Introduction

Copper (Cu) is an essential trace element found in small amounts in a variety of cells and tissues [1]. Its role has been defined for many physiological functions related mostly to its presence in the active sites of cupro-enzymes [2]. Copper is either a cofactor or an integral part of many important enzymes including cytochrome c oxidase, tyrosinase, p-hydroxyphenyl pyruvate hydrolase, dopamine beta hydroxylase, lysyl oxidase, and copper-zinc superoxide dismutase (Cu-Zn SOD). These enzymes are involved in a number of vital biological processes related to growth, development, maintenance, and production [1, 2].

Copper is routinely added to poultry diets at prophylactic concentrations because of its antimicrobial activity and growth-promoting effects [3]. Common copper sources used in poultry diets are inorganic copper salts, primarily the sulfate form, because of their low cost and commercial availability. The influence of supplemental inorganic copper salts on performance of poultry species has been well documented [4–9].

Inorganic copper compounds have been added to poultry diets at excess levels (100–250 mg/kg) of the requirements of broilers (8 mg/kg) recommended by National Research Council. (NRC) [10]. However, copper requirement of the laying hens was not specified in the NRC [10]. When included at higher levels to diets, the so-called pharmacological levels, a high proportion of ingested copper is excreted through feces [2, 11]. Therefore, there is an increasing concern that feeding high levels of copper to poultry will have a negative impact on the environment. With increasing concern for environmental pollution from minerals in excreta, there is an interest in the poultry industry to find alternative sources of copper to improve the biological availability by using organic trace minerals in animal diet [12]. Most of the organic minerals are classified as complexes, chelates, or proteinates [13]. Chelated products or proteinates have superior bioavailability over inorganic copper sources [14–16]. The purpose of a chelated product is to protect the compound of interest from digestion or complex formation thereby enabling the protected compound to reach its target site [17]; thus, lower dietary inclusion levels are required due to the better absorption [16], which enhances the efficiency. Although, animals vary by species in their sensitivity, poultry can tolerate many times their usual daily intakes of copper [1, 2]. However, various levels of organic copper used in diet should be studied for safety because exposure to elevated concentration of copper is damaging. Furthermore, determination of copper residue in the egg may be of importance for health concern of humans. Therefore, this study was performed to determine the effects of three levels of copper proteinate on body weight, egg production, egg weight, food consumption, food efficiency, and serum glucose, proteins, lipids, enzymes, minerals, and malondialdehyde (MDA), which is an indicator of lipid peroxidation status, liver and kidney histology, and copper content in liver and egg.

Materials and Methods

Animals, Diets, and Management

Seventy-two 30-week-old Bovans laying hens were used in this study. Hens were weighed to provide the equal live weight in all groups in the beginning of the study and distributed to four groups with three replicates containing six hens in each after 10 days of acclimation. The control group was fed commercial basal diet containing average 17% crude protein and 2,800 kcal/kg metabolizable energy (ME); treatment groups were fed basal diet supplemented with 150, 300, and 450 mg/kg Cu as copper proteinate (Bioplex Copper, Alltech, Nicholasville, KY, USA) for 4 weeks (Table 1). Hens were allowed ad libitum access to food and water. Animals were reared at 18–20°C at17 h lighting schedule.

Table 1 Ingredients and Chemical Composition of Basal Diet Fed to Laying Hen
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Measurements of Performance

The body weight of animals was recorded in the beginning and in the end of the study. Egg production was recorded daily, and food consumption and egg weight were recorded at weekly intervals. Feed efficiency was calculated by determination of the amount of food consumed for 1 kg of egg.

Sample Collection and Analysis

In the end of the study, 12 blood samples (four per replicate) were collected from each group for biochemical analysis. The sera were separated by centrifugation at 1,300×g after 1 h incubation at room temperature and stored at −20°C until the analysis. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), gamma glutamyl transpeptidase (GGT) activities and triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL), total protein, glucose, calcium (Ca), inorganic phosphorous (Pi), and magnesium (Mg) concentrations were determined with commercially available kits (Beckman, Ireland) by an auto-analyzer (Beckman LX-20 Coulter, Ireland). Serum MDA level, which is an end product of thiobarbituric-acid-reactive substances, was determined according to the method described by Moreno et al. [18].

Nine animals (three per replicate) from each group were neck dislocated, and liver and kidney samples were collected at the end of the feeding trial. Livers were wiped with paper tissues after removal of gall bladders. Liver and kidneys were weighed, and weights were recorded as grams per 100 g body weight.

Parts of liver samples were frozen individually in sealed plastic bags for copper analysis. Remaining parts of liver samples and kidney samples were preserved in buffered formalin for histopathological processing. Tissue samples were embedded in paraffin, sectioned in 5–6 μm, stained with hematoxylin–eosin (HE), and examined under light microscope [19].

Chemical composition of diet was analyzed by the methods of Association of Official Analytical Chemists (AOAC) [20]. Copper levels in tissue and food samples were determined with atomic absorption spectrophotometer (Perkin Elmer, Analyst 700) after microwave digestion by the method described in AOAC [21].

Statistical Analysis

Statistical analyses of data were performed by SPSS 10.0 version for Windows. One-way analysis of variance was used for the differences between groups. When the F values were significant, Duncan’s multiple range test was performed. All data were expressed as means ± SEMs.

Results

No differences were found in body weight, ratio of liver and kidney to body weight (Table 2), food consumption, feed efficiency, and egg weight. Supplementation of 150 and 300 mg/kg Cu increased, whereas 450 mg/kg Cu decreased egg production (p < 0.001; Table 3).

Table 2 Effects of Dietary Copper Proteinate Supplementation on Body Weight and Liver and Kidney Weight in Laying Hens
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Table 3 Effects of Dietary Copper Proteinate Supplementation on Performance in Laying Hens
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Liver copper contents were elevated in 300 and 450 mg/kg Cu supplemented groups (p < 0.001). Egg copper contents were increased by all supplemental copper levels (p < 0.01; Table 4). There were no differences between all groups with regard to serum lipase and AST activity, total protein, TG, Ca, Pi, and Mg levels. Copper supplementation to diet at the levels of 150 and 300 mg/kg had no effects on serum ALT, ALP, GGT, LDH, MDA, and HDL levels. Serum glucose levels were increased (p < 0.001), whereas albumin (p < 0.01) and TC levels were decreased (p < 0.05) by the supplemental levels of 300 and 450 mg/kg Cu. However, only 450 mg/kg Cu supplementation increased ALP and GGT activities (p < 0.05) and MDA and HDL levels (p < 0.01), and this level decreased ALT and LDH activities (p < 0.01; Table 5).

Table 4 Effects of Dietary Copper Proteinate Supplementation on Copper Concentrations of Egg Yolk and Liver in Laying Hens
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Table 5 Effects of Dietary Copper Proteinate Supplementation on Biochemical Parameters in Laying Hens
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No gross and microscopic changes were observed by the histological examinations of liver and kidneys (Figs. 1 and 2).

Fig. 1
figure 1

Normal appearance of the livers of laying hens supplemented with copper proteinate; a 150 mg/kg Cu, b 300 mg/kg Cu, c 450 mg/kg Cu, d control group (HE, ×200)

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Fig. 2
figure 2

Normal appearance of the kidneys of laying hens supplemented with copper proteinate; a 150 mg/kg Cu, b 300 mg/kg Cu, c 450 mg/kg Cu, d control group (HE, ×200)

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Discussion

Copper has become a very popular feed additive in poultry diets because of its possible growth-promoting and antimicrobial effects with the moving away of antibiotics from diets [3]. Inorganic copper compounds are often fed to poultry at the levels of 100–250 mg/kg diet, which are quite above the recommended level of 8 mg/kg for broiler requirements by NRC [10]. Organic products have superior bioavailability over inorganic copper compounds [14, 16]. Although the enhanced bioavailability may relate to less susceptibility of organic compounds to various physiochemical factors which adversely affect the absorption of inorganic minerals [2], different organic copper complexes have different bioavailability [15]. To the author’s knowledge, there are very few studies investigating the effects of organic copper compounds in laying hens. Controversies exist in these studies concerning the levels of organic copper complexes recommended for laying hens without having deleterious effects on performance and metabolism [17, 22, 23]. Determination of possible changes due to copper proteinate, which is one of the organic copper compounds at high levels, in performance and metabolism as well as its accumulation in the liver and residue in the egg yolk may be of importance for health concern in both animals and humans. Therefore, in this study, as low as 150 mg/kg and as high as 450 mg/kg Cu were supplemented to diet of laying hens.

In the present study, supplementation of all levels of copper proteinate had no effects on body weight [5, 17], food consumption [5, 7, 24–26], feed efficiency [24, 26], and egg weight [7, 9, 17, 22, 24, 26] as indicated in previous studies in which layer diets had been supplemented with different sources and levels of organic and inorganic copper compounds. In addition, any level of supplemental copper did not influence serum total protein and major minerals (Ca, Pi, Mg) as reported by Mondal et al. [27].

It was suggested that copper improved the activities of digestive enzymes such as total protease, amylase, and lipase [28]. However, in the present study, similar effect of copper proteinate on serum lipase was not found. Elevated serum ALP and GGT activities were partly consistent with the result of Almansour [29], who found increases in ALP, AST, and ALT activities. The activity of ALT enzyme can be influenced by food restriction [30]. A reduction found in ALT activity confirming the findings of Pearce et al. [4] may be due to the slightly reduced food consumption, which is also confirmed by the slightly reduced body weight. Furthermore, the reduced albumin concentrations by 300 and 450 mg/kg Cu supplementations, consistent with the decreased ALT activity, may suggest an alteration in protein synthesis [30]. Changes in blood chemistry variables occur before the physiological and morphological lesions. Although gross and histopathological examinations revealed no pathological lesion in the kidneys and liver, consistent with no changes in the ratio of kidney and liver weights to the body weight, presence of alterations in the serum enzyme activities, especially in the animals fed diet supplemented with 450 mg/kg Cu, may suggest that when the animals are exposed to this level and higher amounts of copper for prolonged period, potential hazardous effects may arise.

Supplementation of 150 mg/kg Cu did not increase the copper content of liver as in the studies that used similar copper levels from different sources in poultry [16, 23, 31]. A remarkable increase in copper content in liver of hens supplemented with 300 and 450 mg/kg Cu confirmed the results of previous studies [11, 16, 32, 33] that used similar or higher copper levels. The liver copper content is the indicator of the copper status of the animal [2]. Increasing liver copper content consistent with the dietary copper level reflects a dose-dependent manner of copper accumulation in the liver. However, even at the highest dietary copper supplementation, liver copper concentration reached up to 19.63 mg/kg, which is quite a low level than the European Union limit of 80 mg/kg as reported by Skrivan et al. [11]. Lim and Paik [23] found no significant influence of 100 mg/kg dietary copper as copper chelates on the copper content of egg yolk. In contrast, in the present study, a linear increase in egg yolk copper content in all copper-supplemented groups confirmed the results of Pesti and Bakalli [7], who reported increased egg yolk copper content with 125 and 250 mg/kg Cu, and also the results of Skrivan et al. [11], who used a range of copper from 25 up to 240 mg/kg in the form of copper sulfate. Idowu et al. [22] found higher accumulation of inorganic copper than organic form in liver and egg yolk. The copper concentration of liver and egg yolk may depend on the sources and levels of copper used as supplement.

In some previous studies, no effects of copper sulfate from 50 up to 250 mg/kg on egg production were reported [9, 24, 26]. On the other hand, Al Ankari et al. [5] reported reduced egg production by the supplementation of diet with 250 mg/kg Cu as sulfate or acetate. However, in the studies with organic copper compounds, increases in egg production were reported with 100 mg/kg Cu in the form of methionine chelates [23] and with 125 and 250 mg/kg Cu in the form of copper proteinate [22]. Similarly, Pesti and Bakalli [7] reported positive effect of copper sulfate at a level of 250 mg/kg on egg production. In the present study, egg production was increased with the supplementation of copper proteinate at levels of 150 and 300 mg/kg consistent with the results of the studies in which similar levels and sources of copper were used [22, 23].

In this study, improvements in egg production were determined by 150 and 300 mg/kg copper supplementation. However, the reduced egg production by 450 mg/kg copper supplementation, which was consistent with reductions in the serum triglycerides and total cholesterol, may result from the lowered circulating level of 17 beta-estradiol, hence reproductive physiology due to the slightly reduced food consumption resulting in depressed hepatic lipid synthesis as found in the previous studies. In those studies, the serum triglycerides [5, 22, 27] and total cholesterol [5, 6, 9, 22, 25, 27] were also reduced by copper supplementation. In the present study, the reduced plasma cholesterol by supplemental copper may be due to the high liver Cu concentration that regulates cholesterol biosynthesis by reducing hepatic reduced glutathione concentration, which in turn reduces the activity of 3-hydoxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme of cholesterol biosynthesis [5, 27, 33]. The decreases in total cholesterol may also be associated with the increased HDL synthesis. Although in the present study, the low-density lipoprotein (LDL) level could not be determined, the increased blood HDL concentration may result from the synthesis of HDL from LDL by modulation of HMG-CoA reductase activity by copper [27]. The elevated HDL concentration confirmed findings of Lien et al. [9], who fed the copper-sulfate-supplemented diet to laying hens, and Mondal et al. [27], who used copper proteinate and copper sulfate in broiler diets at the levels of 200 and 400 mg/kg.

Although normally bound to proteins, copper may be released and become free to catalyze the formation of highly reactive hydroxyl radicals. Data obtained from in vitro and cell culture studies are largely supportive of capacity of copper to initiate oxidative damage and interfere with important cellular events. It has been suggested that oxidative damage has been linked to chronic copper overload and/or exposure to excess copper [1, 34, 35]. The remarkable increase in copper content in liver of hens supplemented with 300 and 450 mg/kg Cu may reflect higher absorption of copper proteinate, thus animals may be exposed to excess copper. The increased glucose level confirmed the results of Almansour [29] who found the elevated serum glucose in quail fed diet supplemented with copper sulfate above the level of 750 mg/kg. It has been suggested that the increases in lipid peroxidation is associated with the elevated plasma glucose [36]. The increased MDA level in the present study might be resulted from the auto-oxidation of glucose [36] as well as copper itself [34].

The results of this study have shown that supplementation of 150 and 300 mg/kg Cu in the form of copper proteinate increased egg production without having marked adverse effects on the investigated parameters. However, alterations in some blood chemistry variables and reduction in egg production may suggest an adverse effect of copper proteinate at the supplemental level of 450 mg/kg in laying hens. Therefore, further more detailed and prolonged investigations with different organic copper compounds at various levels are needed to better understand the effects of this essential, but potentially toxic, trace element.