Effects of Various Copper Sources and Concentrations on Performance, Skeletal Growth, and Mineral Content of Excreta in Broiler Chickens

The experiment was designed to study the effect of supplemental sources and concentrations of copper on the performance and development and mineralization of tibia bones in broiler chickens. A 42-day feeding experiment was conducted utilising three copper sources, including copper sulphate (CuS), copper chloride (CuCl), and copper propionate (CuP), each with four different concentrations, i.e. 8, 100, 150, and 200 mg/kg. The body weight gain with 200 mg Cu/kg food was noticeably higher during the first 4–6 weeks of age. Due to the interaction between Cu sources and levels, there was no significant change in the body weight gained. The feed intake during various growing phases did differ significantly neither the main effect nor the interaction between different copper sources and levels. A CuP-supplemented diet (200 mg/kg food) considerably (P ≤ 0.05) improved the feed conversion ratio between 4–6 and 0–6 weeks. At the end of the experiment, a total of 72 tibia bones, i.e. six for each treatment were collected. A metabolic trial was conducted to look into mineral retention in broiler chickens on the final 3 days of the trial (40–42 days). Increased tibia bone zinc (Zn) levels were seen with the addition of 8 mg Cu/kg of Cu chloride, 100 mg Cu/kg of Cu propionate, 8 mg Cu/kg of Cu sulphate, and 8 mg/kg of Cu propionate to the diet. At higher levels of Cu (150 and 200 mg/kg diet), there was a significantly (P ≤ 0.01) reduced tibia Zn content. Cu sulphate treatment group had higher (P ≤ 0.01) tibia Cu content (8 mg Cu/kg diet). Cu sulphate supplemented diet had a greater excreta Zn content (P ≤ 0.01) than Cu chloride supplemented diet, and Cu propionate supplemented diet had the lowest excreta Zn content. Excreta with a higher Fe concentration were found in diets supplemented with copper sulphate and copper chloride (P ≤ 0.05) than in diets supplied with copper propionate. Thus, it may be concluded that feeding dietary Cu concentrations up to 200 mg Cu/kg diet, regardless of the different sources, had no negative effects on bone morphometry and mineralization parameters with the exception of a decrease in the tibia’s zinc content.


Introduction
Copper (Cu) is a vital trace element for the maintenance, growth, health, and survival of livestock and chickens.Cu is involved in a wide range of biological functions as a cofactor for several metalloenzymes, including hormone secretion pathways, connective tissue development, erythropoiesis, mitochondrial respiration, and immune system defences [1,2].Cu deficiency affects immune and skeletal system development, and performance in growing broiler chickens [3].The best possible production of broiler chickens continues to be hampered by leg disorders, including deformities and weakness.Several physiological processes, including bone growth and development in chicken, depend on copper (Cu), an essential trace mineral [4,5].In broiler chickens, Cu deficiency causes brittle bones and lameness.The growth and mineralization features of the tibia are reduced when dietary Cu is insufficient.Removal of trace minerals had a negative effect on bone strength [6].
The National Research Council's (NRC) Poultry Subcommittee determined that copper requirements for broiler were 8 mg/kg feed to prevent deficiency symptoms and sustain growth in the last revision of Nutrient Requirements of Poultry [7].Cu sources and levels had no effect on length or width (proximal and distal) in tibia bones [8].However, dietary Cu levels resulted in significantly greater mid shaft width at 12 mg Cu/kg diet compared to the other two levels (8 and 16 mg Cu/ kg diet).A dietary combination of 40 mg Zn/kg and 16 mg Cu/ kg using organic mineral sources was found to be sufficient to provide optimal bone morphometry and mineralization features in broiler chickens [9].Cu deficiency (less than 1 ppm) has been shown to reduce collagen production as well as mineralization.The body's ability to absorb and retain a mineral from the diet is an indicator of a mineral's bio-availability [10].Numerous dietary parameters, such as the kind of diet or component, the sources and concentration of minerals, and the relative quantities of different elements in the diet, all have an impact on the retention of minerals [11].Cu accumulation in soil and water is a problem for the environment in today's civilization.The chicken feed sector continues to formulate feeds with a high safety margin, which results in copper build up in the soil and a disruption of the natural fermentation process [12,13].
Thus, the present study was designed to examine the effects of feeding various sources and concentrations of Cu on the morphometry and mineralization of the tibia bone and utilisation of minerals in broiler chickens.

Animal Ethics Compliance
This experiment was approved and carried out according to the guidelines of the Institutional Animal Ethics Committee (IEAC) and CPCSEA approval (No.CARI/IAEC/17/twelve) of the Central Avian Research Institute, Izatnagar.

Experimental Design, Population, and Diets
Day-old coloured broiler straight-run chicks (n = 360) were distributed at random on the basis of initial body weight in 36 groups of 10 chicks each reared in battery brooders.Twelve dietary treatments with three dietary Cu sources (copper sulphate-CuS, copper chloride-CuCl, and copper propionate-CuP) each at four levels of copper (8, 100, 150, and 200 mg/ kg) in the following factorial design 3 × 4 was conducted during starting (0-3 weeks) and finishing (4-6 weeks) growth phases.The ingredients and chemical composition of basal diets for starting and finishing phase are presented in Table 1.

Production Performance
Weekly body weight and feed intake were recorded and feed conversion ratio (unit feed intake/unit body weight gain) was calculated.

Metabolic Trial
During the feeding trial, last 3 days (40-42 days), a metabolic trial was conducted to study the minerals retention/utilisation in broiler chickens.The net feed consumed by each bird in the respective dietary group was recorded and the dropping voided over the same period was collected quantitatively.On the last day, the feeders were removed to determine the net feed intake and faecal trays were removed to collect the faeces in aluminium dishes.

Faecal Excreta
The dropping collected were dried for 4-5 days in oven at 60 ± 5 °C till a constant weight was attained which represented the net dried faecal output.The dried and pooled excreta samples were ground and stored in air tight container for further analysis of different minerals.The experimental diet and excreta samples were analysed for calcium content [14], phosphorus [15], copper, zinc, manganese, and iron content were analysed by Atomic absorption spectrophotometer (Varian Spectra AA220 Model).

Bone Mineralization
At the conclusion of the 42-day trial, six birds from each feeding regimen (126 = 72 birds) were slaughtered, and their left tibia bones were extracted and freed of any attached soft tissues.According to Deo et al. [16] bone length and width were measured.Using Vernier callipers, the maximum width of the mid shaft, proximal and distal widths, as well as the overall length of each tibia bone, were measured.Each tibia bone was defatted for 18 h using the Soxhlet apparatus and petroleum spirit extraction to analyse the bone mineralization.The bones were then oven dried at 100° for 24 h before being ashed at 650° for 6 h in a muffle furnace.The tibia ash was measured and expressed as percentage of dry bone weight.The calcium and phosphorus content was estimated by method of Talapatra et al. [14] and AOAC [15], respectively.The bone zinc, copper, manganese, and iron content in tibia bone were determined by method of AOAC [15] using AAS (Varian Spectra AA 220 Model).

Statistical Analysis
The data obtained from the experiment were analysed statistically by the method of Snedecor and Cochran [17], and significant means were separated using Duncan's multiple range test (DMRT) described by Duncan [18].

Production Performance
The body weight gain was noticeably higher at 200 mg Cu/kg diet during the first 4-6 weeks of life.Due to the interaction of Cu sources and levels, there were no appreciable differences in body weight gain.Due to neither the main effect of copper sources and levels nor their interaction, the feed intake during various growth phases did not differ significantly.The feed conversion ratio was significantly (P ≤ 0.05) higher in the CuP-supplemented diet (200 mg/kg diet) during the 4-6 and 0-6 weeks.

Bone Morphometry
The effects of feeding different sources and concentrations of copper on bone length, width (proximal, mid shaft, and distal) are summarised in Table 2.The various bone

Bone Mineralization
The effect of feeding different dietary sources and concentrations of copper on bone mineralization traits is presented in

Discussion
Present results get support from works reported by earlier workers that the addition of supernormal levels (125-250 mg/kg) of copper (Cu) in the form of sulphate improve growth rate and feed efficiency in broilers chickens [19].Similarly, Ruiz et al. [20] stated that higher dietary Cu level promoted growth and increased performance of broiler chickens, while Nys [21] stated that the Cu promoted the growth of chicken when it was administered at higher doses.The feed intake during different growth phases did not differ significantly due to either main effect or interaction between different copper sources and levels.Present results are in agreement with work reported by Iqbal et al. [22] who also reported that feed intake of broilers chicks did not influenced significantly due to feeding different sources and concentrations of copper.The feed conversion ratio was significantly (P < 0.05) better in CuP supplemented diet than other sources of copper during 4-6 and 0-6 weeks of age.During (0-3 weeks) of age, feed conversion ratio did not differ significantly due to different dietary copper sources.The feed conversion efficiency was better (P < 0.05) at 200 mg Cu/kg diet than those recorded at other dietary copper levels different growth phases.There was no significant difference in feed conversion ratio due to interaction between copper sources and levels.Improvement of feed conversion ratio has been reported when broilers were fed diets supplemented with 125-250 ppm of copper in the form of sulphate [23].Similarly, Paik [24] reported that supplementation of Met-Cu chelate at the level of 100-125 ppm Cu improved growth performance of broilers and pigs.It was also reported that supplementation of 100 ppm of Cu as Met-Cu or Met-Cu-Zn improved performance in broilers  [25] and egg production was increased by supplementation of 100 ppm of Cu as Met-Cu chelates in layers [26].Mortality was not affected by the treatments.Present results get strength from the previous finding reported by Deo et al. [27] who reported that Cu sources (copper propionate and copper sulphate) and levels (8, 12, and 16 mg/kg) could not bring any significant change in length and width (proximal, mid shaft, distal) of tibia bone of broiler chicks.Similarly, Zheng et al. [28] reported that measurement evaluating in terms of length, proximal width, mid shaft width and distal width of femurs and tibiae did not reveal substantial benefit to skeletal integrity from above 40 mg Zn/kg supplementation.Iqbal et al. [9] also reported that bone length, proximal width, and mid shaft width of bone did not alter statistically due to Cu sources and levels.
Present results get support from earlier observation reported by Hashish et al. [29] who also observed a non-significant effect on tibia bone weight, calcium and phosphorus content of tibia bone by feeding 0 to 200 mg copper/kg diet.In the present finding lower tibia Zn content was recorded at higher levels of Cu (150 and 200 mg/kg diet) than those recorded at lower levels of Cu (8 and 100 mg/kg diet).The reason may be due to the interaction between Cu and Zn contents in the diet, because excess concentration of dietary copper will decrease the availability of zinc due to antagonistic effect.Thus, deposition of zinc in the bone will decrease at higher levels of copper.In the present finding tibia bone Cu content was changed significantly due to copper sources [16].
These results received support from the earlier work reported by Leeson [30] who also reported that when the bioavailability of reagent grade Cu sulphate was set as 100% the relative bioavailability of Cu was 111.63% in 14 days and 110.7% in 35 days of broiler chicks.Similarly, organic Cu sources, such as proteinate and amino acid chelate, have been shown to have higher relative bio-availability than that of inorganic mineral sources, such as oxide and sulphate [13].In contrary to present finding Iqbal et al. [9] who reported that tibia bone copper content was varied due to different levels of copper in the diet.These results partially support by earlier finding reported by Ao et al. [31] who observed that organic form of mineral sources such as proteinate and amino acid chelate have been shown to have relative bioavailability than that of inorganic mineral sources, such as oxide and sulphate which may influenced the higher iron content in the tibia bone in copper propionate supplemented diet.Reeves et al. [32] who observed that copper facilitates the absorption of iron in the body.
The concentrations of calcium (Ca), phosphorus (P), Cu, manganese (Mn), Zn, and iron (Fe) in excreta did not differ significantly due to interaction between different Cu sources and concentrations (Table 4).However, as increasing dietary Cu concentration, increased excreta Cu concentration with dose response.The present result is in close agreement with earlier observation reported by Bao et al. [33] who reported that the excretion of Cu increased linearly with increasing intake of the element through the diet.In the present finding excreta Cu content did not change significantly due to copper sources, but numerically less excreta copper content was observed in organic source of copper than inorganic sources of copper, which indicate higher retention of copper in organic source than inorganic source of copper.These results received support from the earlier observation of Lee et al. [34] also reported that excreta Cu content was greatly reduced when organic source of mineral supplemented in broiler diet.Similarly, Shamsudeen et al. [35] reported that a higher retention of Cu in Cu chelate group than its counterpart Cu-inorganic group in broiler chickens.Iqbal et al. [9] also found non-significant effect on excreta calcium and phosphorus content when diet fed variable levels of Cu, Zn, and their sources in broiler diets.These results also support present finding.Higher excreta Mn content was recorded in Cu chloride supplemented diet than Cu sulphate supplemented diet and significantly lowest excreta Mn content was recorded in Cu propionate supplemented diet.The lower excreta Mn content found in Cu propionate supplemented diet which suggesting that organic source of Cu had higher retention of minerals than inorganic source copper chloride and copper sulphate.Lee et al. [34] also reported that excreta Cu content was greatly reduced when organic source of mineral supplemented in broiler diet Shamsudeen et al. [35] reported that a higher retention of Cu in Cu chelate group than its counterpart Cu-inorganic group in broiler chickens.Excreta Mn content was recorded higher at 200 mg/kg than 150 mg/kg diet and lower at 8 and 100 mgCu/kg diet.Contrary to the present finding El-Damrawy et al. [36] who reported that manganese retention was unaffected by source and dose of copper in the diet.Excreta Zn content was higher in Cu sulphate supplemented diet than Cu chloride supplemented diet and lowest excreta Zn content was recorded in Cu propionate supplemented diet than those observed in Cu sulphate and Cu chloride supplemented diet.Higher excreta Fe content was observed in Cu sulphate and Cu chloride supplemented diet than that recorded in Cu propionate supplemented diet.The lower excreta Zn and Fe content found in Cu propionate supplemented diet which suggesting that organic source copper propionate had higher retention of Zn and Fe than inorganic sources copper sulphate and copper chloride.El-Damrawy et al. [36] also observed that supplementation of copper propionate had higher Zn and Fe retention than its counterpart copper sulphate supplemented diet (Table 5).
Therefore, it can be concluded that feeding dietary Cu concentrations up to 200 mg Cu/kg diet, effective in promoting growth, feed conversion efficiency regardless of different sources, and had no negative effects on bone morphometry and mineralization features, with the exception of a decrease in tibia zinc content.However, compared to Cu sulphate and Cu chloride supplemented diets; Cu propionate considerably increased the retention of trace minerals.

Table 1
Ingredients and nutrients composition (gm/kg) of basal diet fed during starting (0-3 wk) and finishing (4-6 wk) of age to broiler chickens *

Table 2
Growth performance and serum cholesterol contents in broiler chicks as influenced by different sources and levels of copperMean carrying difference superscript in column differ significantly (P < 0.05)

Table 3 .
Various bone mineralization traits such as dried bone weight, bone ash, bone calcium, phosphorus, and manganese content remained statistically unchanged due to either main effect or interaction between different sources and concentrations of copper in the diet.Significantly (P ≤ 0.05) higher tibia bone zinc (Zn) was recorded at 8 mg Cu/kg diet with Cu chloride followed by 100 mg Cu/kg diet with Cu propionate, 8 mg Cu/kg diet with Cu sulphate, and 8 mg/kg diet with Cu propionate supplemented diet than those observed in other dietary combinations.Significantly (P ≤ 0.05) higher tibia Zn content was observed in Cu propionate supplemented diet than that recorded with Cu sulphate supplemented diets.However, Cu chloride supplemented diet, the tibia Zn content was found intermediary.Significantly (P ≤ 0.01) lower tibia Zn content was recorded at higher levels of Cu (150 and 200 mg/kg diet) than those recorded at lower levels of Cu (8 and 100 mg/kg diet).Significantly (P ≤ 0.01) higher tibia Cu content was recorded at 8 mg Cu/kg diet with Cu sulphate followed by 150 mg Cu/kg diet with Cu propionate and 100 mg Cu/kg diet with Cu propionate than those recorded in other dietary combinations (Table4).

Table 4
Tibia bone mineralization of broiler chickens as influenced by feeding different sources and concentrations of copper

Table 5
Excreta mineral contents of broiler chickens as influenced by feeding different sources and concentrations of copper Values bearing different superscripts within a column differ significantly, P ≤ 0.05, P ≤ 0.01; NS non significant