaction (Table 4). However, chicks fed diets supplemented with 200 mg/kg Cu tended to have a higher (P = 0.07) liver Cu concentrations than those fed the control diet. Chicks fed TBCC diets tended to have a lower (P = 0.07) liver Cu concentration than those fed Cu sulfate diets.

Table 4 Effects of Dietary Copper Source and Level on Vitamin E Contents in Plasma and Liver, and Liver Copper Concentrations of 21-day-old Broilers (n = 6; GLM of Log10 Transformed Liver Cu Concentration)
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Oxidation Stability of Vitamin E

During storage, vitamin E contents in feeds decreased linearly (P < 0.05) with time regardless of Cu treatment (Table 5). However, vitamin E contents in the feed fortified with 200 mg/kg Cu as TBCC were always higher (P < 0.01) than those in the feed added with 200 mg/kg Cu as Cu sulfate (Table 6). When the feeds were stored at room temperature for 10, 21, 31, and 41 days, vitamin E contents in the control feed decreased by 12.8%, 19.8%, 26.4%, and 29.1%, and vitamin E contents in the feed supplemented with Cu sulfate decreased by 35.5%, 58.0%, 76.9%, and 78.2%, whereas vitamin E contents in the feed fortified with TBCC reduced by 11.5%, 27.1%, 26.6%, and 26.4%, respectively, in comparison with the value at the beginning of this experiment. Therefore, the loss percentages of vitamin E contents in the feed fortified with TBCC at days 10, 21, 31, and 41 of feed storage were 24.0%, 30.9%, 50.3%, and 51.8% less than those in the feed fortified with CuSO4, respectively.

Table 5 Regressions of Vitamin E Contents or Phytase Activities in Feed (Y) on Time of Storage (X)
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Table 6 Vitamin E Contents and Phytase Activities in Feeds Supplemented with 200 mg/kg Copper as Either TBCC or Cu Sulfate During Different Time of Storage
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Liver vitamin E contents were affected (P < 0.05) by Cu source or an interaction between Cu source and level, but not affected (P > 0.05) by added Cu level (Table 4). Liver vitamin E contents of broilers fed 100 and 150 mg/kg Cu as TBCC were 17.9% (P < 0.01) and 0.7% (P > 0.05) higher than those of the chicks consuming the same levels of Cu as Cu sulfate, respectively. Copper source and added Cu level affected (P < 0.05) plasma vitamin E contents (Table 4). Plasma vitamin E contents of broilers in the treatments of 100, 150, and 200 mg/kg Cu as TBCC were 15.0% (P < 0.01), 7.7% (P > 0.05), and 8.8% (P > 0.05) higher than those of the chicks consuming the same level of Cu as Cu sulfate, respectively. The results of vitamin E contents in plasma and liver further confirmed that prooxidant activity of TBCC was significantly lower than that of Cu sulfate.

Phytase Activities in Feeds

During feed storage, phytase activities in feeds decreased linearly with time regardless of Cu treatments (Table 5). Phytase activities in feeds at day 41 decreased by 41.4% in comparison with the value at the beginning of this experiment. Though the statistical analysis was not significant (P > 0.05), phytase activities in the feed fortified with 200 mg/kg Cu as TBCC were always numerically higher than those in the feed fortified with 200 mg/kg Cu as Cu sulfate (Table 6).

Discussion

The results from Cu contents of sources and uniformity of Cu mixing in feeds indicate that TBCC was advantageous for better mixing quality in feeds. This was attributable to the smaller particles and better flow characteristic of TBCC than those of Cu sulfate.

After Braude [16] first reported that the addition of a high level (ten times the requirement level) of Cu improved the growth performance of fattening piglets, high levels of Cu have been widely used as a growth promoter in animal production. Spears et al. [3] and Cromwell et al. [5] found that TBCC was a new supplemental Cu source to replace Cu sulfate in livestock production. The results from the current study indicate that supplemental TBCC improved ADG without increasing ADFI, and the addition of 200 mg/kg Cu was optimal for improving ADG of broilers, which was in agreement with the above findings.

In this study, the supplemental Cu level intervals were so small that the log10 transformed liver Cu concentrations had no linear relations with Cu intake; thus, relative bioavailability value of Cu as TBCC was not able to be determined by slope ratio comparison from multiple linear regressions. The results from this study showed that chicks fed TBCC had a lower Cu residue in liver than those fed Cu sulfate, indicating that TBCC might be a safer product than Cu sulfate.

Compared with CuSO4, the prooxidant activity and water solubility of TBCC was lower [2, 17]. Hooge et al. [17] and Luo et al. [10] have shown a reduction in vitamin degradation in mixed feed when TBCC rather than Cu sulfate was included as the Cu supplement. In the current study, TBCC was less active than Cu sulfate in promoting the oxidation of vitamin E in feeds, which is consistent with the results of previous research [10, 17]. Luo et al. [10] also reported that both serum and liver vitamin E of chicks mirrored the trends in the feed sample. The results of vitamin E contents in plasma and liver in this study further confirmed that prooxidant activity of TBCC was significantly lower than that of Cu sulfate.

The results from this study indicate that TBCC tended to maintain a higher phytase activity than CuSO4 in broiler feeds, but no significant differences were observed. This might be due to the following reasons: (1) the study was conducted in September and October when the environmental temperature was relatively low, (2) the phytase used in this study was well coated, and (3) Cu sources were added to the complete diets where Cu had fewer chances to get access to the phytase. Therefore, a further study will be needed to address the effect of Cu source on the stability of the uncoated phytase in premixes under a high temperature in order to demonstrate TBCC’s significant advantage over Cu sulfate in maintaining the phytase stability in the feed.