Effects of Different Trace Elements and Levels on Nutrients and Energy Utilization, Antioxidant Capacity, and Mineral Deposition of Broiler Chickens

Abstract

This study investigated the effects of inorganic trace elements (IEs) and sucrose chelated trace elements (SEs) on the growth performance, nutrients and energy utilization, antioxidant capacity, and mineral deposition in broiler chickens, and the efficiency of IEs replaced by SEs at different levels was also evaluated. A total of 448,21-day-old male Arbor Acres broiler chickens with similar body weights were randomly assigned into 6 dietary treatments (8 cages/treatments) in a complete randomized design. Treatments were a basal diet including 2.0 g/kg of IE (IE-2.0) premix, and SE-2.0, SE-1.5, SE-1.0, SE-0.5, and SE-0 were basal diets in which IEs were replaced by SE premix at 2.0, 1.5, 1.0, 0.5, and 0 g/kg, respectively. In general, there was a linear and quadratic decrease in growth performance including average daily feed intake (ADFI) and average daily gain (ADG), apparent and true availability of nutrients (DM, OM, and CP), GE, trace elements (Cu, Zn, Mn, Fe, I, and Se), essential AA (Lys, Met, Arg, His, Phe, Thr, and Val), non-essential AA (Asp, Ser, Glu, Gly, and Cys), superoxide dismutase (SOD), and trace elements (Fe, Zn, Cu, and Mn) in the liver, and an increase in feed-to-gain ratio (F/G) and liver malondialdehyde (MDA), with decreasing SE levels (p < 0.05). In conclusion, under the conditions of this experiment, using half of the sucrose chelated trace elements (Cu, Fe, Zn, and Mn) instead of inorganic trace elements did not affect the growth performance, nutrients and energy utilization, antioxidant capacity, and liver trace element deposition in broiler chickens.

1. Introduction

Trace elements are essential nutrients to improve the growth performance, reproduction, and immunity of fast-growing broiler chickens [1]. It has been well known that trace elements are cofactors of enzymes that participate in hormone secretion and the immune defense system [2]. Additionally, trace elements are involved in bone development, feathering, and regulating the appetites of broiler chickens [3]. It has been reported that the deficiencies of trace elements such as iron [4], zinc [5], copper [6], and manganese [7] increased the risks of anemia, parakeratosis, critical dysfunctions, bone abnormalities, or growth retardation in poultry. According to the recommendations of the National Research Council [8], excessive or high levels of inorganic trace elements are supplemented into poultry diets to ensure birds reach their genetic growth potential and prevent diseases. However, excessive or high levels of inorganic elements to maximize birds’ performance may result in excessive mineral excretion due to low availability, which can be harmful to the environment and cause wastes of nutrients [9].

The supplementations of organic trace elements in poultry diets have been proven to minimize the adverse effects of inorganic elements due to their better bioavailability [10,11]. Previous studies reported that organic trace minerals could improve growth performance, enhance immunity, reduce trace element excretions, and possess antioxidant and antimicrobial abilities [12,13,14,15]. Sucrose chelated multi-elements (SEs) are new organic trace elements prepared from sugar cane molasses and chelated with copper sulfate, zinc, manganese, and iron. We proposed that the matrix of carbohydrates of SEs could isolate the trace elements from the environment, minimizing mineral excretion without compromising the growth performance and nutrient digestibility. In this study, the effectiveness of sucrose chelated trace elements on growth performance, nutrient availabilities, contents of minerals in livers, and antioxidative enzyme activities were evaluated in broiler chickens.

2. Materials and Methods

2.1. Trace Elements Premix

The inorganic trace element (IE) premix includes 4 g/kg of Cu (CuSO₄·5H₂O), 35 g/kg of Zn (ZnSO₄·H₂O), 45 g/kg of Mn (MnSO₄·H₂O), 25 g/kg of Fe (FeSO₄·H₂O), 250 mg/kg of I (KIO₃), and 150 mg/kg of Se (Na₂SeO₃).

The sucrose chelated trace element (SE) premix includes 4 g/kg of Cu (sucrose chelated Cu, C₁₂H₂₂O₁₅SCu), 35 g/kg of Zn (sucrose chelated Zn, C₁₂H₂₂O₁₅SZn), 45 g/kg of Mn (sucrose chelated Mn, C₁₂H₂₂O₁₅SMn), 25 g/kg of Fe (sucrose chelated Fe, C₁₂H₂₂O₁₅SFe), 250 mg/kg of I (KIO₃), and 150 mg/kg of Se (Na₂SeO₃).

SE and IE premixes were both provided by Nanning Zeweier Feed Co., Ltd. (Nanning, China) and all materials used were of feed grade. The zeolite powder provided by Taihang mineral feed premix Co., LTD (Taian, China) was selected for the diluent.

2.2. Experimental Design

A total of 448,21-day-old male Arbor Acres broiler chicks with similar body weight (BW) (953.21 ± 26.97 g) were purchased from a commercial chicken farm (Xiling Family Farm, Tai’an, China), individually weighed, and then randomly distributed into 6 dietary treatments with 8 replicates (8 birds per replicate/cage) in a completely randomized design: basal diet including 2.0 g/kg of IEs (IE-2.0), and the SE-2.0, SE-1.5, SE-1.0, SE-0.5, and SE-0 treatments were IE-2.0 in which IEs were replaced with 2.0, 1.5, 1.0, 0.5, and 0 g/kg of SEs, respectively. Zeolite powder was used to supplement the insufficient quantity in the formula. The rest of the 64 (8 replicates, 8 birds per replicate/cage) birds were randomly assigned for endogenous measurement.

The basal diet and nutrient levels were formulated according to the standards of Arbor Acres growing broilers (Feeding Standard of Chicken of the People’s Republic of China, NY/T 33-2004 [16],

Table 1. Compositions and nutrient levels of basal diet (DM basis) %.

Items	Ingredients	Nutrient Levels 2	Value
Corn	64.45	ME, MJ/kg	12.77
Soybean meal	25.10	CP	19.34
Soybean oil	2.50	Ca	0.79
Corn gluten powder	3.75	Available phosphorus	0.32
CaHPO4	1.20	Lys	1.00
Limestone	1.20	Met	0.40
NaCl	0.30	Met+Cys	0.71
L-Lys	0.20
Met	0.10
Inorganic trace elements premix	0.20
Premix 1	1.00
Total	100.00		12.77

¹ The premix provided the following per kg of the diet: VA 11 500 IU; VD₃ 3 500 IU; VE 30 mg; VK₃ 3 mg; VB₁ 3.38 mg; VB₂ 9.00 mg; VB₆ 8.96 mg; VB₁₂ 0.025 mg; choline chloride 800 mg; calcium pantothenate 13 mg; niacin 45 mg; biotin 0.08 mg; folic acid 1.20 mg. ² Metabolizable energy was calculated according to the total excreta collections method, crude protein and calcium were analyzed values, and the other nutrient levels were calculated values.

). To ensure the consistency of experimental diets, all diets were formulated and manufactured 1 week prior to the trial and stored in sealed containers at approximately 9 °C. The nutrient components of all feeds were analyzed according to the methods described by the AOAC [17] and the trace elements were measured by flame atomic absorption spectrometry (GB 2009.90-2016).

2.3. Animals and Management

Birds from each replicate (8 birds) were housed in individual wire cages equipped with water troughs and feeders in an environmentally controlled house. The temperature was maintained at 20–22 °C and the relative humidity was kept at about 65% during the experimental period. All birds were vaccinated according to the normal immunization program and had ad libitum access to feed (except the endogenous treatment) and water under a 23-hs-on-1-h-off lighting regime. The experiment spanned 28 days after the 7 days of adaptation (21 d to 27 d of age). All birds were inspected at least twice per day and any mortalities or culls were removed or sacrificed in accordance with the guidelines for the care and use of laboratory animals prescribed by the Animal Nutrition Research Institute of Shandong Agricultural University and the Ministry of Agriculture of China. The BW was measured at the end of the adaptation period (28 d) and test period (55 d), feed intake was recorded daily from each cage, and the average daily gain (ADG, g), average daily feed intake (ADFI, g) and feed-to-gain ratio (F/G) were calculated.

2.4. Sample Collections

The nutrient availability experiment was determined based on the total excreta collecting methods by collecting trays. All excreta from 8 birds per cage were collected continuously on d 30–33. Before the collections for endogenous measurement, all selected birds were fasted for 24 h with only access to water, and feces were then collected for the next 48 h. Feathers and shredded dry skin in excreta were removed carefully, and then excreta were weighed, pooled by replicate, and stored at −20 °C until analysis.

2.5. Nutrient Availability

The excreta samples were dried at 65 °C for 72 h, and the dried samples were finely ground using a mortar and pestle, passed through a 1 mm screen, and then stored in sealed containers for the subsequent analysis of dry matter (DM), organic matter (OM), crude protein (CP), gross energy (GE), and trace element according to AOAC (2012). The CP was analyzed by the Kjeldahl method and calculated based on nitrogen content (CP = nitrogen × 6.25). The DM was analyzed by drying at 103 ± 2 °C for 72 h, the OM was determined by 550 °C ash in a muffle furnace (SX2-4-10; Longkou electric furnace manufacturer, Yantai, China), and the GE was determined using the Parr adiabatic bomb calorimeter (Model 6200, Parr Instruments Co., Moline, IL, USA) [18]. After freeze-drying, samples for amino acid (AA, except tryptophan) analysis were measured using an automatic amino acid analyzer (Hitach-835; Hitachi Limited, Japan) by high-performance liquid chromatography (HPLC) according to the method described by [19]. Trace elements were analyzed by flame atomic absorption spectrophotometry (SpectrAA 220, Mulgrave, Victoria, Australia) according to [20]. All analyses were performed in triplicate. The measured dietary trace elements are shown in

Table 2. Measured values of trace elements in diets (DM basis) mg/kg.

Treatments 1	Cu	Zn	Mn	Fe	I	Se
IE-2.0	13.84 ± 0.13	135.2 ± 0.14	127.5 ± 0.13	248.7 ± 0.19	1.35 ± 0.01	0.48 ± 0.01
SE-2.0	13.72 ± 0.15	138.2 ± 0.13	134.3 ± 0.14	259.4 ± 0.21	1.46 ± 0.01	0.51 ± 0.02
SE-1.5	12.61 ± 0.12	125.3 ± 0.12	108.6 ± 0.11	238.9 ± 0.22	1.40 ± 0.01	0.36 ± 0.01
SE-1.0	10.73 ± 0.11	109.3 ± 0.11	88.6 ± 0.09	224.3 ± 0.21	1.39 ± 0.01	0.25 ± 0.01
SE-0.5	8.37 ± 0.09	89.5 ± 0.08	64.8 ± 0.07	212.6 ± 0.20	1.25 ± 0.01	0.22 ± 0.01
SE-0	5.92 ± 0.05	70.7 ± 0.06	39.1 ± 0.04	198.1 ± 0.20	0.95 ± 0.01	0.13 ± 0.01

¹ IE-2.0: basal diet (2.0 g/kg of inorganic trace element premix); SE-2.0, SE-1.5, SE-1.0, SE-0.5, and SE-0 in which IEs were replaced with 2.0, 1.5, 1.0, 0.5, and 0 g/kg of SEs, respectively, and 0, 0.5, 1.0, 1.5, and 2.0 g/kg of zeolite powder was added to supplement the insufficient quantity in the formula.

. And the measured values of trace elements in diets satisfy the feeding standard of Chicken of People’s Republic of China, NY/T 33-2004 (Supplementary Table S1).

The apparent and true availabilities of DM, OM, CP, GE, AA, and trace elements were calculated using the following equations:

Apparent availability = [(TNI − TNE)/TNI] × 100, and

True availability = [(TNI − TNE + TNEE)/TNI] × 100,

where TNI was the total nutrient intake (g) of DM, OM, CP, GE, AA, and trace elements daily, TNE was the total nutrients in excreta of DM, OM, CP, GE, AA, and trace elements daily, and TNEE was the total nutrients in endogenous excreta of DM, OM, CP, GE, AA, and trace elements daily.

2.6. Liver Trace Minerals and Antioxidant Enzymes

At the end of the experiment (d 55), 8 birds (1 per replicate) were randomly selected from each treatment and killed by cervical dislocation after fasting for 24 h. Livers from each bird were immediately collected and kept at −20 °C for analyzing trace minerals and antioxidant enzymes. About 100 g of liver samples were used for analyzing trace mineral contents including iron (Fe), zinc (Zn), copper (Cu), and manganese (Mn) by inductively coupled plasma–atomic emission spectrometry (ICP-AES, Optima 8300, Perkin Elmer, Waltham, MA, USA), which has been well described by [21].

Approximately 10 g of liver samples were used for evaluating the antioxidant enzyme activity of superoxide dismutase (T-SOD) and the content of malondialdehyde (MDA) described by [22]. Briefly, the activity of SOD was measured by a SOD Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China), which was determined by the amount of SOD required to obtain 50% inhibition of the rate of nitrite production measured by a spectrophotometer (UV-2000, Unico Instruments Co. Ltd., Shanghai, China) at optical density (OD₅₅₀) [18]. Additionally, the concentration of MDA was determined by an MDA Assay Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). The principle of MDA measurement was the reaction between Thiobarbituric acid (TBA) and MDA to generate a stable pink color that is evaluated by a spectrophotometer (UV-2000, Unico Instruments Co., Ltd., Shanghai, China) at OD₅₃₂ [23].

2.7. Statistical Analysis

The data were analyzed as a completely randomized design and cages were considered as the experimental units. All data were evaluated by one-way ANOVA using PROC GLM followed by Tukey’s multiple comparison test (SAS 9.4) with the model Yi_j = µ + T_i + e_ij, where µ is the total means, Ti is the fixed treatment effects (i = 1, 2, 3, 4, 5, 6), and eij is the residual of the model. Orthogonal polynomial contrasts were used to analyze the linear and quadratic effects of SE levels on growth performance and nutrient availability. A p-value less than or equal to 0.05 was used to declare significance.

3. Results

3.1. Growth Performance

In general, there was a linear and quadratic decrease in ADFI and ADG and increase in F/G with the decrease in SE levels (p < 0.05) (

Table 3. Effects of different trace element levels on the growth performance of broiler chickens ¹.

Treatments 2		ADFI, g	ADG, g	F/G
IE-2.0		150.76 a	77.31 a	1.95 b
SE-2.0		151.68 a	79.01 a	1.92 b
SE-1.5		151.00 a	77.44 a	1.95 b
SE-1.0		150.77 a	76.15 a	1.98 b
SE-0.5		145.37 ab	71.97 ab	2.02 ab
SE-0		144.34 b	66.82 c	2.16 a
SEM 3		1.328	0.582	0.02
p-value
Treatment		0.037	0.041	0.071
SE	Linear	<0.001	0.022	0.016
	Quadratic	<0.001	0.031	0.023

ADG = Average daily gain; ADFI = Average daily feed intake; F/G = ADFI, g/ADG, g. ¹ Data are means for 8 replicates of 4 broilers per replicate. ² IE-2.0: basal diet (2.0 g/kg of inorganic trace element premix); SE-2.0, SE-1.5, SE-1.0, SE-0.5, and SE-0 in which IEs were replaced with 2.0, 1.5, 1.0, 0.5, and 0 g/kg of SEs, respectively, and 0, 0.5, 1.0, 1.5, and 2.0 g/kg of zeolite powder were added to supplement the insufficient quantity in the formula. ³ Total standard error of the means. ^a,b,c Means within a column with different letters are significantly different (p < 0.05).

). Only SE-0 decreased ADFI and ADG, and increased F/G compared to IE-2.0 (p < 0.05). Interestingly, birds fed a lower SE level (SE0.5 and SE-1.0) had similar effects on ADFI, ADG, and F/G compared to birds fed higher IE (IE-2.0) levels (p > 0.05).

3.2. Apparent and True Availability of Nutrients and Gross Energy

Overall, with the decrease in SE levels, the apparent and true availability of DM, OM, CP, and GE decreased linearly and quadratically (p < 0.05) (

Table 4. Effects of different trace element levels on the availability of nutrients and gross energy of broiler chickens ¹ %.

Treatments 2		Apparent Availability				True Availability
		DM	OM	CP	GE	DM	OM	CP	GE
IE-2.0		80.76 a	81.13 ab	65.27 a	81.1 a	84.16 a	84.65 ab	67.73 a	84.47 a
SE-2.0		81.68 a	82.43 a	66.25 a	82.23 a	85.28 a	86.11 a	68.83 a	85.74 a
SE-1.5		81.00 a	81.23 ab	65.85 a	81.12 a	84.69 a	84.91 ab	68.34 a	84.48 a
SE-1.0		80.77 a	80.18 b	64.86 a	80.58 a	84.33 a	83.68 b	67.31 a	83.93 a
SE-0.5		79.37 ab	80.07 b	62.22 ab	79.68 ab	82.82 ab	83.65 b	64.65 ab	83.09 ab
SE-0		77.34 b	77.90 c	59.86 b	78.48 b	80.85 b	81.43 c	62.18 b	81.79 b
SEM 3		0.328	0.201	0.544	0.326	0.338	0.209	0.563	0.339
p-value
Treatment		0.011	<0.001	0.017	0.048	0.012	<0.001	0.017	0.047
SE	Linear	<0.001	<0.001	0.006	0.006	<0.001	<0.001	0.006	0.006
	Quadratic	<0.001	<0.001	0.002	0.003	<0.001	<0.001	0.002	0.003

DM = dry matter; OM = organic matter; CP = crude protein; GE = gross energy. ¹ Data are means for 8 replicates of 4 broilers per replicate. ² IE-2.0: basal diet (2.0 g/kg of inorganic trace element premix); SE-2.0, SE-1.5, SE-1.0, SE-0.5, and SE-0 in which IEs were replaced with 2.0, 1.5, 1.0, 0.5, and 0 g/kg of SEs, respectively, and 0, 0.5, 1.0, 1.5, and 2.0 g/kg of zeolite powder were added to supplement the insufficient quantity in the formula. ³ Total standard error of the means. ^a,b,c Means within a column with different letters mean significantly different (p < 0.05).

). The SE-0 decreased the apparent and true availability of DM, OM, CP, and GE compared to IE-2.0 (p < 0.05), and SE-0.5 and SE-1.0 only decreased the apparent and true availability of OM compared to SE-2.0 (p < 0.05). Furthermore, birds fed a lower SE level (SE-1.0) had similar effects on the apparent and true availability of DM, OM, CP, and GE compared to birds fed higher IE (IE-2.0) levels (p > 0.05).

3.3. Apparent and True Availability of Trace Minerals

Generally, the apparent and true availabilities of Cu, Zn, Mn, Fe, I, and Se decreased linearly and quadratically with decreasing SE levels (p < 0.05) (

Table 5. Effects of different trace element levels on the availability of trace elements of broiler chickens ¹ %.

Treatments 2		Apparent Availability						True Availability
		Cu	Zn	Mn	Fe	I	Se	Cu	Zn	Mn	Fe	I	Se
IE-2.0		46.10 b	57.14 a	55.24 b	50.0 a	78.75 b	87.55 b	47.18 b	58.51 a	57.01 b	51.18 a	80.73 b	90.08 b
SE-2.0		49.52 a	58.68 a	60.14 a	50.34 a	78.32 b	89.35 a	50.76 a	60.18 a	62.15 a	51.61 a	80.39 b	92.06 a
SE-1.5		42.98 c	50.96 b	48.36 c	38.83 b	77.04 b	86.22 b	44.17 c	52.36 b	50.08 c	39.87 b	79.27 b	89.02 b
SE-1.0		34.89 d	43.51 c	39.71 d	34.58 c	78.41 b	83.57 c	35.80 d	44.60 c	41.02 d	35.43 c	80.47 b	86.07 c
SE-0.5		25.12 e	34.37 d	24.06 e	33.32 c	78.23 b	82.08 c	25.75 e	35.27 d	24.87 e	34.19 c	80.31 b	84.58 c
SE-0		7.38 f	25.59 e	−1.42 f	31.44 c	80.64 a	74.77 d	7.59 f	26.38 e	−1.47 f	32.33 c	82.98 a	77.09 d
SEM 3		0.364	0.554	0.443	0.531	0.323	0.229	0.381	0.569	0.457	0.544	0.334	0.234
p-value
Treatment		<0.001	<0.001	<0.001	<0.001	0.049	<0.001	<0.001	<0.001	<0.001	<0.001	0.042	<0.001
SE	Linear	<0.001	<0.001	<0.001	<0.001	0.029	<0.001	<0.001	<0.001	<0.001	<0.001	0.024	<0.001
	Quadratic	<0.001	<0.001	<0.001	<0.001	0.013	<0.001	<0.001	<0.001	<0.001	<0.001	0.009	<0.001

¹ Data are means for 8 replicates of 4 broilers per replicate. ² IE-2.0: basal diet (2.0 g/kg of inorganic trace element premix); SE-2.0, SE-1.5, SE-1.0, SE-0.5, and SE-0 in which IEs were replaced with 2.0, 1.5, 1.0, 0.5, and 0 g/kg of SEs, respectively, and 0, 0.5, 1.0, 1.5, and 2.0 g/kg of zeolite powder were added to supplement the insufficient quantity in the formula. ³ Total standard error of the means. ^a,b,c,d,e,f Means within a column with different letters are significantly different (p < 0.05).

). Birds fed SE-1.5, SE1.0, SE-0.5, and SE-0 decreased the apparent and/or true availability of Cu, Zn, Mn, and Fe compared to IE-2.0 (p < 0.05); however, SE-2.0 increased the apparent and true availability of Cu, Mn, and Se (p < 0.05). Interestingly, birds fed a lower SE level (SE-0.5) had similar effects on the apparent and true availability of I compared to birds fed higher IE (IE-2.0) levels (p > 0.05).

3.4. Apparent and True Availability of Amino Acids

For essential AA, there was a linear and quadratic decrease in the apparent and true availability of Lys, Met, Arg, His, Phe, Thr, and Val with the decrease in SEs (p < 0.05) (

Table 6. Effects of different trace element levels on the availability of essential amino acids of broiler chickens ¹ %.

Treatments 2		Apparent Availability							True Availability
		Lys	Met	Arg	His	Phe	Thr	Val	Lys	Met	Arg	His	Phe	Thr	Val
IE-2.0		89.48 a	88.95 a	89.33 ab	90.76 ab	89.36 b	83.01 ab	85.70 ab	90.74 b	89.74 a	90.78 ab	92.06 ab	90.99 b	84.47 ab	87.60 ab
SE-2.0		90.77 a	90.58 a	90.09 a	91.72 a	91.37 a	84.94 a	87.14 a	92.25 a	91.49 a	91.54 a	93.09 a	92.90 a	86.13 a	89.05 a
SE-1.5		89.55 a	89.54 a	89.59 ab	90.25 b	89.81 b	83.57 ab	85.13 ab	91.00 b	90.52 a	91.04 ab	91.71 b	91.24 b	84.87 ab	86.86 ab
SE-1.0		89.87 a	89.59 a	89.18 ab	90.50 b	90.06 ab	82.48 ab	85.74 ab	91.23 ab	90.10 a	90.63 b	91.97 ab	91.51 b	83.86 ab	87.26 ab
SE-0.5		89.84 a	89.31 a	88.92 b	90.29 b	89.71 b	81.32 bc	84.70 b	91.33 ab	90.25 a	90.71 ab	91.67 b	90.96 b	82.65 bc	85.91 bc
SE-0		88.05 b	84.36 b	87.88 c	90.07 b	89.16 b	79.44 c	84.21 b	89.47 c	85.34 b	89.72 c	91.63 b	90.27 b	80.64 c	85.04 c
SEM 3		0.173	0.242	0.128	0.151	0.183	0.335	0.265	0.158	0.232	0.109	0.158	0.179	0.333	0.263
p-value
Treatment		0.006	<0.001	0.001	0.051	0.029	0.002	0.06	0.002	<0.001	0.003	0.11	0.008	0.002	0.004
SE	Linear	0.001	<0.001	<0.001	0.006	0.002	<0.001	0.002	0.003	<0.001	<0.001	0.012	0.001	<0.001	<0.001
	Quadratic	0.002	<0.001	<0.001	0.007	0.005	<0.001	0.007	0.001	<0.001	0.001	0.012	0.006	<0.001	0.003

¹ Data are means for 8 replicates of 4 broilers per replicate. ² IE-2.0: basal diet (2.0 g/kg of inorganic trace element premix); SE-2.0, SE-1.5, SE-1.0, SE-0.5, and SE-0 in which IEs were replaced with 2.0, 1.5, 1.0, 0.5, and 0 g/kg of SEs, respectively, and 0, 0.5, 1.0, 1.5, and 2.0 g/kg of zeolite powder were added to supplement the insufficient quantity in the formula. ³ Total standard error of the means. ^a,b,c Means within a column with different letters are significantly different (p < 0.05).

). Birds fed SE-0 decreased the apparent and/or true availability of Lys, Met, Arg, Thr, and Val compared to IE-2.0 (p < 0.05); however, SE-2.0 increased the apparent availability of Phe and the true availability of Lys and Phe (p < 0.05). Interestingly, birds fed a lower SE level (SE-0.5) had similar effects on the apparent and true availability of essential AA compared to birds fed higher IE (IE-2.0) levels (p > 0.05).

For non-essential AA, linear and quadratic decreases in the apparent and true availability of Asp, Ser, Glu, Gly, and Cys were observed with decreasing SE (p < 0.05) (

Table 7. Effects of different trace element levels on the availability of non-essential amino acids of broiler chickens ¹ %.

Treatments 2		Apparent Availability							True Availability
		Asp	Ser	Glu	Pro	Gly	Cys	Tyr	Asp	Ser	Glu	Pro	Gly	Cys	Tyr
IE-2.0		89.29 ab	87.83 a	91.07 b	89.3	83.06 bc	90.39 ab	89.22 a	90.48 ab	89.66 ab	92.78 b	91.16	85.13 ab	92.39 ab	91.60 a
SE-2.0		90.41 a	89.03 a	92.24 a	89.77	85.31 a	90.67 a	89.19 a	91.72 a	90.86 a	94.04 a	91.71	87.34 a	92.34 ab	91.54 a
SE-1.5		89.63 a	87.23 ab	90.96 b	89.5	85.55 a	89.03 bc	87.68 b	90.94 ab	89.03 b	92.84 b	91.84	86.73 a	90.81 bc	89.85 b
SE-1.0		89.17 ab	87.55 ab	90.28 bc	88.81	84.89 ab	89.20 bc	88.20 ab	90.37 ab	89.36 ab	92.15 bc	90.35	86.07 a	91.09 bc	90.16 ab
SE-0.5		89.17 ab	85.95 b	89.31 c	88.83	84.25 abc	89.35 bc	88.64 ab	90.51 ab	88.06 b	91.07 c	90.49	85.48 ab	91.22 bc	90.31 ab
SE-0		87.97 b	84.15 c	87.84 d	88.97	82.43 c	87.85 c	87.94 b	89.39 b	86.30 c	89.75 d	90.91	83.59 b	89.58 c	89.30 b
SEM 3		0.197	0.241	0.154	0.177	0.283	0.182	0.189	0.193	0.229	0.165	0.176	0.298	0.174	0.186
p-value
Treatment		0.045	0.001	<0.001	0.554	0.021	0.002	0.122	0.05	0.002	<0.001	0.11	0.022	0.001	0.008
SE	Linear	0.004	<0.001	<0.001	0.065	0.001	0.008	0.276	0.004	<0.001	<0.001	0.027	0.001	0.001	0.058
	Quadratic	0.002	<0.001	<0.001	0.104	0.001	0.004	0.365	0.002	<0.001	<0.001	0.030	0.004	0.005	0.125

¹ Data are means for 8 replicates of 4 broilers per replicate. ² IE-2.0: basal diet (2.0 g/kg of inorganic trace element premix); SE-2.0, SE-1.5, SE-1.0, SE-0.5, and SE-0 in which IEs were replaced with 2.0, 1.5, 1.0, 0.5, and 0 g/kg of SEs, respectively, and 0, 0.5, 1.0, 1.5, and 2.0 g/kg of zeolite powder were added to supplement the insufficient quantity in the formula. ³ Total standard error of the means. ^a,b,c Means within a column with different letters are significantly different (p < 0.05).

). Compared to IE-2.0, an increased apparent availability of Glu and true availability of Glu and Gly were observed in SE-2.0 (p < 0.05); however, a decreased apparent and/or true availability of Ser and Glu in SE-0.5 and SE-0, and Cys and Tyr in SE-0 were observed (p > 0.05). Overall, birds fed a lower SE level (SE-1.0) had similar effects on the apparent and true availability of non-essential AA compared to birds fed higher IE (IE-2.0) levels (p > 0.05).

3.5. Antioxidant Enzyme Activities and Liver Mineral Deposition Efficiency

In general, there was a linear and quadratic increase in liver MDA and a decrease in SOD with the decrease in SE levels (p < 0.05) (Figure 1). Birds fed SE-0.5 and SE-0 decreased liver SOD compared to IE-2.0 (p < 0.05); however, an increased liver SOD in SE-2.0 and SE-1.5 and decreased MDA in ZE-2.0 were observed (p < 0.05).

Overall, with decreasing SE levels, the contents of Fe, Zn, Cu, and Mn in the liver decreased linearly and quadratically (p < 0.05) (Figure 2). Compared to IE-2.0, decreased liver Fe, Zn, and Mn in SE-0.5 and SE-0 were observed (p < 0.05), and only decreased Cu in SE-0 was observed (p > 0.05). Interestingly, SEs at lower levels (SE-1.0) had similar effects on the contents of liver Fe, Zn, Cu, and Mn compared to higher IE (IE-2.0) levels (p > 0.05).

4. Discussion

Trace elements are indispensable nutrients in poultry, which are required for promoting growth performance and regulating bone development, appetite, and feathering [24]. However, higher levels of trace elements are supplemented to diets to obtain a better growth performance, while the waste cannot be ignored. Therefore, on the basis of meeting the standard required by the feeding standard of Chicken of People’s Republic of China, NY/T 33-2004, we chose several lower levels of SEs (0.5, 1.0, 1.5 or 2.0 g/kg) to find a better solution. It has been reported that broiler chickens supplemented with methionine hydroxy analog chelated manganese (50 mg Mn/kg diet) increased the BW and ADFI compared to those fed the basal diet [25]. However, studies by [26] demonstrated that the feed supplementation of methionine and chelate (1 g methionine chelate/kg diet) including Cu, Fe, Zn, and Mn had no beneficial improvements on the growth performance of broiler chickens. Similarly in our study, there were no significant impacts of SEs (0.5, 1.0, 1.5, 2.0 g/kg feed) and IEs (2.0 g/kg feed) on the ADFI, ADG, and F/G of broiler chickens. Our study at least indicated that the supplementation of SEs at low levels (0.5, 1.0, 1.5 g/kg) could make the birds achieve the same performance as IEs at a high level (2.0 g/kg), which is consistent with those of previous studies [27,28]. This demonstrated that SEs could reduce amounts of in-feed mineral supplementations, which may reduce the amount of excreted minerals and alleviate environmental pollutions. The promised performance could be due to improved nutrient digestibility and absorptions by oligosaccharides in SE [29].

Impacts of different levels of SEs on the availabilities of DM, OM, CP, and GE were evaluated in this study since nutrient availability is an important parameter correlated with growth performance [30]. In our study, feeding birds at the lower SE level (0.5 g/kg) could achieve similar nutrient availabilities when compared to birds fed higher levels of SEs (1.0, 1.5, 2.0 g/kg) and IEs (2.0 g/kg), indicating that organic trace elements could improve nutrient availabilities, which are consistent with previous studies [31,32]. This may be due to the improved gut health of broiler chickens by the supplementation of oligosaccharides in organic trace minerals. It has been reported that organic trace minerals reduced crypt depth, which is related to greater intestinal maturity [33]. The lower crypt depth in broiler chickens is an indicator of decreased requirements of nutrients for maintenance and production that could promote nutrient availability and growth performance [34]. Additionally, previous reports have indicated that organic trace minerals reduced the number of intestinal goblet cells [35]. Goblet cells are responsible for mucin production for the defense of intestinal microorganisms, but it could also lead to reductions in nutrient absorption [36]. Additionally, the improved nutrient availabilities may be due to the modulation of microbiota in the small intestines by organic trace minerals. Gut microorganisms are an essential layer of the brush border with an ability to absorb nutrients, prevent infectious diseases, and enhance immune systems [37]. Interestingly, improved richness and diversity of the chicken intestinal microbiota by organic trace elements have been reported previously [38]. The products for chelating the trace minerals in our research were oligosaccharides. Oligosaccharides that can be fermented by gut microbiota have been well known for enhancing growth performance, elevating nutrient digestibility and absorption, and increasing the relative abundance of beneficial bacterial species such as Lactobacillus crispatus and Anaerostipes butyraticu [39,40]. Despite this, analyses on gut microbiota are still necessary in further experiments to understand the true mechanisms of SEs on nutrient availability.

In addition to nutrient and energy availabilities, the effects of sucrose chelated organic trace elements on the availabilities of minerals and amino acids were evaluated in our study. Antagonistic interactions of inorganic trace minerals can cause low availabilities of minerals, which are often reflected in compromised growth performance, increased mineral excretions, and an elevated economic loss and environmental pollution in poultry farms [41]. It has been reported that the organic trace minerals may be better digested and absorbed compared to inorganic elements due to their minimal interacts with antagonists in chicken feeds such as sulfur in dried distillers’ grains (DDGs) and iron contamination in corns [42]. This could explain the similar digestibility of minerals when birds were fed low levels of SEs (0.5 g/kg) compared to higher levels of SEs (1.0, 1.5, 2.0 g/kg) and IEs (2.0 g/kg). Another interesting finding of this study was the similar amino acid digestibility when birds were fed low levels of SEs (0.5 g/kg) compared to higher levels of SEs (1.0, 1.5, 2.0 g/kg) and IEs (2.0 g/kg). The improved mineral and amino acid digestibility when fed SEs may also be due to the increased stabilities in the gastrointestinal tract and improved antioxidant activities in broiler chickens [43,44]. Another explanation is that the fermentation of oligosaccharides to short-chain fatty acids in the intestines could be beneficial to digestive enzyme activities, gut morphology, and diversity of microbiota [45].

Commercial broiler chickens in intensive poultry systems at high stock density are sensitive to oxidative stressors, inducing compromised growth performance, gut health, and meat quality [46]. The MDA is a product of lipid peroxidation, and its accumulation is the biomarker for reflecting oxidative stress [47]. The SOD is an enzymatic scavenger that neutralizes the reactive oxygen species (ROS), and its concentration can be used to reflect antioxidant status in poultry [48]. Additionally, the liver is an organ that is particularly susceptible to oxidative stress for inducing liver disorders [49]. The results from this study demonstrated that the full replacement of IEs by SEs could increase concentrations of SOD and reduce MDA in livers of broiler chickens, which is consistent with some recent studies [32,50]. Additionally, birds fed a lower level of SEs (1.5 g/kg) exert a comparable or even better antioxidant status than IEs at a higher level (2.0 g/kg). This corresponds to a recent study showing that the replacement of IEs by lower levels of SEs increased activities of liver SOD [32]. The elevated antioxidant enzymes could be due to oligosaccharides in SEs that alleviate antioxidant stress [51]. Trace elements can deposit in broiler chicken organs such as the liver, kidney, bone, and pancreas. The contents of trace elements in the liver can reflect the biological efficacy of dietary trace minerals in broiler chickens [52]. A higher Mn concentration was shown in birds fed SEs at a lower level (1.5 g/kg) compared to IEs at a higher level (2.0 g/kg). Mn is an important activator of essential enzymes such as hydrolases and arginase, involved in many crucial nutrient metabolisms and playing significant roles in the antioxidant system [53]. This may also explain the increased concentrations by SEs compared to IEs.

5. Conclusions

The present study indicated that in broiler chicken diets, feed supplementations of SEs at lower levels (1.0 or 1.5 g/kg) could reach the same results of growth performance and nutrient digestibility compared to high levels of SEs (2.0 g/kg) and IEs (2.0 g/kg). Additionally, the full replacement of IEs (2.0 g/kg) by SEs (2.0 g/kg) reduced MDA and increased SOD. This indicated that our advanced chelating technology can reduce trace element requirements of broiler chickens. This may suggest that SEs can reduce the excretion of minerals in broiler chickens, which may reduce environmental pollution. However, further investigations are still necessary to elucidate the mechanisms of the beneficial effects of SEs on growth performance and nutrient digestibility.