animals

Article
Effects of Different Patterns and Sources of Trace Elements on
Laying Performance, Tissue Mineral Deposition, and Fecal
Excretion in Laying Hens
Kaili Yang 1,2 , Shengjun Hu 1,2 , Rui Mu 1,2 , Yiqing Qing 1,2 , Liang Xie 1,2 , Liyuan Zhou 1,2 , Kolapo M. Ajuwon 3
and Rejun Fang 1,2, *

1 College of Animal Science and Technology, Hunan Agricultural University, No. 1 Nongda Road,
Furong District, Changsha 410128, China; [email protected] (K.Y.); [email protected] (S.H.);
[email protected] (R.M.); [email protected] (Y.Q.); [email protected] (L.X.);
[email protected] (L.Z.)
2 Hunan Co-Innovation Center of Animal Production Safety, Changsha 410128, China
3 Department of Animal Sciences, Purdue University, West Lafayette, IN 47907-2041, USA;
[email protected]
* Correspondence: [email protected]; Tel.: +86-731-861-8177

Simple Summary: This study aims to investigate the effects of laying performance, tissue mineral
deposition, and fecal excretion from laying hens fed dietary treatments with different patterns and
sources of trace elements including zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), and selenium
(Se). A total of 864 healthy laying hens with similar laying rate (Roman, 26-week-old) were randomly





 divided into nine treatments, with six replications of 16 birds per replication, including a control
Citation: Yang, K.; Hu, S.; Mu, R.;
treatment (basic diet without added extra trace minerals) and four patterns with different element
Qing, Y.; Xie, L.; Zhou, L.; Ajuwon, sources (inorganic mineral or organic mineral): 1. NRC (1994) recommended level pattern (NRC-
K.M.; Fang, R. Effects of Different L pattern); 2. NY/T 33-2004 recommended level pattern (NY/T-L pattern); 3. 50% NRC (1994)
Patterns and Sources of Trace recommended level (50% NRC-L pattern); 4. the ratio of minerals in the blood of laying hens was
Elements on Laying Performance, taken as the supplement proportion of trace elements, and Zn was supplemented depended on
Tissue Mineral Deposition, and Fecal the NRC recommended level pattern (TLB pattern). The results indicated that supplemental trace
Excretion in Laying Hens. Animals elements improved laying rate, liver, kidney, pancreas, spleen, pectorals muscle, and tibia mineral
2021, 11, 1164. https://doi.org/
concentrations (Zn, Fe, Cu, Mn, Se). Patterns affected the tissue and fecal mineral concentrations. In
10.3390/ani11041164
addition, supplementation of the minerals with organic trace elements increased tissues and fecal
mineral concentration than those with inorganic minerals. In conclusion, diet supplemented with the
Academic Editor: Colin Scanes
organic trace minerals of 50% NRC-L pattern for laying hens promoted optimum laying performance,
Received: 6 March 2021
mineral deposition, and reduced mineral excretion.
Accepted: 5 April 2021
Published: 19 April 2021 Abstract: This study was conducted to investigate the effects of different patterns and sources of Zn,
Fe, Cu, Mn, and Se on performance, mineral deposition (liver, kidney, pancreas, spleen, pectorals
Publisher’s Note: MDPI stays neutral muscle, and tibia), and excretion of laying hens, then to find an optimal dietary supplemental
with regard to jurisdictional claims in pattern of trace elements in laying hens. A total of 864 healthy laying hens with similar laying rate
published maps and institutional affil- (Roman, 26-week-old) were randomly divided into nine treatments, with six replications of 16 birds
iations. per replication, including a control treatment and four patterns with different element sources
(inorganic or organic): (1) Control treatment (basic diet without added extra trace minerals, CT);
pattern 1, NRC (1994) recommended level (NRC-L): (2) inorganic minerals of NRC-L pattern (IN),
(3) organic minerals of NRC-L pattern (ON); pattern 2, NY/T 33-2004 recommended level (NY/T-L):
Copyright: © 2021 by the authors. (4) inorganic minerals of NY/T-L pattern (IY), (5) organic minerals of NY/T-L pattern (OY); pattern
Licensee MDPI, Basel, Switzerland. 3, 50% NRC (1994) recommended level (50% NRC-L): (6) inorganic minerals of 50% NRC-L pattern
This article is an open access article (IHN), (7) organic minerals of 50% NRC-L pattern (OHN); pattern 4, the ratio of minerals in blood of
distributed under the terms and laying hens was taken as the supplement proportion of trace elements, and Zn was supplemented
conditions of the Creative Commons
depended on NRC recommended level (TLB): (8) inorganic minerals of TLB pattern (IB), (9) organic
Attribution (CC BY) license (https://
minerals of TLB pattern (OB). Two weeks were allowed for adjustment to the conditions and then
creativecommons.org/licenses/by/
measurements were made over eight weeks. Supplementation of trace elements led to increased
4.0/).

Animals 2021, 11, 1164. https://doi.org/10.3390/ani11041164 https://www.mdpi.com/journal/animals

Animals 2021, 11, 1164 2 of 16

daily egg weight (p < 0.05). Patterns of minerals in diets affected the content of liver Mn, pancreas
Mn, tibia Mn, and the tissues Se (p < 0.05). Sources of minerals had positive effects on daily egg
weight (p < 0.05), the concentrations of liver Fe, kidney Cu, tissues Se (except spleen), and fecal Se
(p < 0.05). In conclusion, diet supplemented with the organic trace minerals of 50% NRC-L pattern
(OHN) in laying hens promoted optimum laying performance, mineral deposition, and reduced
mineral excretion.

Keywords: trace element; laying hens; performance; mineral deposition; fecal mineral excretion

1. Introduction
Nowadays, the improvement of diet composition becomes a key factor to improve the
health status and welfare of animals [1] as well as to enhance productivity in livestock [2–4].
Trace elements like zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), and selenium
(Se) play very essential roles in nutrition and physiology of animals [5]. As activators
of various enzymes, these five trace elements participate in metabolism and transport
substances in vivo, including increasing the membrane structure stability, antioxidant
reaction, immune defense metabolism, the processes of reproduction, hormone synthesis,
and bone growth [6–9].
For birds, trace minerals are important, while most feedstuffs do not contain adequate
quantities of most trace elements. In order to fulfill the requirements of mineral elements
for poultry, additional mineral additive elements must be added to the diet. There are
authoritative recommendations like National Research Council (NRC, 1994) and Chinese
Feeding Standard of Chicken (NY/T 33-2004). However, supplementation of trace elements
has been overbalancing [10]. Excessive ingestion of trace elements not only affects the
health of hens, but also increases the excretion of minerals into the soil or/and water,
causing environmental contamination [11]. The utilization of minerals is a complex process
which depends on the animals (breed and age), the bioavailability of mineral forms, the
mineral interaction (synergism and antagonism), and so on [11,12].
Inorganic minerals are commonly used in livestock and poultry production, such as
oxides or sulphates, due to low cost and availability [13]. However, during the passage
through the digestive tract, ions from soluble inorganic trace elements can potentially
be combined with and excreted with other dietary components, making them less avail-
able. [14]. Organic minerals contain metal ions chemically in connection with organics,
which showed uniform stability and high bioavailability. Therefore, the trace elements
seem to be more readily available in the gastrointestinal system due to forming complexes
with other ration components [15]. Numerous studies have reported that organic trace
elements improved laying performance, increased mineral absorption, increased the min-
eral concentrations of tissue, and decreased mineral excretion compared to inorganic trace
elements. Different concentrations of supplemental trace elements also can affect the
above-mentioned parameters [16–18].
Our group has proposed a concept of “Mineral Element Ideal Pattern (MEIP)”, the
composition and proportion of mineral elements in the diet are consistent with the quality
and quantity of mineral elements required by animals, with the expectation that an ideal
pattern of dietary mineral elements will be optimally utilized by animals to support
physiological functions [19]. Therefore, experiments are needed to determine optimum
proportion of minerals in diets of animals to establish the ideal pattern. Determination
of appropriate level and source of trace elements in diets of laying hens is a strategy
for achieving optimal growth performance and reducing mineral excretion in feces. The
mineral requirements of animals can be indicated by the concentration of minerals in tissues
and blood [20]. Some organs are recognized as indicators for assessing the bioavailability
of specific minerals. For example, bone zinc content can be considered as a responsive
criterion for Zn bioavailability [21]. Liver and kidneys are sensitive to the variation
Animals 2021, 11, 1164 3 of 16

of Cu concentration. Mineral metabolism, regulation, and storage can affect mineral

concentration in spleen and liver [22]. It is still unknown whether supplementation of
trace elements in the diets of laying hens at the same ratio in blood is beneficial or not. We
hypothesized that lower supplementation of mineral elements than NRC recommendations
will support normal production performance with no negative influence on the laying
hens, and that organic trace minerals will have higher bioavailability. Therefore, this study
was conducted to investigate the effects of different patterns and sources of trace elements
on laying performance, tissue mineral element retention, and fecal mineral excretion of
laying hens to determine the MELP.

2. Materials and Methods

2.1. Experimental Animals
This experiment was conducted on a commercial poultry farm (Yiyang, China). A total
of 864 healthy laying hens (Roman pink, 26-week-old) with similar laying rate (95 ± 0.5%)
body weight (1.9 ± 0.05 kg) were randomly divided into 9 treatments, with 6 pens of
16 birds per pens. This trial lasted for 10-wk, including 2 weeks for adaptation and 8 weeks
for experiment. During the experiment, the birds were ad libitum to feed and water, with
management of 16 L: 8D. Feeding management and vaccination programs were concordant
to common practice.

2.2. Dietary Treatments

The basic diet was formulated suggested by NRC (1994) (Table 1). In this trial, there
were basic diet and four patterns with two sources (inorganic or organic minerals) which
were divided into nine treatments (Table 2): (1) Control treatment (basic diet without add
extra trace minerals, CT); pattern 1, NRC (1994) recommended level (NRC-L): (2) inorganic
trace minerals of NRC-L (IN) pattern, (3) organic trace minerals of NRC-L pattern (ON);
pattern 2, NY/T 33-2004 recommended level (NY/T-L): (4) inorganic trace minerals of
NY/T-L pattern (IY), (5) organic trace minerals of NY/T-L pattern (OY); pattern 3, 50%
NRC (1994) recommended level (50% NRC-L): (6) inorganic trace minerals of 50% NRC-L
pattern (IHN), (7) organic trace minerals of 50% NRC-L pattern (OHN); pattern 4, the ratio
of mineral elements in blood of laying hens was taken as the supplement proportion of
trace elements, and Zn was supplemented depended on NRC recommended level (TLB):
(8) inorganic trace minerals of TLB pattern (IB) [23,24], (9) organic trace minerals of TLB
pattern (OB). The inorganic Zn (>34.5%), Fe (>30%), Cu (>25.06%) and Mn (>31.8%) were in
the form of feed-grade sulphate, and Se (>1%) was in form of sodium selenite. The organic
Zn (>24%), Fe (>12%), Cu (>12%) and Mn (>14%) were provided as amino-acid minerals
while the Se was as selenium yeast (Se > 0.2%), the organic trace elements (Zn, Fe, Cu, Mn,
Se) were provided by Changsha Xingjia Biotechnology Share Co., Ltd. (Changsha, China).

Table 1. Composition and nutrition of basal diet (as-fed basis).

Ingredient Content (%) Nutrient Level Content

Corn 60.50 Calculated compositions
Soybean meal 24.00 ME, MJ/kg 2 11.08
Soybean oil 1.00 CP, % 3 16.00
Rapeseed meal 2.40 Lysine, % 0.81
Limestone 8.80 Methionine, % 0.36
CaHPO4 1.30 Calcium, % 3.60
Premix 1 2.00 Total phosphorus, % 0.55
Total 100.00 Analyzed compositions
CP, % 16.43
Calcium, % 3.40
Total phosphorus, % 0.54
1 Premix feed provided per kilogram of diet: Vitamin A 6000 IU, Vitamin D3 2775 IU, Vitamin E 25.0 mg, Vitamin
K3 2.25 mg, Vitamin B1 1.8 mg, Vitamin B2 4.50 mg, Vitamin B6 4.0 mg, Vitamin B12 0.20 mg, pantothenic acid
12.0 mg, niacin acid 25 mg, biotin 0.14 mg, folic acid 0.8 mg, phytase 1000 IU. 2 ME: metabolic energy. 3 CP:
crude protein.
Animals 2021, 11, 1164 4 of 16

Table 2. Trace element addition pattern 1 .

Pattern Treatments Zn Fe Cu Mn Se
CT 0 0 0 0 0
100.00 174.43 11.42 85.71 0.29
IN
35.00 45.00 4.00 20.00 0.05
NRC-L
100.00 174.43 11.42 85.71 0.29
ON
35.00 45.00 4.00 20.00 0.05
100.00 75.00 10.00 75.00 0.38
IY
80.00 60.00 8.00 60.00 0.30
NY/T-L
100.00 75.00 10.00 75.00 0.38
OY
80.00 60.00 8.00 60.00 0.30
100.00 174.43 11.42 85.71 0.29
IHN
50% 17.50 22.50 2.00 10.00 0.025
NRC-L 100.00 174.43 11.42 85.71 0.29
OHN
17.50 22.50 2.00 10.00 0.025
100.00 358.21 38.20 21.21 2.57
IB
35.00 125.37 13.37 7.42 0.90
TLB
100.00 358.21 38.20 21.21 2.57
OB
35.00 125.37 13.37 7.42 0.90
1 In each pattern, upper data mean ratio of one trace mineral element to zinc (as 100.00) and down data mean of
the supplemental level of this trace mineral element in diets (mg/kg). Control treatment (basic diet without add
extra trace minerals, CT); NRC recommended level (NRC-L), inorganic trace minerals of NRC-L (IN), organic
trace minerals of NRC-L pattern (ON), NY/T 33-2004 recommended level (NY/T-L), inorganic trace minerals of
NY/T-L pattern (IY), organic trace minerals of NY/T-L pattern (OY); pattern 3, 50% NRC (1994) recommended
level (50% NRC-L), inorganic trace minerals of 50% NRC-L pattern (IHN), organic trace minerals of 50% NRC-L
pattern (OHN), the ratio of mineral elements in blood of laying hens was taken as the supplement proportion of
trace elements, and Zn was supplemented depended on NRC recommended level (TLB), inorganic trace minerals
of TLB pattern (IB), organic trace minerals of TLB pattern (OB).

2.3. Samples Collection and Measurement

2.3.1. Laying Performance
Egg weight and number for each replicate were recorded daily. Feed consumption
was recorded weekly after introduction of the experimental diets. At the end of the trial,
laying rate, average egg weight (g/egg), average daily feed intake (ADFI, g/bird per day),
and feed-to-egg ratio were calculated.

2.3.2. Mineral Concentration

After 8 weeks on experimental diets, a bird was euthanized through cervical disloca-
tion per replicate. Liver, kidney, pancreas, spleen, pectoral muscle, and tibias was collected
and stored at −80 ◦ C.
Tibia was immersed until fat was extracted, and then the traces of flesh and cartilages
were removed. A quarter of feces were collected for 3 consecutive days at the end of the
experiment for each replicate, dried at 65 ◦ C for 48 h, and smashed and sifted to remove
feathers. Then, the fecal sample was grounded to pass through a 1-mm screen after samples
achieved constant weight and packed in self-locked plastic bags and stored at −20 ◦ C
for analysis.
The Zn, Fe, Cu, and Mn content of samples were analyzed by inductively coupled
plasma optical emission spectrometry (ICP-OES, Optima 8300, PerkinElmer, Waltham,
MA, USA) according to the method suggested by Qiu et al. [25]. The Se of samples were
analyzed by atomic fluorescence meter (AFS-920, Titan, Beijing, China) according to the
method suggested by Wang et al. [26].

2.4. Statistical Analysis

The data of the experiment were analyzed using one-way ANOVA by SPSS 23.0
(SPSS. Inc., Chicago, IL, USA) to compare all supplement experiment treatments with the
control. Data were further analyzed as a 4 × 2 (pattern × source) factorial arrangement
of treatments except control treatment by GLM using SPSS 23.0 (SPSS. Inc., Chicago, IL,
Animals 2021, 11, 1164 5 of 16

USA), which included the main effects of pattern and source of trace elements, as well as
their interaction. Dietary treatment was regarded as an independent variable. One dietary
treatment was regarded as the experiment unit, with six replicates, and one replicate was
regarded as a statistical unit. Differences were analyzed by Tukey’s multiple range tests
whose significance levels were defined as p < 0.05, and a trend as 0.05 < p < 0.1. The data
are expressed as means ± Standard Error of Mean (SEM).

3. Results
3.1. Laying Performance
The analyzed composition of trace minerals in diets is shown in Table 3. The concen-
trations of Zn, Fe, Cu, Mn, and Se in basic diets were 16.1, 302, 4.90, 17.9, and 0.06 mg/kg,
respectively. As indicted in Table 4, compared with OB treatment, OHN and OB treatments
increased the average egg weight (p < 0.05). No differences were detected on ADFI, laying
rate, and feed-to-egg ratio (p > 0.10). The average egg weight was increased by organic
minerals compared to inorganic form (p < 0.05). The average daily egg weight of hens
fed dietary of OHN was 2.9% higher compared with the hens from CT group (p < 0.05). A
supplemental trace element pattern did not affect laying performances (p > 0.10). There
was a trend of interaction in average egg weight between pattern and source (0.05 < p <0.1).

Table 3. Trace elements analyzed content of diets (mg/kg, in trace elements).

Patterns NRC-L NY/T-L 50% NRC-L TLB

Items CT IN ON IY OY IHN OHN IB OB
Zn 16.1 45.8 46.3 92.1 94.6 35.5 34.6 46.8 46.2
Fe 302 353 348 379 384 331 328 409 406
Cu 4.90 8.55 8.93 10.7 11.8 7.44 7.41 12.4 12.9
Mn 17.9 34.2 34.4 70.1 73.8 27.2 25.9 23.0 24.4
Se 0.06 0.10 0.13 0.35 0.36 0.08 0.08 1.14 1.20
Control treatment (basic diet without add extra trace minerals, CT); NRC recommended level (NRC-L), inorganic
trace minerals of NRC-L (IN), organic trace minerals of NRC-L pattern (ON), NY/T 33-2004 recommended
level (NY/T-L), inorganic trace minerals of NY/T-L pattern (IY), organic trace minerals of NY/T-L pattern
(OY); pattern 3, 50% NRC (1994) recommended level (50% NRC-L), inorganic trace minerals of 50% NRC-L
pattern (IHN), organic trace minerals of 50% NRC-L pattern (OHN), the ratio of mineral elements in blood of
laying hens was taken as the supplement proportion of trace elements, and Zn was supplemented depended
on NRC recommended level (TLB), inorganic trace minerals of TLB pattern (IB), organic trace minerals of TLB
pattern (OB).

Table 4. Effect of different patterns and sources of mineral elements on laying performance 1 .

ADFI Average Egg Feed-To-Egg

Items Laying Rate (%)
(g/Bird/per Day) Weight (g/Egg) Ratio
CT 107.85 96.18 57.69 ab 1.95
IN 107.53 95.52 57.39 ab 1.98
NRC-L
ON 106.60 95.97 57.17 ab 1.95
IY 106.82 94.55 57.36 ab 1.97
NY/T-L
OY 106.75 94.61 57.65 ab 1.99
IHN 106.49 95.50 57.63 ab 1.94
50% NRC-L
OHN 107.58 95.73 59.34 a 1.93
IB 106.20 95.38 56.34 b 1.98
TLB
OB 110.58 96.39 58.69 a 1.95
SEM 0.38 0.32 0.20 0.07
NRC-L 107.06 95.74 57.28 1.97
NY/T-L 106.79 94.58 57.50 1.98
Patterns
50% NRC-L 107.04 95.61 58.48 1.93
TLB 108.39 95.88 57.51 1.97
Animals 2021, 11, 1164 6 of 16

Table 4. Cont.

ADFI Average Egg Feed-To-Egg

Items Laying Rate (%)
(g/Bird/per Day) Weight (g/Egg) Ratio
Inorganic 106.76 95.24 57.18 † 1.97
Sources
Organic 107.88 95.67 58.21 1.96
CT-trace element
0.164 0.929 0.008 0.808
supplemented
p-value Pattern 0.458 0.579 0.113 0.347
Source 0.155 0.541 0.009 0.548
Pattern × Source 0.094 0.968 0.069 0.841
1 Data are given as treatment means; n = 6 pens per treatment, † different from the corresponding inorganic treatments (p < 0.05). Control treatment
(basic diet without add extra trace minerals, CT); NRC recommended level (NRC-L), inorganic trace minerals of NRC-L (IN), organic trace minerals of
NRC-L pattern (ON), NY/T 33-2004 recommended level (NY/T-L), inorganic trace minerals of NY/T-L pattern (IY), organic trace minerals of NY/T-L
pattern (OY); pattern 3, 50% NRC (1994) recommended level (50% NRC-L), inorganic trace minerals of 50% NRC-L pattern (IHN), organic trace
minerals of 50% NRC-L pattern (OHN), the ratio of mineral elements in blood of laying hens was taken as the supplement proportion of trace elements,
and Zn was supplemented depended on NRC recommended level (TLB), inorganic trace minerals of TLB pattern (IB), organic trace minerals of TLB
pattern (OB). Means for treatments within a row not showing a common small letter superscript differ (p < 0.05), † different from the corresponding
inorganic treatments (p < 0.05).

3.2. Trace Elements Status on Tissues

The effects of trace elements at different patterns and sources on the liver minerals in
Table 5. OHN treatment had higher liver Zn and Fe concentrations than CT and IY groups.
OB treatment had the highest liver Se concentration among the treatments (p < 0.05). Birds
fed organic trace elements diets increased liver Fe and Se content compared with those
fed inorganic mineral diets (p < 0.05). Patterns of minerals affected the liver Mn and Se
concentrations (p < 0.05). The NY/T-L pattern increased the accumulation of liver Mn
compared with the TLB pattern (p < 0.05). The highest liver Se concentration was found in
the TLB pattern (p < 0.05). In kidneys (Table 6), compared with CT treatment, the kidney
Cu content was improved by ON treatment (p < 0.05). Kidney Se content was the highest in
the OB treatment (p < 0.05). In addition, kidney Se concentration was significantly higher
in hens fed diets of OY and IB treatments than other treatments (p < 0.05). Supplementation
of trace elements as organic form increased kidney Cu and Se concentrations compared
to those fed inorganic mineral treatments (p < 0.05). Patterns of minerals significantly
influenced kidney Se concentration (p < 0.001), while it had tendencies for affecting both
Zn and Mn concentrations (0.05 < p <0.1). Kidney Se content in hens fed diet of TLB pattern
was highest. In the pancreas (Table 7), Se concentration in OB groups was the highest
among these groups (p < 0.05). The pancreas of birds in OHN treatment had higher Se
content than that in other treatments except the OB group (p < 0.05). Hens of OY and ON
treatments showed higher pancreas Mn content than OB treatment (p < 0.05). The pattern
of minerals had influences on the pancreas Mn and Se concentrations (p < 0.05). Among
mineral patterns, pancreas Mn content was significantly reduced while Se concentration
was increased in birds fed a diet supplemented with a trace element of the TLB pattern
(p < 0.05). It had a tendency for a 50% NRC-L pattern to increase pancreas Zn content
(0.05 < p <0.1). The Se concentration from IB and OB treatments were higher than that from
other treatments in spleen (p < 0.001). Spleen Se content in OY and OHN treatments were
higher than that from CT, IN, ON, IY, and IHN treatments (p < 0.001) (Table 8). Compared
with the CT group, pectorals muscle Se concentration was increased in ON, OY, OHN,
IB, and OB groups (p < 0.001) (Table 9). The spleen and pectorals muscle Se content was
increased by adding supplemental organic trace elements of TLB patterns (p < 0.001).
There was a trend for spleen Fe concentration in patterns and sources of trace elements
(0.05 < p <0.1). The effects of trace elements at different patterns and sources on the tibia
minerals in Table 10. The Tibia Mn concentration was improved by OY treatment than that
in OB treatment (p < 0.05). Tibia Se concentration was the highest in supplemental trace
elements of OB treatments (p < 0.001). Compared with CT, IN, ON, IY, IHN, and OHN
treatments, birds fed OY and IB diets increased tibia Se content (p < 0.05). Supplementation
Animals 2021, 11, 1164 7 of 16

of the diets with organic trace elements significantly increased tibia Se concentration than
those with inorganic minerals (p < 0.001). Patterns of minerals influenced tibia Mn and Se
concentrations (p < 0.05). The highest Mn and Se content in tibia were found in birds fed
diets supplemented with trace minerals of NY/T-L pattern and TLB pattern, respectively
(p < 0.05). The interaction between trace element patterns and sources was significant in
light of liver Se, kidney Se, pancreas Se and Mn, pectorals muscle Se, and tibia Se (p < 0.05).

Table 5. Effect of different patterns and Sources of mineral elements on liver (mg/kg) 1 .

Items Zn Fe Cu Mn Se
CT 39.44 b 67.34 b 4.75 2.23 0.41 b
IN 49.47 ab 73.81 ab 5.04 2.49 0.34 b
NRC-L
ON 53.52 ab 81.32 ab 5.69 2.69 0.31 b
NY/T- IY 43.33 b 68.37 b 5.33 2.47 0.39 b
L OY 52.29 ab 86.47 ab 5.55 2.75 0.45 b
50% IHN 50.58 ab 70.11 ab 5.21 2.28 0.31 b
NRC-L OHN 63.93 a 90.23 a 5.47 2.52 0.42 b
IB 50.89 ab 81.78 ab 5.41 2.07 0.42 b
TLB
OB 47.02 ab 83.90 ab 5.22 1.98 0.82 a
SEM 1.64 1.85 0.08 0.06 0.03
NRC-L 51.49 77.56 5.36 2.59 A 0.32 B
NY/T-L 47.81 77.42 5.47 2.61 A 0.41 B
Patterns
50% NRC-L 57.26 80.17 5.34 2.40 AB 0.37 B
TLB 49.15 82.84 5.31 2.02 B 0.62 A
Inorganic 48.67 73.5 † 5.26 2.33 0.36 †
Sources
Organic 54.19 85.5 5.48 2.49 0.50
CT-trace element
0.017 0.004 0.192 0.071 <0.001
supplemented
p-value Pattern 0.180 0.667 0.940 0.013 <0.001
Source 0.108 0.001 0.225 0.238 <0.001
Pattern × Source 0.299 0.209 0.460 0.755 0.001
1 Data are given as treatment means; n = 6 pens per treatment. a,b Means for treatments within a row not showing
a common small letter superscript differ (p < 0.05), means for patterns within a row not showing a common
capital letter superscript differ (p < 0.05), † different from the corresponding inorganic treatments (p < 0.05),
the same as below. Control treatment (basic diet without add extra trace minerals, CT); NRC recommended
level (NRC-L), inorganic trace minerals of NRC-L (IN), organic trace minerals of NRC-L pattern (ON), NY/T
33-2004 recommended level (NY/T-L), inorganic trace minerals of NY/T-L pattern (IY), organic trace minerals of
NY/T-L pattern (OY); pattern 3, 50% NRC (1994) recommended level (50% NRC-L), inorganic trace minerals of
50% NRC-L pattern (IHN), organic trace minerals of 50% NRC-L pattern (OHN), the ratio of mineral elements
in blood of laying hens was taken as the supplement proportion of trace elements, and Zn was supplemented
depended on NRC recommended level (TLB), inorganic trace minerals of TLB pattern (IB), organic trace minerals
of TLB pattern (OB).

Table 6. Effect of different patterns and Sources of mineral elements on kidneys (mg/kg).

Items Zn Fe Cu Mn Se
CT 29.33 62.34 3.60 b 2.86 0.27 d
IN 28.32 51.64 3.73 ab 2.40 0.26 d
NRC-L
ON 31.79 58.79 4.17 a 2.60 0.37 cd
NY/T- IY 27.29 63.34 3.89 ab 2.54 0.37 cd
L OY 26.18 53.84 3.87 ab 2.70 0.51 b
50% IHN 25.73 50.63 3.95 ab 2.38 0.29 d
NRC-L OHN 27.37 60.42 4.09 ab 2.18 0.41 c
IB 28.69 53.8 3.97 ab 2.10 0.57 b
TLB
OB 29.06 70.03 4.12 ab 1.97 1.05 a
SEM 0.52 1.86 0.47 0.08 0.05
Animals 2021, 11, 1164 8 of 16

Table 6. Cont.

Items Zn Fe Cu Mn Se
NRC-L 30.06 55.22 3.95 2.50 0.31 C
NY/T-L 26.73 58.59 3.88 2.62 0.44 B
Patterns
50% NRC-L 26.55 55.52 4.02 2.28 0.35 C
TLB 28.88 61.91 4.04 2.03 0.81 A
Inorganic 27.51 54.85 3.89 † 2.36 0.37 †
Sources
Organic 28.60 60.77 4.06 2.36 0.58
CT-trace element
0.167 0.220 0.042 0.089 <0.001
supplemented
p-value Pattern 0.070 0.573 0.500 0.058 <0.001
Source 0.294 0.137 0.044 0.971 <0.001
Pattern × Source 0.449 0.139 0.287 0.722 <0.001
a,b,c,d,A,B,C
Means for treatments within a row not showing a common small letter superscript differ (p < 0.05),
means for patterns within a row not showing a common capital letter superscript differ (p < 0.05), † different from
the corresponding inorganic treatments (p < 0.05), the same as below. Control treatment (basic diet without add
extra trace minerals, CT); NRC recommended level (NRC-L), inorganic trace minerals of NRC-L (IN), organic
trace minerals of NRC-L pattern (ON), NY/T 33-2004 recommended level (NY/T-L), inorganic trace minerals of
NY/T-L pattern (IY), organic trace minerals of NY/T-L pattern (OY); pattern 3, 50% NRC (1994) recommended
level (50% NRC-L), inorganic trace minerals of 50% NRC-L pattern (IHN), organic trace minerals of 50% NRC-L
pattern (OHN), the ratio of mineral elements in blood of laying hens was taken as the supplement proportion of
trace elements, and Zn was supplemented depended on NRC recommended level (TLB), inorganic trace minerals
of TLB pattern (IB), organic trace minerals of TLB pattern (OB).

Table 7. Effect of different patterns and sources of mineral elements on the pancreas (mg/kg).

Items Zn Fe Cu Mn Se
CT 33.13 19.12 1.79 1.96 ab 0.20 e
IN 38.04 24.39 1.84 2.21 ab 0.19 e
NRC-L
ON 39.48 23.73 1.87 2.56 a 0.28 d
NY/T- IY 31.55 41.98 1.73 2.24 ab 0.22 e
L OY 38.71 21.12 1.89 2.75 a 0.38 c
50% IHN 44.40 19.45 1.86 2.30 ab 0.21 e
NRC-L OHN 46.93 21.44 2.1 2.00 ab 0.50 b
IB 40.05 20.85 1.91 1.96 ab 0.30 d
TLB
OB 30.63 24.56 1.88 1.52 b 0.97 a
SEM 1.49 2.42 0.04 0.09 0.01
Patterns NRC-L 38.76 24.06 1.86 2.39 A 0.24 D
NY/T-L 35.13 31.55 1.81 2.49 A 0.30 C
50% NRC-L 45.66 20.45 1.98 2.49 AB 0.37 B
TLB 35.34 22.70 1.89 1.74 B 0.64 A
Sources Inorganic 38.51 26.67 1.83 2.18 0.23 †
Organic 38.94 22.71 1.93 2.20 0.54
CT-trace element
p-value 0.097 0.536 0.681 0.015 <0.001
supplemented
Pattern 0.051 0.528 0.604 0.002 <0.001
Source 0.878 0.481 0.280 0.813 <0.001
Pattern × Source 0.220 0.385 0.711 0.033 <0.001
a,b,c,d,e,A,B,C,D
Means for treatments within a row not showing a common small letter superscript differ (p < 0.05),
means for patterns within a row not showing a common capital letter superscript differ (p < 0.05), † different from
the corresponding inorganic treatments (p < 0.05), the same as below. Control treatment (basic diet without add
extra trace minerals, CT); NRC recommended level (NRC-L), inorganic trace minerals of NRC-L (IN), organic
trace minerals of NRC-L pattern (ON), NY/T 33-2004 recommended level (NY/T-L), inorganic trace minerals of
NY/T-L pattern (IY), organic trace minerals of NY/T-L pattern (OY); pattern 3, 50% NRC (1994) recommended
level (50% NRC-L), inorganic trace minerals of 50% NRC-L pattern (IHN), organic trace minerals of 50% NRC-L
pattern (OHN), the ratio of mineral elements in blood of laying hens was taken as the supplement proportion of
trace elements, and Zn was supplemented depended on NRC recommended level (TLB), inorganic trace minerals
of TLB pattern (IB), organic trace minerals of TLB pattern (OB).
Animals 2021, 11, 1164 9 of 16

Table 8. Effect of different patterns and sources of mineral elements on the spleen (mg/kg)1 .

Items Zn Fe Cu Mn Se
CT 24.41 144.42 2.16 0.05 0.49 e
IN 26.16 121.95 2.01 0.08 0.43 e
NRC-L
ON 26.89 143.67 2.14 0.06 0.53 de
NY/T- IY 25.41 152.66 2.14 0.10 0.53 de
L OY 23.17 129.15 2.01 0.07 0.65 bc
50% IHN 25.38 141.62 2.04 0.06 0.52 de
NRC-L OHN 24.35 144.09 2.04 0.08 0.60 cd
TLB
IB 25.18 152.65 2.52 0.15 0.72 b
OB 23.61 172.98 2.11 0.06 0.94 a
SEM 0.66 3.84 0.05 0.01 0.03
NRC-L 26.52 132.81 2.08 0.07 0.48 C
Patterns NY/T-L 24.29 140.91 2.08 0.08 0.59 B
50% NRC-L 24.86 142.86 2.04 0.07 0.56 BC
TLB 24.40 162.81 2.32 0.11 0.83 A

Sources
Inorganic 25.53 54.85 2.18 0.10 0.55 †
Organic 24.51 60.77 2.07 0.07 0.68
CT-trace element 0.963 0.081 0.552 0.364 <0.001
supplemented
p-value Pattern 0.753 0.063 0.265 0.510 <0.001
Source 0.539 0.488 0.337 0.180 <0.001
Pattern × Source 0.926 0.150 0.356 0.388 0.224
a,b,c,d,e,A,B,C
Means for treatments within a row not showing a common small letter superscript differ (p < 0.05),
means for patterns within a row not showing a common capital letter superscript differ (p < 0.05), † different from
the corresponding inorganic treatments (p < 0.05), the same as below. Control treatment (basic diet without add
extra trace minerals, CT); NRC recommended level (NRC-L), inorganic trace minerals of NRC-L (IN), organic
trace minerals of NRC-L pattern (ON), NY/T 33-2004 recommended level (NY/T-L), inorganic trace minerals of
NY/T-L pattern (IY), organic trace minerals of NY/T-L pattern (OY); pattern 3, 50% NRC (1994) recommended
level (50% NRC-L), inorganic trace minerals of 50% NRC-L pattern (IHN), organic trace minerals of 50% NRC-L
pattern (OHN), the ratio of mineral elements in blood of laying hens was taken as the supplement proportion of
trace elements, and Zn was supplemented depended on NRC recommended level (TLB), inorganic trace minerals
of TLB pattern (IB), organic trace minerals of TLB pattern (OB).

Table 9. Effect of different patterns and sources of mineral elements on the pectoral muscles (mg/kg).

Items Zn Fe Cu Mn Se
CT 3.96 8.36 1.48 0.08 0.08 e
IN 3.59 8.42 1.39 0.05 0.09 de
NRC-L
ON 3.25 6.87 1.45 0.07 0.12 cd
NY/T- IY 3.90 8.08 1.38 0.06 0.11 cde
L OY 3.37 7.01 1.33 0.05 0.20 b
50% IHN 3.98 7.39 1.36 0.06 0.09 de
NRC-L OHN 3.81 6.58 1.35 0.05 0.15 c
IB 3.67 7.13 1.33 0.05 0.12 cd
TLB
OB 4.11 7.44 1.4 0.05 0.42 a
SEM 0.12 0.2 0.02 0.003 0.02
NRC-L 3.42 7.55 1.42 0.06 0.10 C
NY/T-L 3.63 7.55 1.36 0.06 0.16 B
Patterns
50% NRC-L 3.89 6.99 1.36 0.06 0.12 BC
TLB 3.89 7.29 1.37 0.05 0.27 A
Inorganic 3.78 7.75 1.37 0.06 0.10 †
Sources
Organic 3.63 6.98 1.38 0.05 0.22
Animals 2021, 11, 1164 10 of 16

Table 9. Cont.

Items Zn Fe Cu Mn Se
CT-trace element
0.738 0.343 0.668 0.156 <0.001
supplemented
p-value Pattern 0.496 0.700 0.690 0.436 <0.001
Source 0.552 0.072 0.752 0.792 <0.001
Pattern × Source 0.558 0.443 0.745 0.168 <0.001
a,b,c,d,e,A,B,C
Means for treatments within a row not showing a common small letter superscript differ (p < 0.05),
means for patterns within a row not showing a common capital letter superscript differ (p < 0.05), † different from
the corresponding inorganic treatments (p < 0.05), the same as below. Control treatment (basic diet without add
extra trace minerals, CT); NRC recommended level (NRC-L), inorganic trace minerals of NRC-L (IN), organic
trace minerals of NRC-L pattern (ON), NY/T 33-2004 recommended level (NY/T-L), inorganic trace minerals of
NY/T-L pattern (IY), organic trace minerals of NY/T-L pattern (OY); pattern 3, 50% NRC (1994) recommended
level (50% NRC-L), inorganic trace minerals of 50% NRC-L pattern (IHN), organic trace minerals of 50% NRC-L
pattern (OHN), the ratio of mineral elements in blood of laying hens was taken as the supplement proportion of
trace elements, and Zn was supplemented depended on NRC recommended level (TLB), inorganic trace minerals
of TLB pattern (IB), organic trace minerals of TLB pattern (OB).

Table 10. Effect of different patterns and sources of mineral elements on the tibia (mg/kg) 1 .

Items Zn Fe Cu Mn Se
CT 108.01 78.42 1.85 2.43 ab 0.11 c
IN 124.02 69.84 1.82 3.62 ab 0.11 c
NRC-L
ON 122.51 81.94 1.96 3.27 ab 0.14 c
NY/T- IY 128.29 77.79 1.82 4.07 ab 0.14 c
L OY 120.86 64.58 2.06 4.37 a 0.20 b
50% IHN 131.4 79.73 1.95 3.30 ab 0.13 c
NRC-L OHN 115.66 72.75 1.87 3.84 ab 0.14 c
IB 122.97 88.88 1.84 2.93 ab 0.23 b
TLB
OB 116.21 72.03 1.76 2.26 b 0.40 a
SEM 1.91 2.02 0.05 0.16 0.02
NRC-L 123.26 75.90 1.89 3.57 AB 0.12 C
NY/T-L 124.57 71.19 1.94 4.22 A 0.17 B
Patterns
50% NRC-L 123.53 76.24 1.91 3.57 AB 0.13 BC
TLB 119.59 80.46 1.80 2.60 B 0.31 A
Inorganic 126.67 70.06 1.86 3.48 0.15 †
Sources
Organic 118.81 72.83 1.91 3.43 0.22
CT-trace element
0.156 0.277 0.943 0.025 <0.001
supplemented
p-value Pattern 0.807 0.448 0.838 0.011 <0.001
Source 0.052 0.144 0.244 0.882 <0.001
Pattern × Source 0.665 0.100 0.496 0.500 <0.001
a,b,c,A,B,C
Means for treatments within a row not showing a common small letter superscript differ (p < 0.05),
means for patterns within a row not showing a common capital letter superscript differ (p < 0.05), † different from
the corresponding inorganic treatments (p < 0.05), the same as below. Control treatment (basic diet without add
extra trace minerals, CT); NRC recommended level (NRC-L), inorganic trace minerals of NRC-L (IN), organic
trace minerals of NRC-L pattern (ON), NY/T 33-2004 recommended level (NY/T-L), inorganic trace minerals of
NY/T-L pattern (IY), organic trace minerals of NY/T-L pattern (OY); pattern 3, 50% NRC (1994) recommended
level (50% NRC-L), inorganic trace minerals of 50% NRC-L pattern (IHN), organic trace minerals of 50% NRC-L
pattern (OHN), the ratio of mineral elements in blood of laying hens was taken as the supplement proportion of
trace elements, and Zn was supplemented depended on NRC recommended level (TLB), inorganic trace minerals
of TLB pattern (IB), organic trace minerals of TLB pattern (OB).

3.3. Fecal Mineral Concentration

The effects of different patterns and sources on fecal mineral excretion are presented
in Table 11. Supplementation of the basal diet with trace minerals increased fecal Zn
concentration (p < 0.05). IY and OY treatments significantly enhanced the Zn content
compared to other groups (p < 0.05). The fecal Fe content in hens fed diets supplemented
with trace minerals of IB treatment was higher than that in CT, IN, ON, IY, IHN, and
OHN treatments (p < 0.05). Supplementation of the basic diet with trace minerals of ON,
Animals 2021, 11, 1164 11 of 16

IY, OY, IB, and OB treatments significantly increased fecal Cu content compared with CT
treatment (p < 0.05). Fecal Cu content in IY and OY treatments significantly increased
compared with other treatments (p < 0.05), since the fecal Mn content was found to be
significantly higher in hens fed diets of IN, ON, IY, OY, and IHN treatments than CT
treatment (p < 0.05). Compared with CT treatment, fecal Se concentration was increased
with IY, OY, IB, and OB treatments (p < 0.05) while decreased with IN, ON, IHN, and OHN
treatments (p < 0.05). The patterns of minerals influenced fecal Zn, Fe, Cu, Mn, and Se
content (p < 0.001). Among these patterns, the mineral concentration of feces was lower in
patterns of 50% NRC-L and NRC-L treatments. The highest fecal Zn and Mn concentrations
were detected in supplementation of trace minerals of NY/T-L patterns (p < 0.05), while
Fe and Se concentrations were highest in TLB pattern groups (p < 0.05). Cu content in
the excreta of hens were significantly increased (p < 0.001) with supplementation of trace
mineral of NY/T-L and TLB pattern compared with the hens fed NRC-L and 50% NRC-L
pattern (p < 0.001). Mn concentration was significantly decreased with TLelement increased
Cu and Se concentration of feces (p < 0.05). However, the lowest Cu and Se concentration in
feces was found in birds fed a diet of an organic 50% NRC-L pattern (OHN), and the highest
fecal Se content was found in organic TLB pattern treatment. The interaction between
the patterns and sources was significant in terms of fecal Zn, Fe, and Se concentration
(p < 0.05).

Table 11. Effect of different patterns and sources of mineral elements on fecal mineral concentration
(mg/kg).

Items Zn Fe Cu Mn Se
CT 56.20 d 996.36 b 16.03 d 60.99 d 0.40 d
IN 111.16 bc 918.92 b 22.46 cd 94.17 b 0.19 e
NRC-L
ON 136.57 b 931.43 b 24.16 bc 104.42 b 0.16 e
IY 221.05 a 1011.9 b 33.27 a 186.76 a 0.63 c
NY/T-L
OY 231.91 a 1045.78 ab 35.52 a 180.61 a 0.55 c
50% IHN 121.60 bc 893.42 b 21.51 cd 93.09 bc 0.17 e
NRC-L OHN 95.64 c 1024.76 b 21.09 cd 82.36 bcd 0.11 e
IB 117.33 bc 1219.53 a 30.51 ab 64.14 cd 0.81 b
TLB
OB 125.60 b 1065.96 ab 36.05 a 60.62 d 1.55 a
SEM 7.73 17.84 1.05 6.64 0.09
NRC-L 123.86 B 925.18 B 23.31 B 99.29 AB 0.18 C
Patterns NY/T-L 226.48 A 1028.86 AB 34.40 A 183.69 A 0.59 B
50%
NRC-L 108.62 B 959.09 B 21.30 B 87.73 AB 0.14 C
TLB 121.46 B 1142.75 A 33.28 A 62.38 B 1.18 A

Sources
Inorganic 142.78 1010.96 26.94 † 109.54 0.45 †
Organic 147.43 1016.98 29.21 107.00 0.59
CT-trace
element
supple- <0.001 <0.001 <0.001 <0.001 <0.001
p-value mented
Pattern <0.001 <0.001 <0.001 <0.001 <0.001
Source 0.342 0.837 0.043 0.566 <0.001
Pattern × 0.004 0.013 0.303 0.365 <0.001
Source
a,b,c,d,e,A,B,C
Means for treatments within a row not showing a common small letter superscript differ (p < 0.05),
means for patterns within a row not showing a common capital letter superscript differ (p < 0.05), † different from
the corresponding inorganic treatments (p < 0.05), the same as below. Control treatment (basic diet without add
extra trace minerals, CT); NRC recommended level (NRC-L), inorganic trace minerals of NRC-L (IN), organic
trace minerals of NRC-L pattern (ON), NY/T 33-2004 recommended level (NY/T-L), inorganic trace minerals of
NY/T-L pattern (IY), organic trace minerals of NY/T-L pattern (OY); pattern 3, 50% NRC (1994) recommended
level (50% NRC-L), inorganic trace minerals of 50% NRC-L pattern (IHN), organic trace minerals of 50% NRC-L
pattern (OHN), the ratio of mineral elements in blood of laying hens was taken as the supplement proportion of
trace elements, and Zn was supplemented depended on NRC recommended level (TLB), inorganic trace minerals
of TLB pattern (IB), organic trace minerals of TLB pattern (OB).
Animals 2021, 11, 1164 12 of 16

4. Discussion
Compared with Zn, Fe, Cu, Mn, and Se supplemental recommended content of NRC
(1994) to laying hens, mineral concentration of basial diet in current study exhibited that
Zn and Mn concentrations were lower, Fe and Cu content was higher, and Se concentration
was approximate. Zn content was lower while Fe, Cu, Mn, and Se content was higher in
basic diets than in supplemental recommendation of trace minerals with the 50% NRC
pattern. Compared with the supplemental trace minerals of recommendation of NY/T
33-2004, Zn, Cu, Mn, and Se concentrations were deficient while Fe was rich in basial diets.
Zn, Cu, and Se concentrations were lower while Fe and Mn concentrations were higher in
basial diets compared with recommended trace elements amount of the TLB pattern.
The performance of laying hens is mediated by several factors, including the metabolism
of protein, carbohydrate and energy in cells, and tissues and organs which are directly or
indirectly involved in Zn, Fe, Cu, Mn, and Se [27]. The different adding amount and forms
of trace elements have different influences on the growth performance [28]. Dikmen [29]
showed that the addition of amino acid compound Zn and Mn in the diets of laying hens
could significantly improve the egg weight and egg yield. Liu et al. [30] observed Se levels
(0.3 and 0.5 mg/kg) and sources (Yeast selenium and sodium selenite) had no difference
in egg weight and feed conversion, but ADFI was improved by 0.3 mg/kg Se of yeast
selenium, and an interaction of Se sources and levels was in ADFI. Our results exhibited
that different sources of trace elements (Zn, Fe, Cu, Mn, and Se) had a significant influence
on average egg weight of laying hens, and the organic forms of trace elements showed a
higher laying performance. In the present study, OHN treatment had the highest average
egg weight, but had no influence on feed intake, laying rate, egg ratio, and our results
are in accordance with Gheisari et al. [31]. In contrast, in the study of Gheisari et al. [31],
feed intake and feed conversion ratio were significantly affected by forms and dosage of
Mn, Zn, and Cu. Ramos-Vidales et al. [17] reported that the treatments of organic trace
elements (Carbo-Amino-Phosphate-Chelates) had no significant effect on the ratio of feed
to egg, egg yield, and egg weight production performance of laying hens. The reasons for
the difference may attributed to composition of basal diet, the concentration and sources of
trace elements, interaction between minerals, and daily management of layers [32,33].
Trace elements can deposit into the liver, kidney, pancreas, bone, and other internal
organs of the animal body. The physiological changes of the body over a certain period
and the biological efficacy of dietary trace elements usually reflected the content of trace
elements in tissues and organs. [11,34]. As Abedini et al. [12] investigated, Zn content
in the tibia, liver, pancreas, and eggs of laying hens was significantly increased with the
organic Zn (Zn-methionine) and nano Zn treatments, compared with the control group
(basic diet without mineral supplementation). Wang et al. [26] showed that the addition
of 62.5% commercial recommended amounts (Cu, Zn, Fe, Mn, and Se, 10, 80, 30, 80, and
0.3 mg/kg, respectively) of organic trace elements (metal proteinates were sequestered
with enzymatically hydrolyzed soybean protein) promoted the deposition of trace elements
in liver, pancreas, and other tissues. Similar to the previous study, our findings indicated
that the organic mineral treatments could increase the trace elements content in tissues.
This is because inorganic trace elements have poor bioavailability compared with the
organic form. The high bioavailability of mineral chelates prevents chemical actions in
the gastrointestinal tract and retains stability depending on their structure. Compared
with organic mineral, chelates of trace elements pass the intestinal wall more effectively
because of negative charge and amino acid transport of intestinal wall [11,15]. The different
trace element patterns and sources had significant effects on the trace elements content in
diverse tissues. Trace mineral homeostasis is maintained in tissues which may serve as
biomarkers of intake and status [34]. In this study, different levels of Cu, Zn, Fe, Mn, and
Se were found in various tissues. It also observed Zn and Fe content in liver, Cu content
in kidney, Mn content in pancreas and tibia, and Se in tissues had significantly changed
under the influence of different patterns and sources. The Se and Cu content in diets of
NRC-L (IN and ON) and 50% NRC-L (IHN and OHN) patterns, Zn and Fe concentrations
Animals 2021, 11, 1164 13 of 16

in NY/T-L (IY and OY) diets of pattern, and Fe, Cu, and Se concentrations in the diets
of TLB patterns (IB and OB) were higher, while dietary Mn content in the diets of the
TLB pattern was lower. The Zn, Fe, Cu, Mn, and Se concentrations in the basic diet were
the lowest. Corresponding to the supplemental dietary mineral concentration, the TLB
pattern increased tissues Se concentration, while it decreased the Mn content relatively.
However, the highest tissues Cu concentration were found in NY/T-L pattern treatment. In
general, the present study showed that the OHN treatment increased the mineral content
in tissues. Similarly, Qiu et al. [25] mentioned that low doses of trace elements in organic
forms (proteinated trace minerals) improved minerals (Fe, Mn, Zn) deposition. There are
interactions among elements. The previous results revealed that Zn is the first limiting
mineral among Cu, Fe, Mn, and Zn [35]. Cu and Fe both have antagonistic and synergistic
effects [36]. An excess of Cu will hinder the absorption of Fe and Mn in the body. Se and
Mn in the body are negatively correlated [37,38]. This might be the reason that the diets of
TLB pattern with the high Fe and Cu content had no significant influence on the Fe and
Cu deposition in the body. The 50% NRC pattern had the same ratio as the NRC pattern,
which exhibited different performances due to antagonistic and synergistic of minerals
possibly. Moreover, it’s better to reduce the Cu and Se content of TLB diet, while increasing
the Mn content of the TLB diet.
In this study, the Zn, Fe, Cu, Mn, and Se content in the feces of laying hens were
significantly affected by different patterns of trace elements (Zn, Fe, Cu, Mn, and Se). It was
observed that fecal trace elements were reduced with a lower dosage of trace minerals in the
feedstuff. Zn, Fe, Cu, and Se content of hen excreta were lower in the treatments of NRC-L
and 50% NRC-L pattern than other patterns. Fecal Cu concentration was reduced by the
TLB pattern. Previous investigations suggested that a positive correlation exists between
the excretion and feedstuff of trace element content [26,39]. Similarly, the research of Zhu
et al. [40] found that Cu, Zn, Fe, and Mn content in chicken feces were significantly reduced
by low dose trace elements (Cu, Zn, Fe, and Mn are 3, 24, 12, and 20 mg/kg, respectively).
However, there was no significant difference between organic (amino plex) and inorganic
(sulphates) forms. In the current study, Zn, Fe, and Mn concentrations were not affected by
mineral sources while Cu and Se did. Compared with organic mineral, fecal Cu content
was reduced while Se concentration was increased with inorganic form. However, on the
contrary, supplemental organic trace elements of 50% NRC-L pattern (OHN) reduced Cu
content in the excreta of hens, while organic trace elements of TLB (OB) pattern increased
fecal Se content. Among these patterns, Cu concentration was lower in the diets of the 50%
NRC-L pattern. On the other hand, Se concentration was higher in diets with TLB patterns.
It showed a lower dosage of supplemental organic minerals in the scope of supplemented
concentration of trace elements for diets of hens, which may promote absorption of trace
elements while reducing fecal mineral content. If mineral supplementation is out of range,
fecal trace elements will be increased in hens fed diets supplemented with organic mineral
than those fed inorganic minerals. Organic trace elements are supplemented at lower
levels in diets of animals because they have higher bioavailability compared with inorganic
forms [34,41]. Yenice et al. [15] showed that the Cu, Mn, and Zn content in the feces of
laying hens were significantly reduced by organic Cu, Mn, and Zn (methionine chelated
mineral) in lower levels (2.5, 40, and 30 mg/kg) compared with inorganic forms (Cu, sulfate
source; Zn, Mn, oxide sources). This research revealed that the OHN diet significantly
reduced Zn, Cu, and Se concentrations of excretion, and the IHN treatment had the lowest
fecal Fe content. As reported in other studies, the organic minerals reduced fecal Fe, Cu,
Mn, and Zn concentrations of laying hens because of the high bioavailability compared
with inorganic trace elements [15,25,42]. This study showed that the use of lower levels of
organic trace minerals may lead to lower excretion of minerals in feces, helping to reduce
the negative impact of poultry production on the environment.
Animals 2021, 11, 1164 14 of 16

5. Conclusions
This study aimed to provide a basic guide for the effective utilization of minerals on
poultry production. We found that the diets of supplemental trace element of NY/T-L
pattern increased tissue Mn content while trace elements of TLB pattern increased Se
concentration. Organic trace minerals increased laying performance, mineral deposition in
tissues, and reduction of fecal mineral excretion. In addition, OHN improved egg weight,
OY and OHN improved mineral deposition in tissues, and OHN reduced fecal mineral
excretion in laying hens. In conclusion, the organic trace minerals of 50% NRC-L (OHN) in
laying hen diets promoted optimal laying performance, mineral deposition, and reduced
mineral excretion. In poultry production, adding low dosage minerals with organic forms
can improve bird performance and mineral bioavailability within limits, but it should pay
more attention to the interactions among the trace elements.

Author Contributions: Conceptualization, K.Y.; methodology, K.Y. and S.H.; data curation, R.M. and
Y.Q.; writing—original draft preparation, K.Y. and L.X.; writing—review and editing L.Z. and K.M.A.;
supervision, R.F.; project administration, R.F. All authors have read and agreed to the published
version of the manuscript.
Funding: National Key R&D Program of China (2018YFD0500605, 2018YFD0501403) and Hunan
Provincial Innovation Foundation for Postgraduate( CX20190511).
Institutional Review Board Statement: This study was approved by the Committee of Laboratory
Animal Management and Animal Welfare of Hunan Agricultural University (Changsha, China), and
all experimental produces conformed to the Ethical Committee of Hunan Agricultural University
(201905-10, 10 October 2019).
Data Availability Statement: The data presented in this study are available upon request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Abbate, J.M.; Macri, F.; Capparucci, F.; Iaria, C.; Briguglio, G.; Cicero, L.; Salvo, A.; Arfuso, F.; Ieni, A.; Piccione, G.; et al.
Administration of Protein Hydrolysates from Anchovy (Engraulis Encrasicolus) Waste for Twelve Weeks Decreases Metabolic
Dysfunction-Associated Fatty Liver Disease Severity in ApoE(-/-)Mice. Animals 2020, 10, 2303. [CrossRef] [PubMed]
2. Avondo, M.; Pagano, R.I.; Guastella, A.M.; Criscione, A.; Di Gloria, M.; Valenti, B.; Piccione, G.; Pennisi, P. Diet selection and
milk production and composition in Girgentana goats with different alpha(s1)-casein genotype. J. Dairy Res. 2009, 76, 202–209.
[CrossRef] [PubMed]
3. Armato, L.; Gianesella, M.; Morgante, M.; Fiore, E.; Rizzo, M.; Giudice, E.; Piccione, G. Rumen volatile fatty acids x dietary
supplementation with live yeast and yeast cell wall in feedlot beef cattle. Acta Agric. Scand. Sect. A Anim. Sci. 2016, 66, 119–124.
[CrossRef]
4. Monteverde, V.; Congiu, F.; Vazzana, I.; Dara, S.; Di Pietro, S.; Piccione, G. Serum lipid profile modification related to polyunsatu-
rated fatty acid supplementation in thoroughbred horses. J. Appl. Anim. Res. 2017, 45, 615–618. [CrossRef]
5. Cohen, J. Trace Mineral Nutrition Symposium: Exploring recent developments in swine and ruminants. J. Anim. Sci. 2014, 92,
414–415. [CrossRef]
6. Aschner, J.L.; Aschner, M. Nutritional aspects of manganese homeostasis. Mol. Asp. Med. 2005, 26, 353–362. [CrossRef]
7. Choi, S.; Liu, X.; Pan, Z. Zinc deficiency and cellular oxidative stress: Prognostic implications in cardiovascular diseases. Acta
Pharmacol. Sin. 2018, 39, 1120–1132. [CrossRef]
8. Han, O. Molecular mechanism of intestinal iron absorption. Metallomics 2011, 3, 103–109. [CrossRef]
9. Echeverry, H.; Yitbarek, A.; Munyaka, P.; Alizadeh, M.; Cleaver, A.; Camelo-Jaimes, G.; Wang, P.; O, K.; Rodriguez-Lecompte, J.C.
Organic trace mineral supplementation enhances local and systemic innate immune responses and modulates oxidative stress in
broiler chickens. Poult. Sci. 2016, 95, 518–527. [CrossRef]
10. Leeson, S.; Taylorpickard, J.A.; Tucker, L.A. Redefining Mineral Nutrition; Nottingham University Press: Nottingham, UK, 2005; pp.
107–117.
11. Mezes, M.; Erdélyi, M.; Balogh, K. Deposition of Organic Trace Metal Complexes as Feed Additives in Farm Animals. Eur. Chem.
Bull. 2012, 1, 410–423.
12. Abedini, M.; Shariatmadari, F.; Karimi Torshizi, M.A.; Ahmadi, H. Effects of zinc oxide nanoparticles on the egg quality, immune
response, zinc retention, and blood parameters of laying hens in the late phase of production. J. Anim. Physiol. Anim. Nutr. 2018,
102, 736–745. [CrossRef]
Animals 2021, 11, 1164 15 of 16

13. Stefanello, C.; Santos, T.C.; Murakami, A.E.; Martins, E.N.; Carneiro, T.C. Productive performance, eggshell quality, and eggshell
ultrastructure of laying hens fed diets supplemented with organic trace minerals. Poult. Sci. 2014, 93, 104–113. [CrossRef]
14. Aksu, T.; Aksu, M.; Yoruk, M.A.; Karaoglu, M. Effects of organically-complexed minerals on meat quality in chickens. Br. Poult.
Sci. 2011, 52, 558–563. [CrossRef]
15. Yenice, E.; Mızrak, C.; Gültekin, M.; Atik, Z.; Tunca, M. Effects of Organic and Inorganic Forms of Manganese, Zinc, Copper, and
Chromium on Bioavailability of These Minerals and Calcium in Late-Phase Laying Hens. Biol. Trace Elem. Res. 2015, 167, 300–307.
[CrossRef]
16. Lin, X.; Yang, T.; Li, H.; Ji, Y.; Zhao, Y.; He, J. Interactions Between Different Selenium Compounds and Essential Trace Elements
Involved in the Antioxidant System of Laying Hens. Biol. Trace Elem. Res. 2020, 193, 252–260. [CrossRef]
17. Ramos-Vidales, D.; Gómez-Verduzco, G. Organic trace minerals on productive performance, egg quality and immune response in
Bovans White laying hens. J. Anim. Physiol. Anim. Nutr. 2019, 103, 1484–1491. [CrossRef]
18. Matte, J.J.; Girard, C.L.; Guay, F. Intestinal fate of dietary zinc and copper: Postprandial net fluxes of these trace elements in portal
vein of pigs. J. Trace Elem. Med. Biol. 2017, 44, 65–70. [CrossRef]
19. Fang, R.X.; Yang, Y.; Cao, M. Mineral Element Ideal Pattern—From Theory to Practice. Chin. J. Anim. Nutr. 2013, 25, 891–898.
20. King, J.C.; Shames, D.M.; Woodhouse, L.R. Zinc Homeostasis in Humans. J. Nutr. 2000, 130, 1360S. [CrossRef]
21. Boerboom, G.M.; Busink, R.; Smits, G.H.; Hendriks, W.H.; Martin-Tereso, J. Efficacy of L-glutamic acid, N,N-diacetic acid to
improve the dietary trace mineral bioavailability in broilers. J. Anim. Sci. 2020, 98, 9. [CrossRef]
22. Burrow, K.; Young, W.; McConnell, M.; Carne, A.; Barr, D.; Reid, M.; Bekhit, A.E.D. The Effect of Sheep and Cow Milk
Supplementation of a Low Calcium Diet on the Distribution of Macro and Trace Minerals in the Organs of Weanling Rats.
Nutrients 2020, 12, 594. [CrossRef]
23. Dobrzañski, Z.; Korczyñski, M.; Chojnacka, K.; Górecki, H.; Opaliñski, S. Influence of organic forms of copper, manganese and
iron on bioaccumulation of these metals and zinc in laying hens. J. Elementol. 2008, 13, 309–319.
24. Li, J.; Wang, X. Effect of dietary organic versus inorganic selenium in laying hens on the productivity, selenium distribution in
egg and selenium content in blood, liver and kidney. J. Trace Elem. Med. Biol. 2004, 18, 65–68.
25. Qiu, J.; Lu, X.; Ma, L.; Hou, C.; He, J.; Liu, B.; Yu, D.; Lin, G.; Xu, J. Low-dose of organic trace minerals reduced fecal mineral
excretion without compromising performance of laying hens. Asian Australas. J. Anim. Sci. 2020, 33, 588–596. [CrossRef]
[PubMed]
26. Wang, G.; Liu, L.; Wang, Z.; Pei, X.; Tao, W.; Xiao, Z.; Liu, B.; Wang, M.; Lin, G.; Ao, T. Comparison of Inorganic and Organically
Bound Trace Minerals on Tissue Mineral Deposition and Fecal Excretion in Broiler Breeders. Biol. Trace Elem. Res. 2019, 189,
224–232. [CrossRef] [PubMed]
27. Saleh, A.A.; Eltantawy, M.S.; Gawish, E.M.; Younis, H.H.; Amber, K.A.; Abd El-Moneim, A.E.E.; Ebeid, T.A. Impact of Dietary
Organic Mineral Supplementation on Reproductive Performance, Egg Quality Characteristics, Lipid Oxidation, Ovarian Follicular
Development, and Immune Response in Laying Hens Under High Ambient Temperature. Biol. Trace Elem. Res. 2020, 195, 506–514.
[CrossRef] [PubMed]
28. Zapata, N.K.R. Effect of Increasing Levels of Dietary Zinc (Zn), Manganese (Mn), and Copper (Cu) from Organic and Inorganic Sources on
Egg Quality and Egg Zn, Mn, and Cu Content in Laying Hens; Louisiana State University: Baton Rouge, LA, USA, 2016.
29. Dikmen, B.Y. Effects of Supplementary Mineral Amino Acid Chelate (ZnAA-MnAA) on the Laying Performance, Egg Quality
and Some Blood Parameters of Late Laying Period Layer Hens. Kafkas Univ. Vet. Fak. 2015, 21, 155–162.
30. Liu, H.; Yu, Q.; Tang, X.; Fang, C.; Fang, R. Effect of Selenium on Performance, Egg Quality, Egg Selenium Content and Serum
Antioxidant Capacity in Laying Hens. Pak. J. Zool. 2020, 52. [CrossRef]
31. Gheisari, A.A.; Sanei, A.; Samie, A.; Gheisari, M.M.; Toghyani, M. Effect of diets supplemented with different levels of manganese,
zinc, and copper from their organic or inorganic sources on egg production and quality characteristics in laying hens. Biol. Trace
Elem. Res. 2011, 142, 557–571. [CrossRef]
32. Lim, H.S.; Paik, I.K. Effects of Supplementary Mineral Methionine Chelates (Zn, Cu, Mn) on the Performance and Eggshell
Quality of Laying Hens. Asian Australas. J. Anim. 2003, 16, 1804–1808. [CrossRef]
33. Burrell, A.L.; Dozier, W.A., 3rd; Davis, A.J.; Compton, M.M.; Freeman, M.E.; Vendrell, P.F.; Ward, T.L. Responses of broilers to
dietary zinc concentrations and sources in relation to environmental implications. Br. Poult. Sci. 2004, 45, 255–263. [CrossRef]
34. Michael, H. Biomarkers of Trace Mineral Intake and Status. J. Nutr. 2003, 133, 948S–955S.
35. Bao, Y.M.; Choct, M.; Iji, P.A.; Bruerton, K. Trace mineral interactions in broiler chicken diets. Br. Poult. Sci. 2010, 51, 109–117.
[CrossRef]
36. Arredondo, M.; Mendiburo, M.J.; Flores, S.; Singleton, S.T.; Garrick, M.D. Mouse divalent metal transporter 1 is a copper
transporter in HEK293 cells. BioMetals 2014, 27, 115–123. [CrossRef]
37. Mackenzie, B.; Takanaga, H.; Hubert, N.; Rolfs, A.; Hediger, M.A. Functional properties of multiple isoforms of human divalent
metal-ion transporter 1 (DMT1). Biochem. J. 2007, 403, 59–69. [CrossRef]
38. Monedero-Prieto, M.J.; González-Pérez, J.M.; González-Reimers, E.; Hernández-Pérez, O.; Monereo-Muñoz, M.; Galindo-
Martín, L.; Quintero-Platt, G.; Abreu-González, P. Effects of selenium on liver and muscle contents and urinary excretion of zinc,
copper, iron and manganese. Biol. Trace Elem. Res. 2014, 158, 224–229. [CrossRef]
39. Idowu, O.M.O.; Ajuwon, R.O.; Oso, A.O.; Akinloye, O.A. Effects of Zinc Supplementation on Laying Performance, Serum
Chemistry and Zn Residue in Tibia Bone, Liver, Excreta and Egg Shell of Laying Hens. J. Poult. Sci. 2011, 10, 225–230. [CrossRef]
Animals 2021, 11, 1164 16 of 16

40. Zhu, Z.; Yan, L.; Hu, S.; An, S.; Lv, Z.; Wang, Z.; Wu, Y.; Zhu, Y.; Zhao, M.; Gu, C.; et al. Effects of the different levels of dietary
trace elements from organic or inorganic sources on growth performance, carcass traits, meat quality, and faecal mineral excretion
of broilers. Arch. Anim. Nutr. 2019, 73, 324–337. [CrossRef]
41. Suttle, N.F. Mineral nutrition of livestock. Cabi Bookshop 2009, 215, 1–8.
42. Pierce, J.; Ao, T.; Charlton, P.; Tucker, L.A. Organic minerals for broilers and laying hens: Reviewing the status of research so far.
World Poult. Sci. J. 2009, 65, 493. [CrossRef]