Organic trace minerals and calcium levels in broilers' diets to 21 days old

This study was undertaken to evaluate the effects of dietary calcium levels and supplementation with organic trace minerals selenium, copper, iron, zinc and manganese on performance, tissue deposition and litter mineral concentration. A total of 2,496 one-day-old male Cobb 500 broilers were randomly assigned to a 3 × 4 factorial experimental design with three levels of dietary Ca [8, 10 and 12 g kg–1, while maintaining the same Ca:nPP (non-phytate phosphorus) ratio (2:1)] and four levels of micromineral supplementation (0.62, 0.72, 0.82 and 0.92 g kg–1). There was a total of 12 treatments, with eight replicates of 26 birds per pen. Micromineral supplementation (MS) was achieved by adding different levels of the product Bioplex TR Se® and Ca supplementation was achieved by adding increasing levels of limestone and dicalcium phosphate. An interaction between Ca and MS levels was observed (p 0.05) for Ca, P or ash concentrations in the tibia, which were influenced only by dietary Ca levels (p < 0.05). The Ca level of 10 g kg–1 promoted higher Ca and P concentration in the tibia and lower micromineral excretion in the litter. The combination of MS level of 0.82 g kg–1 with Ca level of 10 g kg–1 led to the best BWG response. The supplementation conditions that led to higher micromineral levels in the liver and breast varied for each mineral.


Introduction
Inadequate mineral supplementation during the growth phase of birds results in an imbalance in mineral homeostasis and improper development of bones, i.e., abnormal bone calcification. However, excess calcium (Ca) may act as an antagonist, making it difficult to absorb trace minerals such as iron (Fe), copper (Cu), zinc (Zn) and other minerals such as magnesium (Mg), sodium (Na) and potassium (K) (Smith and Kabaija, 1985;Waldroup, 1996). High Ca levels in broiler chicken feed increase the need for phosphorus (P) because Ca interferes with phosphorus absorption. Ca and P form complexes in the intestine, making P less available hindering the absorption of phytin phosphorus by the bird (Wise, 1983).
Trace minerals such as selenium (Se), Cu, Fe, manganese (Mn) and Zn are essential to chicken development because they are active in several metabolic pathways. These minerals are involved in physiological functions that are essential to the maintenance of life, including reproduction, growth, immune system function, bone formation and energy metabolism (Dieck et al., 2003;Bao et al., 2007;Dibner et al., 2007). They are usually supplemented in the form of inorganic salts, such as sulphates, oxides and carbonates, to ensure healthy development and greater productivity. However, these inorganic forms are thought to interact with other minerals, such as Ca. This negative effect can be minimized by supplementing these minerals in organic form.
The aim of the present study was to evaluate the effects of dietary Ca levels (while maintaining the same Ca:P ratio) and different supplementation levels of the organic trace minerals Se, Cu, Fe, Zn and Mn on the performance, tissue deposition and mineral excretion in the litter.

Materials and Methods
The study was approved by the Ethics Committee for the Use of Production Animals (CEUAP) of the Universidade Federal de Viçosa (UFV; Minas Gerais, Brazil), with number 100/2014.
A total of 2,496 one-day-old male Cobb 500 broiler chicks with a mean initial weight of 44 g were housed up to 21 days of age in 96 pens (experimental units) with 3m 2 each, which were lined with wood shavings and located within a masonry shed.
The experimental design was fully randomized, with 12 treatments (Table 1) in a 3 × 4 factorial design that consisted of three levels of dietary Ca [8, 10 and 12 g kg -1 , with a constant Ca:nPP (non-phytate phosphorus) ratio of 2:1] and four levels of micromineral supplementation (0.62, 0.72, 0.82 and 0.92 g kg -1 ). Each treatment consisted of eight replicates of 26 birds.
The corn-soybean meal basal diets (Table 2) were formulated so as to be adequate in all nutrients according to Rostagno et al. (2011) with the exception of the microminerals (Mn, Zn, Fe, Cu and Se) and Ca levels, which were added based on respective treatments. The diets were supplemented with a Escherichia coli -derived Organominerals and calcium for broilers Sci. Agric. v.77, n.1, e20180071, 2020 phytase (5,000 FTU g -1 of phytase, providing 1000 FTU per kg of diet) to simulate a commom comercial diet.
Trace mineral supplementation was achieved by adding different levels of a commercial micromineral supplement (MS), which contains 50 g Mn, 40 g Zn, 30 g Fe and 6 g Cu (as proteinates), 180 mg Se (derived from yeast enrichment) and 2 g I (as inorganic source) per kilogram of the product.
Ca supplementation was performed by adding increasing levels of limestone (377 g of Ca per kg of limestone). The Ca:nPP ratio was maintained in the different treatments by adding different levels of dicalcium phosphate (CaHPO 4; with 245 g of Ca and 185 g of P available per kg of dicalcium phosphate), limestone and an inert material (washed sand).
Each experimental unit was equipped with a nipple and feeder to provide water and feed ad libitum. The feed was given according to the consumption of the animals in each experimental unit. The birds and leftover feed were weighed at the beginning of the experiment Table 1 -Experimental treatments and organic trace minerals  levels 1 .   Treatment  Ca  nPP  MS  Mn  Zn  Fe  Cu  Se   ---------------g kg -1 ------------------------------------------------ppm ---------------------------------T1   8  The minerals were supplied by the micromineral supplementation (MS), which contained 50 g kg -1 manganese (Mn), 40 g kg -1 zinc (Zn), 30 g kg -1 , iron (Fe), 6 g kg -1 copper (Cu), 180 mg kg -1 selenium (Se) and 2 g kg -1 iodine (I). Ca supplementation was achieved by adding increasing levels of limestone (377 g of Ca per kg of limestone) and dicalcium phosphate (245 g of Ca and 185 g of P available per kg of dicalcium phosphate). and at 21 days of age to determine body weight gain (BWG, kg), feed intake (FI, kg) and feed-to-gain ratio (F:G, kg kg -1 ). The number of birds dying throughout the experimental period was quantified to calculate viability. F:G was determined considering the correction of feed intake by birds alive in each unit. For this each feeder unit was weighed after a dead bird was detected. Temperature was measured daily. In the first week, the mean minimum and maximum temperatures were 25 °C and 32 °C, respectively. These temperatures were 21 °C and 31 °C from day 8 to 21. At 21 days of age, one bird per pen (eight birds per treatment) was selected for slaughter based on the mean weight of its experimental unit. The birds were slaughtered using the cervical displacement method followed by exsanguination.
To collect the liver, an incision was made in the abdominal cavity of the bird to expose the viscera.
After plucking and cleaning, a single sample of breast muscle weighing approximately 30 g was collected from each experimental unit.
Litter samples were collected from a previously established site at the centre of each experimental unit (pen) to determine the mineral (Mn, Zn, Fe, Cu and Se) concentration in the litter. All of the material contained within a 90-cm 2 piece of plastic lining was collected.
Samples of the right tibia, liver, and right portion of the breast musculature from each bird and litter of each experimental unit were collected and placed separately in plastic bags labelled with the corresponding treatments and stored in a freezer at -20 °C.
The tibia were washed, cleaned of all residual tissue and dried at 60 °C for 72 h. Subsequently, pre-degreasing was performed for 4 h with petroleum ether in a glass vessel. The tibia were then ground, and a sample of each was removed (eight samples per treatment, totalling 96 samples), dried for 12 h at 105 °C to determine dry mass and then ashed in a muffle furnace (600 °C for 4 h) to determine ash content. Calcium and P were determined after wet-ash digestion with nitric and perchloric acids according to the 935.13 method (AOAC, 2000). Calcium in wet-ashed samples was determined by the atomic absorption spectrophotometric method 968.08 (AOAC, 2000) using an atomic absorption spectrometer (AAnalyst 300). Phosphorus concentration was determined using a colorimetric assay (Fiske and Subbarow, 1925). Acid molybdate and Fiske's SubbaRow reducer solution were added to wet-ash samples to perform a phosphor-molybdenum complex. Color intensity was proportional to P concentration and was determined with a spectrophotometer using absorbance at 620 nm (SpectraCount, Model #AS1000). The analyses were performed at the Animal Nutrition Laboratory in Viçosa (20°45'14" S, 42°52'53" W and 648.74 m of altitude), in the state of Minas Gerais, Brazil.
Samples of diets, liver, breast and litter were sent to a laboratory (54'23" S, 47°3'42" W and 760 m of altitude, Campinas, in the state of São Paulo, Brazil) to determine the concentrations of Mn, Zn, Fe, Cu and Se. For this the frozen samples of liver and breast were packed with ice in a Styrofoam box to maintain their physiochemical characteristics. The litter samples were dried for 72 h in a ventilated oven at 60 °C and were then ground (0.5 mm).
Zinc, Mn, Fe, Cu and Se concentrations in liver, breast, litter and diet samples were determined following the methods described by AOAC (1984). An aliquot of 0.5 g of diets, tissues and litter samples were weighed on the analytical balance and added 5 ml of 4:1 nitroperchloric acid solution (4 parts nitric acid and 1 part perchloric acid). The samples were heated and digested at 200 °C in the digester block and the residue was filtrated through quantitative paper (7.5 μm pore) and completed for 50 mL with distilled water. This solution was analyzed in an atomic absorption spectrophotometer (AAS) to obtain the mineral concentrations (Mn, Fe, Zn and Cu) and for Se concentrations the graphite furnace atomic absorption spectroscopy (GFAAS) was used.
Data were analysed using the GLM procedure (SAS v. 9.3). Pens containing 26 birds were considered as the experimental units. The model included the main effects of Ca level and micromineral supplementation and their interaction, as described below: The isolated effects of mineral supplementation were analysed by orthogonal polynomial contrasts, and the isolated effects of Ca level were compared using Tukey's means test. The effects were considered significant at p < 0.05.

Results
The interaction between MS and Ca level was significant (p < 0.05) for BWG, FI and F:G (Table 3). At the Ca level of 10 g kg -1 , BWG showed a quadratic response to MS, with Y = 0.435 + 1.141X -0.622X 2 (R 2 = 0.86). The greatest BWG was observed at the MS level of 0.917 g kg -1 . MS did not influence BWG at Ca levels of 8 and 12 g kg -1 .
At a Ca level of 8 g kg -1 , FI showed a quadratic response to MS, with Y = 2.617 -3.708X + 2.481X 2 (R 2 = 0.89). The lowest feed intake was observed at the MS level of 0.747 g kg -1 . However, at the Ca level of 10 g kg -1 , FI showed a linear relationship with MS, represented by the equation Y = 1.024 + 0.293X (R 2 = 0.84). At the MS level of 0.92 g kg -1 , FI was lower at dietary Ca levels of 10 and 12 g kg -1 .
At the Ca level of 8 g kg -1 , F:G showed a quadratic response to MS, according to the equation Y = 2.429 -2.959X + 1.981X 2 (R 2 = 0.97). The lowest feed-to-gain ratio was observed at the MS level of 0.747 g kg -1 . In the same way, at the MS level of 0.92 g kg -1 , F:G was lower at dietary Ca levels of 10 and 12 g kg -1 .
Viability did not interact with and was not independently affected by the factors analysed (p > 0.05).
Additionally, MS had no independent or interactive effect on the percentages of Ca, P and ash in the tibia (p > 0.05). However, increasing the dietary Ca level resulted in increased Ca, P and ash concentrations in this bone (p < 0.05) ( Table 4).
The MS and Ca independently influenced the concentrations of Mn and Zn in the liver and MS levels influenced the Se liver concentration (p < 0.05) ( Table  5). The MS level was positively and linearly correlated with Se and Mn concentrations (p < 0.05) in the liver, while a quadratic relationship was observed for the Zn concentration (p < 0.05). These three relationships can be represented by the equations Y = 0.189 + 0.912X (R 2 = 0.99), Y = 5.284 + 6.020X (R 2 = 0.94) and Y = 38.937 + 162.288X -78.00X 2 (R 2 = 0.83), respectively. According to this equation, the highest Zn deposition in the liver occurs with 1.04 g kg -1 of MS. However, as this level is outside the MS range tested here, this equation is not representative of high Zn deposition in the liver. Higher Mn concentrations were observed at Ca levels of 8 and 10 g kg -1 .
The concentrations of Cu and Fe were affected by interaction between the MS and Ca levels (p < 0.05; Table 5). At Ca levels of 8 and 10 g kg -1 , MS produced a linear increase in liver Cu concentration, according to the equations Y = -65.497 + 137.007X (R 2 = 0.96) and Y = -43.278 + 116.803X (R 2 = 0.95), respectively. At the lowest MS level (0.62 g kg -1 ), the highest liver Cu concentration was observed at a Ca level of 12 g kg -1 .
For Fe in liver, at a Ca level of 12 g kg -1 , MS had a quadratic effect on the liver Fe concentration, with Y = 2124.465 -4544.524X + 3106.109X 2 (R 2 = 0.92), with the lowest Fe deposition observed at 0.73 g kg -1 MS. At a 0.72 g kg -1 MS, the highest liver Fe deposition was observed at 8 g kg -1 Ca.       v.77, n.1, e20180071, 2020 The MS levels independently influenced the Mn and Zn concentrations in the breast muscle (p < 0.05) ( Table 6). The Mn concentration in the breast showed a quadratic response to MS, where Y = -0.723 + 3.536X -2.10X 2 (R 2 = 0.84). The highest Mn deposition was observed at 0.84 g kg -1 MS. Moreover, the concentration of Zn in the breast increased linearly with MS, with Y = 26.430 + 6.807X (R 2 = 0.77). The dietary Ca level influenced independently the Fe and Mn concentration in the breast, with the lowest levels observed at 10 g kg -1 Ca.
The Se and Cu concentrations in the breast were affected by the interaction between MS and Ca levels (p < 0.05; Table 6). At 8 g kg -1 Ca, the Se concentration in the breast showed a quadratic response to MS, with Y = -0.831 + 2.666X -1.625X 2 (R 2 = 0.62). The highest concentration was observed at 0.83 g kg -1 MS. At the Ca levels of 10 and 12 g kg -1 , MS produced a linear increase in breast Se concentrations, according to the equations Y = 0.057 + 0.254X (R 2 = 0.88) and Y = 0.032 + 0.315X (R 2 = 0.96), respectively.
At 12 g kg -1 Ca, the breast Cu concentration showed a quadratic response to MS, according to the equation Y = -140.908 + 388.932X -236.347X 2 (R 2 = 0.95). The highest Cu concentration was observed at 0.82 g kg -1 MS.
At the MS level of 0.92 g kg -1 , the highest breast Se concentrations were observed at 10 and 12 g kg -1 Ca. At the MS levels of 0.72, 0.82 and 0.92 g kg -1 , the highest breast Cu concentrations were found at 12 g kg -1 Ca.
The Ca level affected the Fe concentration in the litter (p < 0.05), with the lowest value observed at 10 g kg -1 Ca ( Table 7).
The Se, Mn and Zn concentrations in the litter showed interactive effects with MS and the Ca level (p < 0.05) ( Table 7).
For Se, at 10 g kg -1 Ca, MS showed a quadratic effect, Y = -1.245 + 3.678X -2.219X 2 (R 2 = 0.99), with the highest concentration found at 0.829 g kg -1 MS. At     At 0.62 and 0.92 g kg -1 MS, the lowest litter Se concentration was observed at 10 g kg -1 Ca.
The lowest litter Mn concentrations at 0.62 and 0.72 g kg -1 MS were also observed at 10 g kg -1 Ca. At 0.82 g kg -1 MS, the lowest Mn concentrations were found at 8 and 10 g kg -1 Ca. At 0.92 g kg -1 MS, the lowest litter Mn concentration was found at 8 g kg -1 Ca.
At 0.72 g kg -1 MS alone, the lowest Zn concentration was found at 10 and 12 g kg -1 Ca.

Discussion
At all MS levels in the diets used in this study, the micromineral concentrations per kilogram of feed were lower than those recommended by the NRC, (1994) and Rostagno et al., (2011).
Research into minerals from inorganic sources has demonstrated an antagonistic relationship between the macromineral Ca and certain trace microminerals, such as Mn, Fe and Zn. However, many studies have reported that this antagonistic relationship may be reduced by supplementing these microminerals using organic trace minerals sources at levels much lower than those recommended.
In the present study, the observed responses of BWG, FI and F:G confirm the hypothesis of an interaction between the levels of dietary Ca and trace micromineral supplementation during the starter phase of broiler production.
When 10 g Ca per kilogram of diet was used, a mineral supplementation level of 0.82 g per kg of diet produced the largest BWG. At Ca levels above or below the recommendation, BWG was not affected, indicating the importance of adequately defining Ca levels as well as supplementing microminerals from organic sources.
The observed FI responses were distinct and also dependent on the levels of Ca and MS. At the lowest Ca concentrations (8 and 10 g kg -1 ), the highest MS level (0.92 g kg -1 ) promoted greater FI, which showed a quadratic response and a linear increase with MS at these levels. At the highest Ca concentration (12 g kg -1 ), this effect was not observed, possibly resulting from excess dietary Ca.
These observed responses of BWG and FI are consistent with those observed for F:G. At the lowest Ca level, the highest MS level stimulated FI, thus decreasing F:G. The observed performance effects confirm the influence of Ca on F:G via the birds' FI.
These results are supported by Bao et al. (2007), who observed better performance in the group of broilers whose diet contained moderate levels of organic mineral, close to those used in the present study and lower than those recommended by the NRC, (1994). Corroborating these results, Peric et al. (2007) and Nollet et al. (2008) found that supplementation with organic minerals souces at levels lower than those currently used with inorganics does not affect the performance of broiler chickens. Both studies also verified that Ca levels lower than 10 and 12 g kg -1 and greater than 8 g kg -1 yielded favorable performances in broiler chickens when used in combination with organic mineral sources.  =7.458 + 248.734X (R 2 = 0.94); 6 Mn litter Ca12 = 145.004 + 124.656X (R 2 = 0.97); 7 Zn litter Ca8 = 72.122 + 102.258X (R 2 = 0.91); 8 Zn litter Ca10 = 20.482 + 148.894X (R 2 = 0.96); 9 Zn litter Ca12 = 32.444 + 146.660X (R 2 = 0.94); z and w, s and t, g and h, p and q Means represents by different letters in the same column are different from each other (p < 0.05) consider the effect of Ca levels in MS levels; *Coefficient of variation. assured the best performance had lower excretion than those in treatments with a higher MS level. Bao et al. (2010) utilized microminerals from organic sources and evaluated the interaction between them, finding in one experiment that adding Cu, Fe, Mn and Zn in combination not only affected the concentrations of the trace minerals but also influenced Ca and P excretion. These authors also showed that Mn supplementation alone increased only Mn excretion and not the excretion of the other minerals. Compared to Mn supplementation, combined supplementation with Zn, Mn, Cu and Fe had no additional effect on Mn excretion but showed decreased Ca excretion and increased Cu and Fe excretion.
Using 50 % of the NRC, (1994) recommendation for the microminerals Zn, Mn and Cu in organic form and 10.7 or 9 g kg -1 Ca in broiler diets, El-Husseiny et al. (2012) found improved performance and carcass yield and lower excretion of these minerals compared to birds whose diet contained 100 % of the recommendation in inorganic form. Manangi et al. (2012) compared low Zn, Cu and Mn levels from organic sources to the industry-standard supplementation levels of the same minerals from inorganic sources. These authors found that the concentration of these microminerals was reduced in the litter of birds supplemented with Zn, Cu and Mn from organic sources by 40, 74 and 35 %, respectively. In contrast, there was no difference in Zn, Cu, Ca and P concentrations in the tibiae of animals that received high dietary supplementation levels from inorganic sources.
The present study demonstrates that micromineral supplementation from organic sources decreases antagonistic interactions with macromineral Ca and reduces binding with nutrients and non-nutritive components of the digesta resulting in less negative interaction in absorption and, consequently, better utilization of these minerals by poultry, favouring adequate tissue deposition (breast and liver) and lower excretion.

Conclusion
The Ca level of 10 g kg -1 combined with an MS level of 0.82 g kg -1 results in greater weight gain, higher deposition of the macrominerals Ca and P in bone tissue and of the microminerals Mn, Zn, Fe, Cu and Se in the liver and breast and lower excretion of these microminerals in the litter.