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Scientia Agricola

On-line version ISSN 1678-992X

Sci. agric. (Piracicaba, Braz.) vol.77 no.1 Piracicaba  2020  Epub July 01, 2019

http://dx.doi.org/10.1590/1678-992x-2018-0071 

Animal Science and Pastures

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

Bruno Damaceno Faria1  * 
http://orcid.org/0000-0002-6099-5244

Leandro Moreira Silva1 
http://orcid.org/0000-0002-3599-7002

Valdir Ribeiro Junior1 
http://orcid.org/0000-0001-6317-9966

Adriana Helena do Nascimento Ferreira2 
http://orcid.org/0000-0002-3980-9371

Horacio Santiago Rostagno1 
http://orcid.org/0000-0003-3222-9718

Luiz Fernando Teixeira Albino1 
http://orcid.org/0000-0002-2753-2010

Melissa Izabel Hannas1 
http://orcid.org/0000-0003-3198-0155

1Universidade Federal de Viçosa – Depto. de Zootecnia, Av. Peter Henry Rolfs, s/n − 36570-900 – Viçosa, MG – Brasil

2Alltech do Brasil, Av. Paranavaí, 2468 − 87070-130 – Maringá, PR – Brasil


ABSTRACT:

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 the parameters of performance, liver Cu concentration, breast Se and Cu concentrations and litter Se, Mn and Zn concentrations. No interactions were 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.

Keywords: chicken; micromineral; tibia; breast muscle

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.

The experiment was conducted in Viçosa (20°45'14” S, 42°52'53” W and 648.74 m of altitude), in the state of Minas Gerais, Brazil.

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 3m2 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.

Table 1 Experimental treatments and organic trace minerals levels1

Treatment Ca nPP MS Mn Zn Fe Cu Se
------------------------------ g kg–1 ------------------------------- ---------------------------------------------------------- ppm -----------------------------------------------------------
T1 8 4 0.62 31 24.8 18.6 3.72 0.112
T2 0.72 36 28.8 21.6 4.32 0.130
T3 0.82 41 32.8 24.6 4.92 0.148
T4 0.92 46 36.8 27.6 5.52 0.166
T5 10 5 0.62 31 24.8 18.6 3.72 0.112
T6 0.72 36 28.8 21.6 4.32 0.130
T7 0.82 41 32.8 24.6 4.92 0.148
T8 0.92 46 36.8 27.6 5.52 0.166
T9 12 6 0.62 31 24.8 18.6 3.72 0.112
T10 0.72 36 28.8 21.6 4.32 0.130
T11 0.82 41 32.8 24.6 4.92 0.148
T12 0.92 46 36.8 27.6 5.52 0.166

1The 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).

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 phytase (5,000 FTU g–1 of phytase, providing 1000 FTU per kg of diet) to simulate a commom comercial diet.

Table 2 Ingredients and nutrient composition of the basal diets. 

Ca (g kg–1)
Ingredient 8 10 12
Corn 537.40 537.40 537.40
Soybean meal 46 379.10 379.10 379.10
Soy oil 35.00 35.00 35.00
Washed sand* 15.93 / 15.83 / 15.73 / 15.632 8.72 / 8.62 / 8.52 / 8.423 1.53 / 1.43 / 1.33 / 1.234
Dicalcium phosphate 9.97 15.37 20.78
Limestone 9.25 11.06 12.84
NaCl 4.80 4.80 4.80
DL-methionine 2.85 2.85 2.85
L-lysine HCl 1.57 1.57 1.57
L-threonine 0.41 0.41 0.41
Choline chloride, 60 % 1.00 1.00 1.00
Vitamin supplement1 1.25 1.25 1.25
Mineral supplement* 0.62 / 0.72 / 0.82 / 0.922 0.62 / 0.72 / 0.82 / 0.923 0.62 / 0.72 / 0.82 / 0.924
Anticoccidial salinomycin 12 % 0.55 0.55 0.55
Antioxidant5 0.10 0.10 0.10
Phytase6 0.20 0.20 0.20
Calculated nutrient content
Crude protein 217.13 217.13 217.13
ME (kcal kg–1)7 3,000 3,000 3,000
Calcium 8.0 10.0 12.0
Non-phytate phosphorus 4 5 6
Na 2.09 2.09 2.09
Dig. lysine 12.00 12.00 12.00
Dig. methionine 5.73 5.73 5.73
Dig. methionine + cysteine 8.68 8.68 8.68
Dig. threonine 7.80 7.80 7.80
Dig. tryptophan 2.47 2.47 2.47
Dig. arginine 13.86 13.86 13.86
Dig. valine 9.24 9.24 9.24

1Supplied per kilogram of diet: vitamin A, 8250 IU; vitamin D3, 2090 IU; vitamin E, 31 IU; vitamin B1, 2.20 mg; vitamin B2, 5.50 mg; vitamin B6, 3.08 mg; vitamin B12, 0.013 mg; pantothenic acid, 11.0 g; biotin, 0.077 mg; vitamin K3, 1.65 mg; folic acid, 0.77 mg and nicotinic acid, 33 mg.

*Supplementation levels of Bioplex TR Se microminerals and amount of washed sand (inert) in the respective treatments:

2T1, T2, T3 and T4;

3T5, T6, T7 and T8;

4T9, T10, T11 and T12;

5Butylated hydroxyl toluene 99 %;

6Concentration: 5,000 FTU g–1 phytase;

7Calculated Metabolizable Energy.

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 (CaHPO4; 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 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-cm2 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:

yijk=μ+Cai+MSj+(Ca×MS)ij+εijk i=1,, a;j=1,, b; k=1,,n,

where: yijk = observation k in level i of factor Ca and level j of factor MS; μ = the overall mean; Cai = the effect of level i of the factor Ca level, MSj = the effect of level j of the factor Mineral supplementation; (Ca × MS)ij = the effect of the interaction of level i of factor Ca with level j of factor MS; εijk = residual random error; a = the number of levels of factor Ca; b = the number of levels of factor MS; n = the number of observations for each Ca × MS combination.

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+1.141X0.622X2(R2=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.

Table 3 Influence of dietary Ca and MS on body weight gain (BWG), feed intake (FI), feed-to-gain ratio (F:G), viability (Viab.)1

Treatments Variables
Ca MS BWG FI F:G Viab.
--------------- g kg–1 ---------------- -------------------- kg --------------------- intake/gain %
83, 5 0.62 0.936 1.268 1.354 97.59
0.72 0.938 1.247 1.330 99.04
0.82 0.926z 1.232 1.330 97.48
0.92 0.946 1.310w 1.385w 97.60
10 2, 1 4 0.62 0.907 1.212 1.336 98.56
0.72 0.922 1.215 1.317 99.04
0.82 0.964w 1.282 1.331 97.12
0.92 0.955 1.286zw 1.348zw 98.56
12 0.62 0.934 1.256 1.346 97.67
0.72 0.929 1.235 1.330 99.04
0.82 0.928z 1.226 1.320 98.56
0.92 0.935 1.233z 1.320z 98.08
MS 0.62 0.926 1.245 1.345 97.94
0.72 0.930 1.232 1.326 99.04
0.82 0.940 1.247 1.327 97.72
0.92 0.945 1.277 1.351 98.08
Ca 8 0.937 1.264 1.350 97.93
10 0.937 1.249 1.333 98.32
12 0.931 1.238 1.329 98.33
ANOVA MS 0.129 0.005 0.004 0.337
Ca 0.727 0.051 0.012 0.754
MS × Ca 0.045 < 0.001 0.032 0.932
CV%* 3.352 3.431 2.157 2.807

1Means represent eight experimental units with 26 birds per experimental unit;

2 BWGCa10=0.435+1.141X-0.622X2(R2=0.86) ;

3 FICa8= 2.617 - 3.708 + 2.481 X2(R2= 0.89) ;

4 FICa10= 1.024 + 0.239X (R2= 0.84) ;

5 F:GCa8= 2.429 - 2.959X + 1.981X2(R2= 0.97) ;

z and wmeans 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.

At a Ca level of 8 g kg–1, FI showed a quadratic response to MS, with Y=2.6173.708X+2.481X2(R2=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(R2=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=2.4292.959X+1.981X2(R2=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).

Table 4 Dry basis percentages of calcium (Ca), phosphorus (P) and ash in the tibia of birds under different treatments1

Treatments Variables
Ca MS Ca P Ash
-------------------- g kg–1 --------------------- --------------------------------------- % ----------------------------------------
8 0.62 16.61 9.48 51.61
0.72 16.67 9.55 51.83
0.82 16.51 9.85 52.10
0.92 16.14 9.47 51.77
10 0.62 16.19 10.08 52.54
0.72 16.61 10.13 52.53
0.82 17.17 9.89 52.23
0.92 17.30 10.11 52.34
12 0.62 17.05 9.65 52.31
0.72 17.07 9.76 52.84
0.82 17.08 10.06 52.55
0.92 17.17 9.84 53.29
MS 0.62 16.64 9.74 52.15
0.72 16.78 9.81 52.40
0.82 16.92 9.93 52.29
0.92 16.87 9.81 52.47
Ca 8 16.37b 9.59c 51.83b
10 16.82a 10.05a 52.41a
12 17.09a 9.83b 52.75a
ANOVA MS 0.194 0.326 0.296
Ca 0.002 < 0.001 < 0.001
MS × Ca 0.242 0.153 0.066
CV%* 4.703 3.740 1.143

1Means represent eight experimental units with 26 birds per experimental unit;

a, b, cMeans within the same column that have different letters are significantly different from each other (p< 0.05);

*Coefficient of variation.

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(R2=0.99) , Y=5.284+6.020X(R2=0.94) and Y=38.937+162.288X78.00X2(R2=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.

Table 5 Treatment effects on the dry basis concentrations of the micronutrients selenium (Se), copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn) in broiler livers1

Treatments Minerals
Ca MS Se Cu Fe Mn Zn
------------ g kg–1 ----------- ----------------------------------------------- ppm ---------------------------------------------
82 0.62 0.743 22.176 517.785 9.094 111.480
0.72 0.818 27.972 555.503 10.120 108.015
0.82 0.979 49.017 474.627 10.521 115.102
0.92 0.983 60.830 492.504 10.665 123.090
103 0.62 0.779 30.545 502.226 9.208 108.332
0.72 0.829 41.050 436.407 9.998 110.564
0.82 0.919 47.828 525.005 10.569 115.685
0.92 1.031 67.220 542.522 10.855 118.898
124 0.62 0.717 56.151 506.252 8.310 111.773
0.72 0.928 40.976 446.404 9.075 118.865
0.82 0.920 45.765 502.715 10.207 136.517
0.92 1.054 48.944 567.110 10.412 121.812
MS 0.62 0.746 36.291 508.754 8.871 110.529
0.72 0.858 36.666 479.438 9.731 112.481
0.82 0.939 47.537 500.782 10.432 122.435
0.92 1.023 58.998 534.046 10.644 121.267
Ca 8 0.881 39.999 510.105 10.100a 114.422a
10 0.889 46.661 501.540 10.158a 113.370a
12 0.905 47.959 505.620 9.501b 122.242a
ANOVA MS < 0.001L1 < 0.001 0.170 < 0.001L2 0.011Q
Ca 0.832 0.142 0.921 0.008 0.040
MS × Ca 0.669 0.004 0.034 0.878 0.349
CV%* 17.99 38.09 16.67 9.22 12.82

1Means represent eight experimental units with 26 birds per experimental unit;

L1Linear effect (Y=0.189+0.912X) ;

L2Linear effect (Y=5.284+6.020X) ;

QQuadratic effect (Y=+38.937+162.288X-78.00X2) ;

a,bMeans within the same row that have different letters are significantly different from each other;

2 Cu liverCa8= -65.497 + 137.007X (R2= 0.96) ;

3 Cu liverCa10= -43.278 + 116.803X (R2= 0.95) ;

4 Fe liverCa12= 2124.465 - 4544.524X + 3106.109X2(R2= 0.92) ;

*Coefficient of variation.

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(R2=0.96) and Y=43.278+116.803X(R2=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=2124.4654544.524X+3106.109X2(R2=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.

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+3.536X2.10X2(R2=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(R2=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.

Table 6 Treatment effects on the dry basis concentrations of the micronutrients selenium (Se), copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn) in broiler breasts1

Treatments Minerals (ppm)
Ca MS Se Cu Fe Mn Zn
-------------- g kg–1 ------------- --------------------------------------------- ppm --------------------------------------------
82 0.62 0.219 8.633 25.798 0.693 31.356
0.72 0.233 9.332t 28.158 0.727 31.010
0.82 0.307 6.936f 29.149 0.834 31.145
0.92 0.255z 8.875h 26.678 0.793 31.928
103 0.62 0.204 10.893 25.776 0.668 31.730
0.72 0.258 5.821t 23.795 0.677 29.474
0.82 0.263 7.557f 22.710 0.699 32.993
0.92 0.287w 8.564h 22.113 0.639 32.303
124,5 0.62 0.233 9.011 21.285 0.650 30.186
0.72 0.255 17.703s 25.743 0.729 31.474
0.82 0.280 17.994e 26.587 0.830 31.997
0.92 0.329w 17.233g 26.051 0.803 34.459
MS 0.62 0.219 9.512 24.286 0.670 31.091
0.72 0.249 10.952 25.899 0.711 30.653
0.82 0.283 10.829 26.149 0.788 32.045
0.92 0.291 11.557 24.947 0.745 32.896
Ca 8 0.253 8.444 27.449a 0.762a 31.360
10 0.253 8.209 23.599b 0.671b 31.625
12 0.274 15.485 24.916a 0.753a 32.029
ANOVA MS < 0.001 0.594 0.633 0.001Q 0.027L
Ca 0.087 < 0.001 0.023 0.001 0.618
MS × Ca 0.036 0.008 0.365 0.222 0.216
CV%* 16.59 49.39 21.99 14.11 8.65

1Means represent eight experimental units with 26 birds per experimental unit;

QQuadratic effect (Y=0.723+3.536X2.10X2) ;

LLinear effect (Y=26.430+6.807X) ;

a-bMeans within the same column that have different letters are significantly different from each other (p < 0.05);

2 Se breastCa8= -0.831 + 2.666X - 1.625X2(R2= 0.62) ;

3 Se breastCa10= Y = 0.057 + 0.254X (R2= 0.88) ;

4 Se breastCa12= 0.032 + 0.315X (R2= 0.95) ;

5 Cu breastCa12= -140.908 + 388.932X - 236.347X2(R2= 0.96) ;

z and w, s and t, g and h, e and fmeans 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.

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+2.666X1.625X2(R2=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(R2=0.88) and Y=0.032+0.315X(R2=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+388.932X236.347X2(R2=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 Cu and Fe concentrations in the litter increased linearly with MS (p < 0.05) according to the equations Y=20.851+15.915X(R2=0.08) and Y=964.071+768.073X(R2=0.18) , respectively (Table 7).

Table 7 Treatment effects on the dry mass concentrations of the micronutrients selenium (Se), copper (Cu), iron (Fe), manganese (Mn) and zinc (Zn) excreted in litter1

Treatments Minerals (ppm)
Ca MS Se Cu Fe Mn Zn
----------- g kg–1 ------------ --------------------------------------------- ppm -----------------------------------------------
84,7 0.62 0.234w 28.938 1562.19 196.886w 132.331
0.72 0.249 29.455 1547.61 213.264t 148.234w
0.82 0.260 36.155 1599.65 219.481h 160.574
0.92 0.276t 34.100 1704.48 207.558q 162.304
102,5,8 0.62 0.184z 29.630 1304.23 167.561z 116.114
0.72 0.248 29.940 1413.41 182.594s 121.984z
0.82 0.284 35.715 1512.30 201.659h 144.023
0.92 0.259s 37.034 1538.34 244.118p 158.399
123,6,9 0.62 0.239w 35.530 1521.66 222.910w 126.316
0.72 0.244 33.684 1540.56 231.845t 136.503z
0.82 0.229 31.716 1567.33 251.186g 146.951
0.92 0.310t 35.376 1854.10 258.015p 171.720
MS 0.62 0.219 31.366 1462.69 195.786 124.920
0.72 0.247 31.026 1500.53 209.234 135.573
0.82 0.258 34.529 1559.76 224.109 150.516
0.92 0.282 35.503 1698.97 236.563 164.141
Ca 8 0.255 32.162 1603.48a 209.30 150.86
10 0.243 33.080 1442.07b 198.98 135.13
12 0.255 34.077 1620.91a 240.99 145.37
ANOVA MS <0.001 0.024L1 <0.001L2 <0.001 <0.001
Ca 0.083 0.451 <0.001 <0.001 <0.001
MS × Ca <0.001 0.137 0.273 <0.001 0.018
CV%* 11.88 18.24 10.54 7.35 7.22

1Means represent eight experimental units with 26 birds per experimental unit;

L1Linear effect (Y=20.851+15.915X) ;

L2Linear effect (Y=964.071+768.073X) ;

a-bMeans within the same column that have different letters are significantly different from each other (p < 0.05);

2 Se litterCa10 = -1.245 + 3.368X - 2.219X2(R2 = 0.99) ;

3 Se litterCa12 = 0.102 + 0.19X (R2 = 0.48) ;

4 Mn litterCa8=230.792+1127.829X707.531X2(R2=0.99) ;

5 Mn litterCa10=7.458+248.734X(R2=0.94) ;

6 Mn litterCa12=145.004+124.656X(R2=0.97) ;

7 Zn litterCa8=72.122+102.258X(R2=0.91) ;

8 Zn litterCa10=20.482+148.894X(R2=0.96) ;

9 Zn litterCa12=32.444+146.660X(R2=0.94) ;

z and w, s and t, g and h, p and qMeans 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.

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+3.678X2.219X2(R2=0.99) , with the highest concentration found at 0.829 g kg–1 MS. At 12 g kg–1 Ca, increasing MS produced a linear increase in litter Se concentration, according to the equation Y=0.102+0.19X(R2=0.48) .

With respect to the Mn concentration in the litter, MS had a quadratic effect at 8 g kg–1 Ca, represented by Y=-230.792+1127.829X-707.531X2(R2=0.99) . The highest litter Mn concentration was observed at 0.797 g kg–1 MS. At 10 and 12 g kg–1 Ca, MS had a linear effect on litter Mn concentration, according to the equations Y=7.458+248.734X(R2=0.94) and Y=145.004+124.656X(R2=0.97) , respectively.

At all Ca levels (8, 10 and 12 g kg–1), the litter Zn concentration increased linearly with MS, according to the equations Y=72.122+102.258X(R2=0.91) , Y=20.482+148.894X(R2=0.96) and Y=32.444+146.660X(R2=0.94) , respectively.

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.

When evaluating the effect of increasing dietary Ca levels, Qian et al. (1997) and Alves et al. (2002) observed decreased consumption and weight gain as the level of this mineral in the feed increased. This reduction in consumption with increasing dietary Ca may be related to elevated ionic Ca in the blood, which inhibits the birds' appetite (Lobaugh et al., 1981). Reduced consumption results in lower nutrient availability and utilization, which Shafey et al. (1991) attributed to a decrease in nitrogen digestion. However, this lower nutrient utilization can be attributed to an increased intestinal microflora population, causing increasing irritation in the intestinal mucosa. In contrast, Salter (1973) reported an increase in mucosal thickness, which would impede effective intestinal absorption and diminish dietary transit.

The responses of bone tissue development was dependent on the dietary Ca level, with Ca and ash deposition being positively affected and P deposition negatively affected by higher Ca levels. These results are consistent with the fact that an intermediate Ca level (10 g kg–1) is currently recommended, following requirements suggested by Rostagno et al. (2011) that the Ca levels need to be 0.92 g kg–1 (one-to seven days old) and 0.84 g kg–1 (eight to 21d old), and support the higher Ca level (12 g kg–1) and impaired P deposition in the bird tibia.

High dietary Ca levels may impair the efficiency of phytase, which was used in all experimental diets in this study, and interfere with the absorption of Ca, P, Zn and Mn. Very low Ca levels impair P absorption, consequently reducing the concentrations of these minerals in the tibia (Schoulten et al., 2002).

Shafey et al. (1991) found that a high Ca concentration (greater than 15.3 g kg–1) in the feed elevated the pH of the crop and ileum contents but did not affect the pH of the other segments of the chicken's gastrointestinal tract. This result suggests that the elevation of intestinal pH as a function of increasing dietary Ca levels reduces the soluble fraction of minerals and their availability for absorption. According to the NRC, (1994), the Ca requirement for one- to 21-day-old broilers is 1 % for diets of 3,200 kcal kg–1. However, Karunajeewa (1976) concluded that 6 g kg–1 Ca and 5.7 g kg–1 phosphorus are sufficient for adequate growth and bone formation when the minerals are present in highly available forms.

In the present study, the interaction between Ca and MS levels in tissue concentration suggests that although higher Ca levels may affect micromineral utilization, these effects may have been minimized by the use of organic mineral sources with high bioavailability. Consequently, this apparently increased the liver and breast Se and Mn concentrations without reducing the Fe and Zn concentrations. The organic mineral sources of Se and Mn effectively permitted the deposition of these minerals in the tissues. Notably, several studies using inorganic selenium salts have found no increase in the concentration of this mineral in the breast. This micromineral is of interest to the industry because producers are currently seeking to increase its concentration in human food and to advance the productive characteristics of broilers. Thus, Se supplementation is an important strategy to enrich the final product.

Bao et al. (2007) found that trace mineral concentrations (Cu, Fe, Mn and Zn) in the livers of broilers fed a control diet without MS were higher than those in supplemented birds (three levels of organic minerals and one inorganic). Similar results have been attributed to a dilution effect as a result of rapid growth of adequate diets and slow growth in the control diet (Roth, 2003).

Ao et al. (2009) evaluated the effects of different forms of Zn and Cu on broiler performance and tissue mineral content and observed that liver Cu concentration was reduced by Zn supplementation. The Zn content in the duodenal mucosa increased with the addition of organic Zn in the diet, and the Cu content in the duodenal mucosa also increased with the addition of both organic Zn and Cu.

Schoulten et al. (2002) found that increasing the Ca level decreased Mn absorption, while Sebastian et al. (1996) found no effect on the relative retention of Mn in one- to 21-week-old broiler chickens when dietary Ca was increased from 6 to 12.5 g kg–1.

Li et al. (2005) evaluated Mn bioavailability in diets with a high Ca level (18.5 g kg–1) and a normal Ca level (11.7 g kg–1) and found that organic Mn with moderate or strong chelation strength could partially or completely resist the antagonistic effect of increased dietary Ca during digestion and exhibited higher relative bioavailability than under normal dietary Ca conditions.

The organic sources in higher levels of MS (0.82 and 0.92 g kg–1) were responsible for an increase in the mineral concentration of Zn, Mn and Se in tissues. An MS interaction with Ca influenced Fe, Cu and Se. These levels are lower than the current levels practiced for better growth or a better feed-to-gain ratio. These results show the importance of using the sources and levels effectivily to permit higher tissue concentration and are perharps reflected in health or meat quality. The impact of Ca levels needs to be observed in the production system because low levels impaired better growth performance and higher levels interact more with the concentration of microminerals in tissues.

The observed Cu and Fe micromineral concentrations in the litter reflected only the increase in MS, showing higher concentrations with increasing MS in the diet. The interaction between Ca and MS levels confirmed that Ca can interfere with micromineral absorption and, consequently, excretion. Excretion is reduced when the Ca level recommended by Rostagno et al. (2011), i.e., 10 g kg–1, is used, as shown by the results for excreted Se, Mn and Zn.

Bao et al. (2007) also detected a linear increase in Cu, Mn, Zn and Fe excretion with increasing consumption of these microminerals. Thus, birds that were supplemented with 0.82 g kg–1 MS and 10 g kg–1 Ca and 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.

Acknowledgments

The authors would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG), Institutos Nacionais de Ciência e Tecnologia (INCT) and Universidade Federal de Viçosa for important financial support.

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Received: March 09, 2018; Accepted: August 01, 2018

*Corresponding author <brunodfzoo@gmail.com>

Edited by: Antonio Faciola

Authors' Contributions

Conceptualization: Faria, B.D.; Hannas, M.I.; Rostagno, H.S.; Albino, L.F.T.; Ferreira, A.H.N. Data acquisition: Faria, B.D.; Silva, L.M. Data analysis: Faria, B.D.; Hannas, M.I.; Rostagno, H.S.; Albino, L.F.T. Design of methodology: Faria, B.D.; Hannas, M.I.; Rostagno, H.S.; Albino, L.F.T. Software development: Faria, B.D.; Ribeiro Junior, V. Writing editing: Faria, B.D.; Hannas, M.I.; Ribeiro Junior, V.

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