INTRODUCTION
Manganese (Mn) is a bioactive element required for multiple postabsorptive physiological processes in poultry, such as carbohydrate, lipid, and amino acid metabolism, as well as cartilage and bone development, and antioxidant defense [
1]. For fast-growing broilers, Mn deserves concern since its manipulation in practical feeds may attenuate the incidence of certain disorders associated with fast growth and, in turn, may protect against adverse impacts on revenue and profitability of the chicken meat industry. As a cofactor for glycosyltransferase, Mn acts by attaching glucosamines to a protein core during the synthesis of proteoglycans, an important constituent of bone organic matrix and cartilage [
2]. Low concentrations of Mn in broiler feeds have been shown to decrease the activity of glycosyltransferase and, consequently, the synthesis of proteoglycans [
3], which is commonly associated with the incidence of leg abnormalities in broilers fed Mn deficient feeds [
4,
5]. In turn, leg abnormalities in commercial flocks lead to deprivation of locomotive freedom and restricted access to feeds, and consequently to impairments in growth and feed efficiency, which on welfare and economic grounds is undesired in intensive poultry rearing [
6].
Manganese is also a component of Mn-containing superoxide dismutase (SOD), an essential antioxidant metalloenzyme responsible for free radical scavenging in mitochondria by catalyzing the dismutation of superoxide anions produced from the electron transport chain to molecular oxygen and hydrogen peroxide [
7]. Mitochondria are the major oxidative phosphorylation site where carbohydrates and fats are oxidized to produce energy for cell functioning [
8]. Electron and proton leaks across the mitochondrial respiratory chain are believed to increase the generation of free radicals, which may reduce the respiratory chain function or even result in cellular apoptosis [
7,
8]. Apart from their consequences on the energetic efficiency of cells, intracellular oxidation of lipids and proteins caused by free radicals has a severe practical implication for the poultry meat industry, since damage in tissues affects relevant sensory traits of meat such as overall appearance, color, texture, and flavor [
9]. Lu et al [
5] demonstrated that dietary Mn supplementation upregulated Mn-SOD gene expression, increased Mn-SOD activity, and reduced malondialdehyde content, a biomarker of lipid peroxidation, in leg muscles of growing chicks.
Manganese requirements of growing broilers are described by the National Research Council [
10] based on leg abnormalities and growth as 60 mg/kg diet. This value is half the 120 mg Mn/kg recommended by Lu et al [
5] few years earlier. Contrary to NRC [
10], the referred authors [
11] based their estimates on growth performance, tissue mineralization, and Mn-SOD activity in heart. As an attempt to reevaluate growing chick responses to supplemental Mn, Lu et al [
4] found that neither growth performance nor meat quality traits were affected by the supplemental Mn levels under investigation (100 to 500 mg Mn/kg diet as Mn sulphate), indicating that adding 100 mg Mn/kg diet supports broiler carcass traits and meat quality. Even though relevant as references, these findings fail to establish the minimum level of supplemental Mn to optimize chick responses.
From the mid-2000s, organic trace minerals (OTM) received considerable attention due to their potential benefits and advantages compared with inorganic salts. After decades of investments and research efforts, OTM have proved to be more bioavailable than carbonates, oxides, and sulphates traditionally used by industry [
12]. One point commonly ignored in assessments of poultry responses to sources and concentrations of trace minerals, which could potentially lead to misinterpretations of outcomes, is the source of the other trace minerals provided in the supplement in experimental diets. Generally, trace mineral supplement used in assays conducted to determine ideal levels of a given OTM provide all the other trace minerals as inorganic salts. Although widespread, such approach might not be the most accurate one to validate optimum levels of organic minerals for poultry. Firstly, because it fails in mimicking the conditions under which commercial flocks are reared. In intensive broiler rearing, Mn provided as inorganic salts has been typically supplemented with other trace minerals also in inorganic form, whereas Mn as proteinates or chelates has been offered in feeds with other OTMs. Therefore, assessment of the optimal dietary concentrations of supplemental manganese for use by the poultry industry may be most appropriately accomplished in the dietary environment in which each manganese source will typically be used in practice, organic manganese with other organic minerals and inorganic manganese with other inorganic minerals.
Secondly, Mn interacts with other trace minerals in many physiological processes either antagonistically or synergistically. A classic antagonistic interaction of Mn has been reported with iron (Fe). Both minerals share two common transport protein in intestine, the cellular importer divalent metal transporter 1 (DMT1) and the cellular exporter ferroportin 1 [
13,
14]. Evidences have suggested that higher amounts of Fe inhibit the expression of DMT1 in enterocytes, and lead to a depression of Mn uptake [
15]. Because OTMs may be absorbed as peptides and/or amino acids in intestine, it seems reasonable to hypothesize that organic Fe in feeds, for example, could optimize the absorption and utilization of organic Mn by broilers. Therefore, chick responses to organic Mn could be potentially different, and lower levels could be required whether all the trace mineral sources are provided in organic form rather than inorganic form. We hypothesized that the supplemental level of organic Mn required to optimize growing broiler chick responses would be lower than inorganic supplemental Mn. Therefore, we conducted an experiment to evaluate the effects of supplemental levels of Mn provided by organic and inorganic trace mineral (ITM) supplements on growth, tissue mineralization, mineral balance, and antioxidant status of growing broiler chicks.
DISCUSSION
Establishing broiler requirements for Mn has been revealed to be particularly challenging, since estimates and biological responses may be affected by several factors, which include the source of Mn under study, the experimental basal diets used to produce treatments, the concentration, and source of the other trace minerals in experimental diets, as well as the biological response used as a reference to determine the optimal level. In the current research, a semi-purified diet based on dextrose, casein, and albumin was supplemented with Mn levels and sources to produce dietary treatments. As highlighted in the last revised edition of Nutrient Requirements of Poultry [
10], estimates of trace mineral requirements of chicks fed semi-purified diets are expected to be lower than those obtained from cereal-based diets due to poor or nonexistent presence of antinutritional factors such as phytate and fiber. In this experiment, both sodium phytate and cellulose, as well as a microbial phytase, were added to experimental diets to simulate a commercial cereal-based diet. When investigating growing chick requirements for Mn using a semi-purified dextrose-casein diet, Southern and Baker [
20] estimated optimal Mn level at 14 mg Mn/kg diet. In the current research, with the same type of ingredients, we estimated higher requirements, whose values were similar to literature. Such fact indicates fiber and sodium phytate added to experimental diets fulfilled the purpose of simulating practical cereal-based diets.
Our outcomes demonstrated that regardless of the sources assessed, performance traits were influenced by Mn supplementation to the basal feeds, which proves the essentiality of this mineral for growing chicks. Even though chicks fed 25 mg supplemental Mn/kg diet achieved similar performance targets to chicks fed the highest Mn level under study, there was a linear improvement in performance responses as Mn supplementation increased, mainly in FCR (
Table 3). In order to describe responses to supplemental Mn; and estimate the supplemental Mn levels required to optimize the performance traits, ADG, and FCR data were fitted to the LBL regression model. As summarized in
Table 8, in chicks fed MnSO
4·H
2O supplemented diets, ADG increased linearly and reached the plateau at 59.8 mg Mn/kg diet, whereas FCR decreased linearly and achieved a flat line at 74.3 mg Mn/kg. In turn, MnPro fed chicks had the ADG and FCR optimized at 20.6 and 38 mg Mn/kg diet. Our estimates for the inorganic Mn level suggest that NRC [
10] recommendations may support ADG, but higher concentrations are required for FCR optimization.
As expected, our findings clearly demonstrate that com pared with MnSO
4·H
2O, lower amounts of Mn were required to reach maximal chick performance when Mn was provided as MnPro via the OTM supplement. So far as the mechanisms underlying Mn absorption are understood, Mn uptake occurs mainly in the upper small intestine by the transport protein DMT1 [
21,
22], whose expression in intestinal mucosa is modulated by Mn source and dietary level [
13]. Previous findings have demonstrated that complexed or chelated organic Mn increases mRNA expression of DMT 1 in broiler chick small intestine compared with Mn sulphate [
23], which explains, at least in part the fact that lower levels of organic Mn were able to reproduce similar performance to higher levels of inorganic Mn in the current research.
Previous reports have suggested that Mn supplementation did not affect ADG and FCR of growing chicks, and that cereal-based diets containing 19 to 26 mg Mn/kg from cereal grains in the diet without a supplemental Mn source, could support performance objectives [
1,
3,
4]. Our findings indicate that 25 mg supplemental Mn/kg diet was sufficient to support proper growth rates, which considering Mn content in basal diet, i.e. 6 mg Mn/kg, would be equivalent to 31 mg Mn/kg. Despite similarities, a comparison between our requirement estimates and those described in the refereed references cannot be made with confidence due to differences in the Mn sources. The bioavailability of Mn sources is limited in poultry, especially in cereals [
10]. Wedekind et al [
24] reported that only 9% of the Mn provided by a corn and soybean-based diet supplemented with MnSO
4·H
2O (100 mg Mn/kg diet) was absorbed by broiler chicks. Yet, the authors reported that such rate was 2.8% when no Mn source was added to basal diet. Such findings may be clearly supported by our results. As detailed in
Table 5, the amount of Mn retained in chicks relative to Mn intake, i.e. Mn balance, was 56% lower in birds fed basal diets compared with the balance in chicks fed the lowest supplemental Mn level of 25 mg/kg diet. Even though Mn balance differed among Mn-supplemented groups, the difference was narrow between the lowest and highest Mn balance (35.5% vs 36.5%), which suggests that dietary levels higher than those estimated for performance optimization were utilized by chicks and retained in the body, as our data for tissue mineralization show. As detailed in
Table 8, when fitting Mn balance data from chicks fed both Mn sources to LBL regression model, slightly lower levels were estimated maximum Mn balance (18.6 mg Mn/kg). As expected, a lower supplemental Mn level was estimated to maximize Mn balance in chicks fed MnPro (16.6 Mn/kg) compared with MnSO
4·H
2O fed chicks (20.6 mg Mn/kg).
Manganese levels influenced liver Mn concentrations; whose greatest value was at 50 mg supplemental Mn/kg diet. Despite being statistically different, the means of liver Mn content from chicks fed 50 and 100 mg Mn/kg diet were quite similar numerically (11.5 vs 11.3 mg Mn/kg). Likewise, after adding tribasic manganese chloride or MnSO
4·H
2O to a low Mn basal diet, Conly et al [
25] reported that chick liver Mn content increased up to 60 mg Mn/kg diet and remained constant up to 130 mg Mn/kg diet, regardless of the source assessed. When fitting liver Mn concentration data to LBL model, the breakpoints for maximum concentration were estimated at 39.3 mg Mn/kg diet considering both sources, and 33.5 and 43.1 mg Mn/kg diet for chicks fed MnPro and MnSO
4·H
2O supplemented diets, respectively. Liver is the primary organ responsible for regulating Mn body status through biliary excretion [
26]. When provided in concentrations that exceed physiological needs, Mn may be progressively accumulated in different organs and, beyond critical limits, be excreted to avoid toxicity [
20]. Chicks fed higher levels of Mn than the requirements established herein for organic (38 mg Mn/kg diet) and inorganic (74.3 mg Mn/kg diet) stored Mn in extra-hepatic tissues (e.g. breast and tibia). Breast muscle Mn content responded with increasing Mn deposition up to 75 mg Mn/kg diet, whereas Mn deposition in tibia continuously increased up to 100 mg Mn/kg diet, the highest level under study. Our findings support those reported by Yan and Waldroup [
27] who reported that regardless of the source of Mn assessed (MnO, MnSO
4·H
2O, or amino acid chelated Mn), Mn concentrations in broiler tibia were gradually increased up to 800 mg supplemental Mn/kg diet. Similarly, when investigating the Mn supplementation on broiler diets, Conly et al [
25] noticed that Mn was continuously deposited in the tibia of chicks fed tribasic manganese chloride or MnSO
4·H
2O supplemented diets (0, 30, 60, and 130 mg supplemental Mn/kg), achieving the greatest value at the highest level studied. Although Mn content in breast muscle and tibia were unaffected by Mn sources, the analysis of levels in each source suggests that Mn concentration in breast increased until 75 mg Mn/kg only when Mn was supplemented as MnPro. According to polynomial regression model estimates, Mn concentration in breast reached its maximum values at supplemental Mn level of 67.5 mg/kg, and for chicks fed MnPro, maximum deposition was achieved at 62.3 mg/kg. We noticed that Mn concentrations in tibia and liver approximately doubled in chicks fed the highest supplemental Mn level compared with chicks fed diets without supplemental Mn. Such outcomes differ from those reported by Lu et al [
5] that neither amino acid chelated Mn nor MnSO
4·H
2O affected breast muscle Mn content of growing chicks fed diets supplemented at 100 and 200 mg Mn/kg, and from those reported by Yan and Waldroup [
27] who observed a higher Mn content in the tibia of chicks fed amino acid chelated Mn compared with chicks fed MnO and MnSO
4·H
2O. Our results suggest that Mn concentration in breast muscle are mainly influenced by proteinate Mn source, i.e. MnPro.
Although Mn levels did not affect antioxidant enzyme activity, we observed that, curiously, chicks fed inorganic minerals supplemented diets exhibited higher activity of GSH-Px in breast muscle and liver, and higher total-SOD activity in liver compared with chicks fed organic minerals. Organic minerals are potentially more bioavailable than the inorganic forms, so it was expected that they would support higher total-SOD activity. It is worth highlighting, however, that although trace minerals modulate the activity of antioxidant enzymes like SOD, GSH-Px, and catalase, they may also act as pro-oxidant agents [
28,
29]. Free iron and copper, for example, have been described as the major catalyzers of the production of free radicals such as hydrogen peroxide (H
2O
2) and hydroxyl, which disrupt the redox balance in cells, and cause oxidative damage to tissues [
30,
31]. Because ITMs are affected by the variation of pH, they may reach some tissues such as gut mucosa and blood as reactive ions, which may potentially oxidize cytosolic structures and DNA of cells. Therefore, inorganic minerals may be potentially pro-oxidant compared with OTMs, whose chemical structures are more stable and not so easily dissociated. Even though GSH-Px is an enzyme dependent on selenium and not Mn, its activity is expected to increase in response to the increase in total-SOD activity. Whereas SOD acts in a first level, in the dismutation of superoxide radical to H
2O
2, GSH-Px ends the process by detoxifying H
2O
2.