In-feed organic and inorganic manganese supplementation on broiler performance and physiological responses
Article information
Abstract
Objective
A trial was conducted to investigate the effects of supplemental levels of Mn provided by organic and inorganic trace mineral supplements on growth, tissue mineralization, mineral balance, and antioxidant status of growing broiler chicks.
Methods
A total of 500 male chicks (8-d-old) were used in 10-day feeding trial, with 10 treatments and 10 replicates of 5 chicks per treatment. A 2×5 factorial design was used where supplemental Mn levels (0, 25, 50, 75, and 100 mg Mn/kg diet) were provided as MnSO4·H2O or MnPro. When Mn was supplied as MnPro, supplements of zinc, copper, iron, and selenium were supplied as organic minerals, whereas in MnSO4·H2O supplemented diets, inorganic salts were used as sources of other trace minerals. Performance data were fitted to a linear-broken line regression model to estimate the optimal supplemental Mn levels.
Results
Manganese supplementation improved body weight, average daily gain (ADG) and feed conversion ratio (FCR) compared with chicks fed diets not supplemented with Mn. Manganese in liver, breast muscle, and tibia were greatest at 50, 75, and 100 mg supplemental Mn/kg diet, respectively. Higher activities of glutathione peroxidase and superoxide dismutase (total-SOD) were found in both liver and breast muscle of chicks fed diets supplemented with inorganic minerals. In chicks fed MnSO4·H2O, ADG, FCR, Mn balance, and concentration in liver were optimized at 59.8, 74.3, 20.6, and 43.1 mg supplemental Mn/kg diet, respectively. In MnPro fed chicks, ADG, FCR, Mn balance, and concentration in liver and breast were optimized at 20.6, 38.0, 16.6, 33.5, and 62.3 mg supplemental Mn/kg, respectively.
Conclusion
Lower levels of organic Mn were required by growing chicks for performance optimization compared to inorganic Mn. Based on the FCR, the ideal supplemental levels of organic and inorganic Mn in chick feeds were 38.0 and 74.3 mg Mn/kg diet, respectively.
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.
MATERIALS AND METHODS
Animal care
Animals care and use procedures described in this section were in accordance with Brazilian Legislation on Animal Experimentation and Welfare, and the experimental protocol was approved by the Ethics Committee on the Use of Farm Animals (CEUAP-UFV) of the Universidade Federal de Viçosa, (protocol 111/2014) before the initiation of the trial.
Birds and husbandry
A total of five hundred 1-d-old male Cobb 500 chickens obtained from a local commercial hatchery were used in this study. During the first seven days of age, the birds were fed a pre-starter diet based on corn and soybean meal formulated to meet or exceed nutritional recommendations of Rostagno et al [16], except for Mn, whose dietary concentration was provided at 43 mg Mn/kg of feed as manganese sulphate (MnSO4·H2O), which corresponded to 50% of the dose recommended by NRC [10]. The pre-starter diet contained less Mn than typically used in commercial production to avoid excessive storage reserves of this mineral. Throughout the entire pre-experimental period, chicks had free access to water and mash feed. On d 8 post hatch, chicks were housed in an environmentally controlled room and allotted into 49 cm× 27 cm×33 cm (length×height×width) plastic cages with raised wire floors until the end of the feeding assay. Chicks were allowed ad libitum access to feed and demineralized water throughout the 10-d experimental period via plastic feeders and cup drinkers. Photoperiod was set at 12 h natural light/12 h artificial light. Both temperature and humidity were set according to genetic guideline recommendations.
Experimental diets and treatments
Prior to the experimental period, all chicks were weighed and assigned to treatment groups so that initial body weight (BW, 174.6±1.0 g) was similar among experimental treatments. A 2×5 fractional factorial arrangement design was used in this study, where two Mn sources were added to basal Mn deficient diets at the supplemental Mn levels of 0, 25, 50, 75, and 100 mg Mn/kg diet. The organic source of Mn under study was manganese proteinate (MnPro, 13.9% Mn, Bioplex Mn, Alltech, São Pedro do Ivaí, Brazil), whereas inorganic Mn was supplied as manganese sulphate (MnSO4·H2O, 30.3% Mn). Chicks were randomly assigned to ten treatments with 10 replicates of five birds per treatment. A semi purified basal diet was formulated to meet or exceed Rostagno et al [16] recommendations for starting broilers (8 to 21 days of age), except for trace minerals. The basal diet was based on casein, albumin, isolated soybean protein, corn, starch, and dextrose (Table 1), from which 4 different diets were produced. The 4 diets differed from each other with regard to amount and source of Mn supplemented (MnPro or MnSO4·H2O) and the source of other trace minerals (organic or inorganic) as follows: i) basal diet supplemented with OTM supplement without supplemental Mn; ii) OTM+100 mg Mn/kg feed supplemented as MnPro; iii) basal diet supplemented with ITM supplement without supplemental Mn; and iv) ITM + 100 mg Mn/kg of feed supplemented as MnSO4·H2O. Diets supplemented with OTM containing 0 and 100 mg Mn/kg as MnPro were mixed in appropriate ratios to produce five diets with supplemental organic Mn concentrations of 0, 25, 50, 75, and 100 mg Mn/kg of feed. The same procedure was adopted with diets supplemented with ITM, to obtain the five levels of manganese. The OTMs were supplied as Bioplex Fe (15% Fe), Bioplex Zn (15.0% Zn), Bioplex Cu (11.4% Cu), and selenium yeast as Selplex (0.236% Se), whereas ITMs sources included iron sulphate (21.9% Fe), zinc sulphate (22.9% Zn), copper sulphate (25.1% Cu), and sodium selenite (51.6% Se) in ITM, and iodine was added as calcium iodide (86% I) in both trace mineral supplements. Except for Mn, all trace minerals were supplied to meet or exceed NRC [10] recommendations. Sodium phytate and cellulose were added to the semi-purified diet to simulate practical cereal-based-diets. All test diets were supplemented with a commercial microbial phytase enzyme. Prior to the beginning of the assay, diets were analyzed for Mn concentration (Table 2) as described by AOAC [17]. Overall, the analyzed dietary concentrations of Mn were close to those calculated.
Ingredient and composition of the semi-purified basal diet (as-fed basis)
Analyzed concentration of manganese and trace minerals in experimental diets (as- fed basis)
Performance, sampling, and chemical analysis
On d 17 post-hatch, all chicks and feed leftovers from each experimental unit were weighed to determine the final BW, ADFI, average daily gain (ADG), and feed conversion ratio (FCR). From d 13 to 17 post-hatch, trays covered with plastic were placed underneath pens for total excreta collection. Total excreta were collected daily, weighed, and frozen (−20°C). At the end of the collection period, excreta from each pen were homogenized. On d 18 post-hatch, one bird per cage (10 birds/treatment) was randomly selected and sacrificed by cervical dislocation. Subsequently, the breast, liver, and left and right tibia were collected and stored at −20°C. The bird had the breast muscle, liver, and left and right tibia collected, stored at −20°C, and subsequently, were ether extracted for 4 h in a Soxhlet extractor. Posteriorly, these samples, as well the samples of excreta, were freeze-dried for 72 h at −80°C under 800 mbar of pressure (Liobras, São Carlos, SP, Brazil), ground in a stainless ball mill (Micro spray mill, R-TE 350, TECNAL, Ourinhos, SP, Brazil), and finally analyzed in the atomic absorption spectrophotometer (Spctr AA-800; Varian spectrometer, Harbor City, CA, USA) at Animal Nutrition Laboratory (Universidade Federal de Viçosa, Viçosa, MG, Brazil) to obtain the Mn, Cu, Zn, Fe, Ca, and P concentrations as described by AOAC [17]. Manganese retention was calculated through the difference between the amount of Mn consumed and excreted and expressed as mg/kg of BW. The total superoxide dismutase (total-SOD) and glutathione peroxidase (GSH-Px) activity in breast muscle, and liver tissues were performed according to Walsh et al [18] using the kits of Randox Laboratories Ltd. (County Antrim, UK) Ransod and Ransel, respectively, through an automatic biochemical analyzer (Mindray BS-200E, Shenzhen Mindray Bio-Medical Electronics Co., Shenzhen, China) following the manufacturer guidelines.
Statistical analysis
Data were analyzed as a completely randomized design under an incomplete 2-way (source×levels) factorial assay with inorganic and OTMs supplement without Mn supplementation (zero level) and Mn supplement levels as organic Mn in OTM supplement and inorganic Mn in ITM supplement. In this context, the traditional factorial analysis was updated to an incomplete factorial design [19]. This approach is easily accomplished by using common statements from PROC MIXED of SAS (Version 9.4, SAS Institute Inc., Cary, NC, USA) software. According to the previously mentioned analysis, the significance (p<0.05) of source effect (only two levels) was evaluated through F-test; whereas orthogonal contrasts were applied to perform the analysis between linear and quadratic responses of dependent variables in function of increasing Mn levels. Additionally, when Mn levels in each source were significant, means were compared using Tukey’s multiple comparison test. Quadratic polynomial regression model is:
where Y is the dependent variable, X is the dietary Mn concentration, and β0 is the intercept, β1 and β2 are the linear and quadratic coefficients, respectively. Additionally, performance data were fitted to linear broken-line model (LBL) using NLMIXED procedure of SAS (Version 9.4, SAS Institute Inc., Cary, NC, USA) software to estimate the supplemental Mn level which optimized performance responses assessed. The LBL model was expressed as:
where Y is the dependent variable, X is the dietary Mn concentration, β0 is the value at the plateau, β1 is the slope and β2 is the Mn supplemental at the break point (level to optimize the biological response). Statistical significance was considered as 0.05 for all executed analysis. The term “tendency” was used for situations in which p-value is between 0.05 and 0.10.
RESULTS
For both Mn sources under study, the analyzed values of Mn in experimental diets were close to those expected, as well as the concentrations of Fe, Cu, and Zn (Table 2).
Performance responses
Neither interactive effects between sources and supplemental Mn levels (p>0.05) nor main effects of sources (p>0.05) were noticed on chick performance responses assessed (Table 3). Chicks fed diets supplemented with Mn (25 to 100 mg/kg diet) exhibited higher BW, ADG, and better FCR (p<0.05) compared with chicks fed the basal diet with no supplemental Mn. A tendency for higher (p = 0.078) ADFI was noticed on the group of chicks fed Mn supplemented diets. Manganese supplementation improved (p<0.05) linearly all the performance traits assessed and a tendency of quadratic effect of the levels under study was noticed for BW (p = 0.072) and ADG (p = 0.070). The BW and ADG increased (p<0.05) linearly as dietary Mn supplementation as MnPro or MnSO4·H2O (Table 4) increased. Chick FCR decreased (p<0.05) linearly as dietary MnSO4·H2O supplementation increased and decreased (p<0.05) linearly and were quadratically (p< 0.05) affected by increasing dietary MnPro supplementation (Table 4).
Growth performance of broiler chicks fed different supplemental manganese levels of provided by organic and inorganic sources
Linear and quadratic effects of manganese levels supplemented from different sources
Manganese balance
Manganese supplemental levels and sources influenced the intake, excretion, retention, and balance of Mn in broiler chicks (Table 5). No interaction effects (p>0.05) between sources and levels of Mn were noticed on Mn balance responses. Chicks fed MnPro supplemented diets exhibited a lower intake of Mn (p<0.05) compared with MnSO4·H2O fed chicks. Chick intake, excretion, and retention of Mn increased (p<0.05) gradually in each supplemental Mn level, achieving the highest values at 100 mg Mn/kg diet. Manganese intake, excretion, and retention were linearly increased (p<0.05) as supplementation Mn levels increased. Chicks fed diets without supplemental Mn exhibited the lowest Mn balance (p<0.05) compared with the other Mn supplemental levels. Manganese balance was linearly and quadratically influenced (p<0.05) by Mn supplementation. Chicks fed dietary MnPro or MnSO4·H2O supplementation levels exhibit a linear increase (p<0.05) in Mn intake, Mn excretion, and Mn retention (p<0.05) (Table 4). Chicks fed basal diets without supplemental Mn showed a Mn balance strikingly lower (p< 0.05) compared with MnPro or MnSO4·H2O fed chicks. Both linear and quadratic effects of MnPro and MnSO4·H2O supplementation were noticed on chick Mn balance (p<0.05).
Manganese balance in broiler chicks fed different supplemental manganese levels of provided by organic and inorganic sources
Manganese tissue concentrations
No interactive effects between Mn source and Mn supplementation levels (p>0.05) were noticed on Mn tissue concentration (Table 6). The highest concentrations of Mn (p<0.05) in liver, breast muscle, and tibia were noticed at 50, 75, and 100 mg Mn/kg diet, respectively. Chicks fed basal, i.e. without supplemental Mn, exhibited the lowest Mn concentrations (p< 0.05) in the tissues. Manganese supplementation elicited a linear increase (p<0.05) in Mn tissue concentrations in breast muscle, liver, and tibia. A quadratic response to dietary supplemental Mn levels (p<0.05) was noticed for Mn concentration in liver and tibia of chicks, whereas a tendency of quadratic effect (p = 0.074) of levels was noticed for Mn content in breast muscle. Manganese concentration in breast, liver, and tibia were increased (p<0.05) linearly and quadratically in response to increased dietary MnPro supplementation (Table 4). Dietary MnSO4·H2O supplementation elicited a linear increase (p<0.05) in Mn concentration in liver and tibia and quadratic increase (p<0.05) in liver (Table 4).
Manganese concentration (dry matter) on tissues of broiler chicks fed different supplemental manganese levels of provided by organic and inorganic sources
Antioxidant enzyme activity
As detailed in Table 7, no interactive effects between source and supplemental Mn levels were noticed (p>0.05) on antioxidant enzyme activity in chick liver and breast muscle (Table 7). Chicks fed MnSO4·H2O supplemented diets exhibited a higher activity of GSH-Px in the liver and breast muscle (p< 0.05) compared with MnPro fed chicks. Breast muscle total-SOD activity tended to be higher (p = 0.081) in MnSO4·H2O fed chicks. The activity of GSH-Px in breast muscle tended (p = 0.074) to increase linearly as dietary MnPro supplementation increased (Table 4). GSH-Px activity in liver tended to be quadratically affected (p = 0.089) by MnSO4·H2O supplementation.
Antioxidant enzymes activity on breast muscle and liver of broiler chicks fed different supplemental manganese levels of provided by organic and inorganic sources
Optimal supplemental Mn levels for organic and inorganic sources
Data of performance, Mn balance, and Mn tissue concentration were fitted to different regression models as summarized in Table 8. The supplemental Mn levels which optimized performance traits assessed according to LBL regression model are detailed in Table 8. When fitting data collected from both sources to the LBL model, the optimum levels of Mn for BW and ADG was 27.6 mg Mn/kg diet, whereas FCR was optimized at 61.3 mg Mn/kg diet. As MnPro, Mn supplemental levels for optimum BW, ADG, and FCR were estimated as 21.6, 20.6, 38.0 mg Mn/kg diet, respectively. Chicks fed MnSO4·H2O supplemented diets had the BW, ADG, and FCR optimized at supplemental Mn level of 59.9, 59.8, and 74.3 mg Mn/kg diet, respectively. Manganese balance achieved its maximum value at the supplemental level of 18.6 mg Mn/kg diet. When different Mn sources were considered in regression analysis, chicks fed MnSO4·H2O and MnPro supplemented diets had Mn balance maximized at 20.6 and 16.6 mg/kg diet, respectively. The greatest concentration on Mn in breast muscle and liver were observed at the supplemental level of 67.5 and 39.3 mg Mn/kg diet, respectively. Chicks fed MnPro supplemented diets exhibited maximum Mn concentration at breast and liver at the supplemental Mn level of 62.3 and 33.5 mg/kg, respectively. For chicks fed MnSO4·H2O diets maximum Mn concentration in liver were achieved at the supplemental level of 43.1 mg Mn/kg.
Optimum supplemental manganese (Mn) levels for broiler chicks considering organic (MnPro) and inorganic (MnSO4·H2O) sources individually or combined
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 MnSO4·H2O 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 MnSO4·H2O, 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 MnSO4·H2O (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 MnSO4·H2O 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 MnSO4·H2O 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 MnSO4·H2O 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, MnSO4·H2O, 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 MnSO4·H2O 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 MnSO4·H2O 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 MnSO4·H2O. 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 (H2O2) 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 H2O2, GSH-Px ends the process by detoxifying H2O2.
CONCLUSION
To the best of our knowledge, this is the first research where the ideal level of organic Mn for broiler chicks was estimated using a supplement in which the other trace minerals (zinc, copper, iron, and selenium) were provided as organically complexed metals. As hypothesized, when compared with inorganic Mn, lower levels of organic Mn are required by growing chicks for performance optimization. Overall, estimates of supplemental Mn levels required to optimize Mn balance and Mn tissue concentration in chicks were lower than those levels to optimize feed conversion ratio in chicks. Based on the feed conversion response, the ideal supplemental levels of organic and inorganic Mn for broiler chicks were 38.0 and 74.3 mg Mn/kg diet, respectively.
ACKNOWLEDGMENTS
The authors gratefully acknowledge to Alltech, Inc. (Nicholasville, KY, USA), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), Instituto Nacional de Ciência e Tecnologia de Ciência Animal (INCT-CA) for financial support.
Notes
CONFLICT OF INTEREST
James Eugene Pettigrew contracts with Alltech, the supplier of the MnPro used in this experiment, to supervise this research. We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.