MATERIALS AND METHODS

Birds and diets: This study was performed from January 1 to February 20, 2009 at the Isfahan Research Center for Agriculture and Natural Resources, Isfahan, Iran. Three hundred and twelve, day-old mixed sex broiler chicks (Ross 308) were individually weighed and randomly divided into 6 main groups of 52 chicks. To limit differences due to position, these groups were each divided into 4 subgroups of 13 chicks each. The chicks were randomly allocated to floor pens and exposed to continuous light for the first 2 days, then to 23 h light and 1 h darkness until 49 days of age. Diets (in mash form) and water were provided for ad libitum consumption. Corn-soybean experimental diets (Table 1) were formulated to meet broiler chickens nutritional requirements, as recommended by NRC (1994).

These diets consisted of the basal diet supplemented with added Zn, Mn and Cu in combination, from either inorganic or organic sources. The mineral premix used in this experiment was a commercial premix without Zn, Mn and Cu. The inorganic supplementations were zinc sulfate (ZnSO₄), zinc oxide (ZnO), manganese sulfate (MnSO₄), manganese oxide (MnO) and copper sulfate (CuSO₄. 5H₂O). Organic supplementation (Aviala ZMC, Zinpro Animal Nutrition Inc., Eden Prairie, Mn 55344) consisted of a combination of complex amino-acid-metals that contained 40 mg kg^-1 Zn, 40 mg kg^-1 Mn and 7 mg kg^-1 Cu, respectively.

Six dietary treatments based on corn and soybean meal with different mineral premix levels were formulated as follow: A: basal diet supplemented with common mineral premix containing 100, 100 and 10 mg kg^-1 Zn, Mn and Cu from ZnO, MnO and CuSO₄ sources, respectively (control diet); B: basal diet supplemented with 140, 140 and 17 mg kg^-1 Zn, Mn and Cu from inorganic (ZnO, MnO and CuSO₄) and organic (40, 40, 7 mg kg^-1 Zn-Mn-Cu); C: basal diet supplemented with premix providing NRC (1994) requirements for Zn, Mn and Cu supplied by sulfates and oxide (40, 60, 8 mg kg^-1); D: basal diet supplemented with 40, 40 and 7 mg kg^-1 Zn, Mn and Cu from Zn-Mn-Cu-amino acid complex; E: basal diet supplemented with 40, 40 and 7 mg kg^-1 Zn, Mn and Cu supplied by sulfates; F: basal diet supplemented with 60, 60 and 10.5 mg kg^-1 Zn, Mn and Cu from Zn-Mn-Cu-amino acid complex.

Table 1:	Composition of the basal diets
1Provided per kg of diet: vitamin A (as all-trans retinol acetate): 15,000 IU; cholecalciferol: 3900 IU; vitamin E (as all-rac--tocopherol acetate): 30 IU; vitamin K (as menadione sodium bisulfate): 3.0 mg; thiamin (as thiamin mononitrate): 2.4 mg; riboflavin: 9.0 mg; vitamin B6: 4.5 mg; vitamin B12: 0.021 mg; calcium pantothenate: 30 mg; niacin: 45 mg; folic acid: 1.2 mg; biotin: 0.18 mg; choline (as choline chloride): 700 mg; iron (from iron sulphate): 80 mg; iodine (from potassium iodide): 0.35 mg; selenium (from sodium selenite): 0.15 mg

Performance: Body weight was determined at 1, 21, 42 and 49 days of age. Feed intake, weight gain and feed conversion ratio (feed intake: weight gain) were recorded in different periods and overall growth period.

Carcass components: At the end of the experiment (49 days of age), two birds were c randomly chosen from each replicate, slaughtered and the abdominal fat, liver, bursa of Fabercius and spleen were collected, weighed and calculated as a percentage of live body weight.

Zn, Mn and Cu concentrations in Tibia Ash: At 49 days of age, right tibia was removed from two birds per replicate for determination of bone ash percentage and Zn, Mn and Cu concentration according to the method described by Kurtoglu et al. (2005).

Statistical analysis: The data were subjected to analysis of variance procedures appropriate for a completely randomized design using the General Linear Model procedures of SAS Institute (1997). Means were compared using Duncan multiple range test. Statements of statistical significance are based on (p<0.05).

RESULTS

Neither dietary Zn, Mn and Cu concentrations nor Zn, Mn and Cu sources significantly altered body weight at days 21, 42 and 49 (Table 2). The effects of Zn, Mn and Cu levels and sources on feed consumption were significant (p<0.05) only when trace minerals were greater in the diets (B treatment) in starter period, but in grower period E treatment had a higher feed intake. Dietary supplementation of Zn, Mn and Cu did not affect daily feed intake in other periods.

Supplementation of diets with the highest levels of inorganic forms of Zn, Mn and Cu (100, 100 and 10 mg kg^-1, respectively) (A treatment) and the lowest levels of their organic forms (40, 40 and 7 mg kg^-1) markedly improved feed conversion ratio (1.41 and 1.42 g g^-1, respectively) in starter period. Average feed conversation ratio at 21-42 day of age for A treatment and also group fed diet containing 60, 60 and 10.5 mg kg^-1 Zn, Mn and Cu, respectively (F treatment) were significantly better than chickens fed the diet in which Zn, Mn and Cu concentrations were according to NRC recommendation (C treatment) from their inorganic sources. However, the best overall feed conversion ratio was observed for birds fed diets supplemented with the highest levels of inorganic forms of the elements during different periods of the experimental period (A treatment). On the other hand, as shown in Table 2 the highest average of overall feed conversion rate (2.15 g g^-1) was observed for group fed diet containing 40-60-8 mg kg^-1 Zn-Mn-Cu from their oxide, oxide and sulfate sources, respectively (C treatment).

Dietary treatments had only a significant (p<0.05) effect on carcass yield at 49 d of age (Table 3). Birds fed Zn, Mn and Cu, in sulfate forms (E treatment) significantly (p<0.05) had a higher carcass yield than those fed oxide forms of Zn, Mn and Cu. Supplementary diets with different sources and levels of Zn, Mn and Cu did not have any significant effects on abdominal fat, bursa of Fabricius and spleen percentage (Table 3). However, increasing Zn, Mn and Cu levels improved bursa of Fabricius and spleen percentages (treatment B).

Table 2:	Effect of different mineral supplementations on the performance of broiler chickens
1A: 100, 100 and 10 mg kg-1 Zn, Mn and Cu from ZnO, MnO and CuSo4; B: 100, 100 and 10 mg kg-1 Zn, Mn and Cu from ZnO, MnO, CuSo4 and 40, 40, 7 mg kg-1 Zn-Mn-Cu-Amino acid; C: 40, 60 ,8 mg kg-1 Zn, Mn and Cu from ZnO, MnO and CuSO4; D: 40, 40 and 7 mg kg-1 Zn, Mn and Cu from Zn-Mn-Cu-Amino acid; E: 40, 40 and 7 mg kg-1 Zn, Mn and Cu supplied by sulfates. F: 60, 60 and 10.5 mg kg-1 Zn, Mn and Cu from Zn-Mn-Cu-amino acid. Means with different superscript within a row are significantly different (p<0.05)

Table 3:	Effects of dietary Zn, Mn and Cu source and levels on carcass traits and lymphoid organs (percentage of live weight)
Means with different superscript within a column are significantly different (p<0.05)

Tibia ash percentage increased in chicks fed diets containing the highest level of Zn, Mn and Cu (B treatment), but this increase was not significant (Table 4). Tibia Mn and particularly Zn and Cu concentrations increased in chickens fed diets containing high levels of Zn, Mn and Cu from combination of both their organic and inorganic sources (B treatment). On the other hand, among the groups fed diet supplemented with the same concentrations of Zn, Mn and Cu (C, D and E treatments) birds supplemented with organic sources of the minerals tended to have higher levels of tibia ash and also Mn and Cu concentrations in tibia ash at 49 day of age. During the experimental period mortality was within acceptable ranges (less than 3%) and was not related to dietary treatments.

Table 4:	Effects of dietary Zn, Mn and Cu sources and levels on tibia ash and concentration of Zn, Mn and Cu in tibia ash of broiler chickens
Means with different superscript within a column are significantly different (p<0.05)

DISCUSSION

In the present experiment broiler chicks responses to different dietary Zn, Mn and Cu sources and levels were studied. According to the results showed in Table 1 application of only high levels of the experimental minerals (Zn, Mn and Cu) from their inorganic sources (ZnO, MnO and CuSO₄.5H₂O) (B treatment) supported an optimal growth performance of broiler chicks. Whereas, dietary application of Zn, Mn and Cu at equal amounts of NRC recommendation (40, 60 and 8 mg kg^-1, respectively) (C treatment) from the sources same as B treatment caused remarkable negative effects on body weight, feed intake and feed conversion rate during different stages of rearing period. It means that birds fed oxide form of the manganese and zinc faced with their bioavailability, since increasing their dietary concentrations from 40 to 100 mg kg^-1 markedly improved most of the growth performance indices, particularly feed conversion ratio and their concentrations in tibia ash (Table 4). The results are in contradictory with findings of Pimentel et al. (1991) who observed no differences in relative bioavailability between oxide and organic zinc sources in broilers. In this relation, availability of minerals within inorganic sources has been shown to vary from 60 to 96% (Henry et al., 1989; Zanetti et al., 1991) although for a specific ingredient such as Mn oxide, bioavailability has been reported to vary by 60% depending on its source (Wong-Valle et al., 1989).

On the other hand, substitution of oxide sources with their organic (D treatment) or sulfate forms (E treatment) to supply Zn, Mn and Cu requirements suggested by NRC (40, 60 and 8 mg kg^-1, respectively) supported growth performance rate similar to that of A treatment as and more better than C treatment. This result is in agreement with Ledoux et al. (1991), Smith et al. (1995), Pesti and Bakalli, (1996) and Sandoval et al. (1997) who reported that sulfates forms of the trace minerals have higher bioavailability than their oxides. Thereby, some researchers (Wong-Valle et al., 1989; Wedekind and Baker, 1990) have shown that using minerals in sulfate form at 40% of current oxide form inclusions, supports bird performance. Regarding bioavailability of organic sources of minerals, Mn proteinates have been shown to be 50-70% more available than oxides (Smith et al., 1995), while Henry et al. (1989) suggested 30 to 40% greater bioavailability of a manganese-methionine chelate compared to an oxide source. While, Miles et al. (2003) showed reduced potency of amino acid chelates of Cu and Mn compared to sulfates.

On the other hand, some studies indicated small or no differences in bioavailability of Zn between inorganic and organic Zn sources (Pimentel et al., 1991; Baker and Ammerman, 1995). However, variability in availability of organic minerals may relate to their ability to remain chelated during solubilization (Guo et al., 2001). Interestingly, use of additional dosages of even organic forms of the minerals more than 100, 100 and 10 mg kg^-1 Zn, Mn and Cu existing in A treatment (B treatment) or 40, 40 and 7 mg kg^-1 existing in D treatment (F treatment) did not any significant improvement in growth performance parameters (Table 2) or their concentrations in tibia ash (Table 4). It means that with inclusion of additional dosage of trace elements into diets improvement of performance would not be expected. Meanwhile, according to current researches on trace minerals it is suggested that broilers fed diets supplemented with trace minerals far in excess of their actual requirements.

In current experiment, dietary supplementation of the trace minerals did not significantly affect lymphoid organ weights. Nevertheless, the relative weights of bursa of Fabricius and spleen in group fed diet containing high levels of zinc, manganese and copper (B treatment) were higher than the other groups. Virden et al. (2004) also showed that dietary supplementation of Zn and Mn increased relative weight of spleen and bursa Fabricius in progeny chicks.

The increase in tibia zinc observed when broiler diets were supplemented with a combination of Zn-AA+ZnSO₄ (B treatment) may be due in part to increased zinc absorption, which by supplying both organic and inorganic sources of zinc could involve more absorption sites or transporters in the intestine, hence increasing zinc retention (Burrell et al., 2004). Irrespective of dietary concentration impact of the minerals on their deposition rate in tibia, our results showed that with feeding different diets containing same concentrations of Zn, Mn and Cu (C, D and E treatments) birds will be able to deposit organic form of minerals more efficient than their inorganic forms. Henry et al. (1989) also reported that the estimated bioavailability based on bone Mn accumulation was 108% for Mn-Met relative to Mn sulfate in chicks fed corn-soybean meal diets.

In conclusion, present study shows that NRC (1994) recommendation for supplementation of corn-soybean diet with 40-60-8 mg kg^-1 Zn-Mn-Cu cannot support maximum broiler chicken's performance. In addition, corn-soybean diets supplied with 40-40-7 mg kg^-1 Zn-Mn-Cu from their sulfate or organic (metal amino acid complexes) sources instead of application of 100-100-10 mg kg^-1 Zn-Mn-Cu from their oxide sources can support performance of broiler chickens properly.

ACKNOWLEDGMENTS

The authors are grateful to Zinpro Co. (Zinpro Animal Nutrition Inc., Eden Prairie, Mn 55344) and Sana Dam Pars Co. (No.125, Mirdamad blvd., Tehran, Iran) for part of research funding support. Appreciation is expressed to Pejman Ajilchi, Mohsen Heidari and Mehdi Toghyani for their valuable assistance in this research project.