Eects of feed supplementation with various zinc sources on mineral concentration
and selected antioxidant indices in tissues and plasma of broiler chickens
Oksana Ivanišinová, Ľubomíra Grešáková, Miroslav Ryzner, Vladimíra Oceľová,
Klaudia Čobanová
Slovak Academy of Sciences, Institute of Animal Physiology, Košice, Slovak Republic
Received November 6, 2015
Accepted August 31,2016
Abstract
The aim of this study was to compare the eects of organic dietary zinc (Zn) sources and
zinc sulphate on mineral deposition, activity of total superoxide dismutase (SOD) and copper/
zinc SOD in tissues of broiler chickens. The performance indicators and lipid peroxidation by
measuring the contents of thiobarbituric acid reactive substances (TBARS) in tissues and plasma
were also evaluated. Broilers were assigned to 4 treatment groups, each replicated × 6, with
9 birds per replicate. The control group was fed conventional basal diet (BD); the three other
groups received identical BD supplemented with 120 mg Zn/kg in the form of zinc sulphate, zinc
chelate of glycine hydrate (Zn-Gly), and zinc proteinate (Zn-Pro), respectively. After 5 weeks of
dietary treatment, feed supplementation with Zn sulphate resulted in signicantly higher average
daily weight gain and nal body weight, as well as improved feed conversion ratio compared to
the Zn-Gly group. Intake of Zn-Pro signicantly increased SOD activity (P < 0.05) in erythrocytes
and lipid peroxidation (P < 0.01) in plasma. Activities of total SOD and Cu/Zn SOD in liver and
kidney were not aected by Zn supplementation. Addition of Zn supplements to broiler diets did
not inuence concentrations of zinc, manganese and copper in plasma, liver, kidney or breast
muscle, with the exception of Zn deposition in the liver being signicantly higher (P < 0.05) in
the Zn-Pro supplemented group. Results of our study show that organic zinc sources have eects
comparable to inorganic zinc sulphate in broilers fed diets containing a higher Zn content.
Zinc chelate, tissue deposition, superoxide dismutase, lipid oxidation, poultry
Appropriate mineral feed supplementation is required for many physiological functions
and can improve growth performance and health of chickens for fattening. Zinc is
known to be an essential microelement inuencing immune functions, gene expression,
cell proliferation, growth, and fertility. Being an essential part of more than 300 known
enzymes, zinc directly participates in metabolic pathways and is one of major components
of cell defence against oxidative stress as an integral part of cytosolic Cu/Zn superoxide
dismutase (Cu/Zn SOD) (Zago and Oteiza 2001; McDowell 2003).
Recently, organic sources of Zn have been introduced into animal nutrition due to
their potentially higher bioavailability compared to the traditionally used inorganic
forms. However, study results regarding the bioavailability of organic chelates remain
controversial. Several studies show that organic Zn sources are more available to animals
(Yenice et al. 2015; Sahraei et al. 2013; Yu et al. 2010; Rupić et al. 1997), contribute
to elevation of Cu/Zn SOD activity in chicken liver (Ma et al. 2010) and improve growth
performance of broilers (Feng et al. 2010). In contrast, other results indicate that organic
chelates are comparable to standard Zn sulphate (Cao et al. 2000). Use of organic
sources of trace elements (i.e. chelated amino acids, proteinates) in animal nutrition may
prevent minerals from creating indigestible complexes with some dietary compounds, and
reciprocal mineral antagonisms in the intestine which could reduce their absorption rate
(Swiatkiewicz et al. 2014).
The current study was conducted to compare the eects of organic Zn sources with
ACTA VET. BRNO 2016, 85: 285-291; doi:10.2754/avb201685030285
Address for correspondence:
RNDr. Klaudia Čobanová, PhD.
Institute of Animal Physiology
Slovak Academy of Sciences
Šoltésovej 4-6, 040 01 Košice, Slovak Republic
Phone: +421 557 922 965
E-mail: boldik@saske.sk
http://actavet.vfu.cz/
inorganic Zn sulphate supplemented to conventional broiler diet for 5 weeks on the
deposition of Zn, Cu and Mn as well as the activities of total and Cu/Zn SOD in tissues.
Moreover, the extent of lipid peroxidation in plasma, liver and kidney measured as TBARS
level was determined in broilers fed with a higher content of dietary Zn, and performance
indicators were monitored during the whole experiment as well.
Materials and Methods
The experiment was carried out on a total of 216 broiler chickens (ROSS 308) obtained from a commercial
hatchery (Budmerice, Slovakia). One-day-old chicks of both sexes were weighed and assigned to 4 treatment
groups with each containing 6 replicates. Based on the body weight (BW), nine chicks were allotted to each
replicate cage resulting in a similar mean initial BW per replicate. Birds of all groups received identical basal
diet (BD) formulated to meet the requirements for broiler chickens (NRC 1994). All chicks were fed the starter
diet from 1 to 19 days of age followed by the grower diet from 20 to 35 days of age. The BD applied in our
286
Table 1. Ingredients and chemical composition of basal diet fed to broiler chickens.
Item Starter diet (Days 1 to 19) Grower diet (Days 20 to 35)
Ingredient (%)
Maize, ground (9% CP) 41.96 42.13
Soybean meal, extracted (48% CP) 28.00 30.00
Wheat, ground (12% CP) 22.00 22.00
Maize gluten (67% CP) 3.00 -
Limestone 1.50 1.40
Monocalcium phosphate 1.30 1.30
Sunower oil 1.00 2.00
Premix
a
0.30 0.30
Feed salt 0.40 0.40
Lysine 0.35 0.25
DL-Methionine 0.17 0.20
Allzyme SSF-poultry
b
0.02 0.02
Nutrient composition
Dry matter (g/kg) 884.0 889.3
Crude protein (g/kg) 198.0 187.0
Crude bre (g/kg) 31.8 30.3
Zinc (mg/kg) 84.4 64.6
Manganese (mg/kg) 135.4 134.7
Copper (mg/kg) 22.7 21.5
Calcium (g/kg) 9.8 9.5
Phosphorus (g/kg) 4.7 4.7
Lysine (g/kg) 13.7 13.1
Methionine (g/kg) 5.4 5.3
Methionine + cysteine (g/kg) 9.3 9.1
Metabolizable energy (MJ/kg) 12.6 12.7
Dry matter, crude protein, crude bre, zinc, manganese and copper are analysed data.
Experimental diets were supplemented with 120 mg Zn/kg in the form of zinc sulphate, zinc chelate of glycin
hydrate (Zn-Gly) or zinc proteinate (Zn-Pro).
a
The vitamin/mineral premix provided per kg of complete diet: vitamin A 13 500 IU, vitamin D
3
5 000 IU,
vitamin K 4.2 mg, vitamin E 60.0 mg, thiamine 5.4 mg, riboavin 7.5 mg, pyridoxine
4.8 mg, cyanocobalamin
0.03 mg, vitamin C 75.0 mg, niacin 54.0 mg, pantothenic acid 16.5 mg, biotin 0.2 mg, folic acid 1.8 mg, choline
90.0 mg, betaine 195.0 mg, I 1.2 mg, Zn 55.0 mg, Mn 115.0 mg, Cu 16.5 mg, Se 0.3 mg, Co 0.5 mg, Fe 81.6 mg.
b
Allzyme
®
SSF Enzyme Complex, Altech, Inc., Nicholasville, Kentucky USA
experiment was a conventional diet commonly used in the nutrition of broiler chickens, and its composition and
nutrient values are shown in Table 1. No supplemental zinc was added to BD for the control group, whereas the
three other groups were fed identical BD supplemented with equal amounts of 120 mg Zn/kg complete feed from
either Zn sulphate (ZnSO
4
·H
2
O, reagent grade, Sigma-Aldrich, USA), or Zn chelate of glycine hydrate (Zn-
Gly; Glycinoplex-Zn 26%, Phytobiotics Futterzusatzstoe GmbH, Eltville, Germany) or Zn proteinate (Zn-Pro;
Bioplex
®
-Zn 15%, Alltech Inc., Nicholasville, KY, USA), respectively. Feed and drinking water were oered ad
libitum.
All birds were kept under similar conditions of management throughout the experiment in accordance with
the guidelines for the care of fattening broiler chickens (Aviagen’s Manual, 2014). The lighting schedule was
maintained at 23 h of light and 1 h of darkness during the rst 7 days, followed by 6 h of a continuous period of
darkness. Relative humidity varied from 60 to 70% and the temperature regimen was adjusted to the particular
age of the chickens according to breeding recommendations.
All procedures were in accordance with the European Community guidelines (Directive 2010/63/EU)
on the protection of animals used for scientic purposes and the experimental protocol was approved by the
Ethics Committee of the Institute of Animal Physiology SASci and by the State Veterinary and Food Oce
(Ro-4160/13-221).
Weekly measurements of body weight and feed intake for each replicate of the treatments were taken to
calculate the average daily feed intake, average daily weight gain and feed conversion ratio (FCR). On day 35, all
birds were weighed individually and the nal average body weight was calculated for each treatment. Mortality
within each replicate was monitored daily throughout the experiment.
At the end of the experiment, two randomly selected chickens from each replicate (a total of 12 birds from each
treatment) were slaughtered by electrical stunning followed by decapitation. Blood was collected into heparinised
tubes and centrifuged for plasma samples at 1,180 × g for 15 min. Tissue samples (5–8 grams) were collected
from identical areas of the liver, kidney and breast muscle, immediately ushed with ice-cold saline and quickly
frozen. All tissue and plasma samples were stored at –70 °C until analysis.
Dry matter (DM) of diets and tissues was determined using the standard method of drying samples at 105 °C
for 48 h. Feed was also analyzed for crude protein and crude bre using standard procedures (AOAC 2005;
methods 976.05, 973.18).
Superoxide dismutase (SOD; EC 1.15.1.1) activity in erythrocytes and the content of haemoglobin (Hb) were
analyzed in fresh blood using a commercial kit from Randox (Randox Laboratories, UK). Liver and kidney
samples for analysis of total SOD and Cu/Zn SOD activities were homogenized in ice-cold buer (pH 7.4)
containing 10 mM Tris and 0.25 M sucrose. Subsequently, homogenates were centrifuged at 10 000 × g for 30 min
at 4 °C and supernatants were used for analysis of total SOD activity using the spectrophotometric method based
on the autoxidation of pyrogallol (Marklund and Marklund 1974). For each sample, a parallel determination
was performed in the presence of 1 mM KCN and the activity of Cu/Zn SOD was calculated as the activity
inhibited by KCN. Protein concentration in the sample supernatants was measured using the spectrophotometric
method published by Bradford (1976). The TBARS levels in plasma, liver and kidney were measured using the
modied uorometric method in accordance with Jo and Ahn (1998).
The contents of Zn, Mn, and Cu in samples were determined using a double-beam atomic absorption
spectrometer (AAS-7000 series, Shimadzu, Kyoto, Japan). All samples except plasma were dried (at 105 °C
for 48 h) and then ground for subsequent wet digestion in concentrated nitric acid and hydrogen peroxide
(3:1) in a microwave digestion system (MWS-4, Berghof, Germany). Mineral deposition in tissue samples, Zn
and Cu contents in plasma and the mineral content in diets were determined using ame atomic absorption
spectrophotometry (FAAS). The AAS equipped with a graphite furnace atomizer GFA-7000 and deuterium lamp
for background correction was used to measure the concentration of Mn in plasma.
Statistical analysis was done by one-way analysis of variance (ANOVA) with post hoc Tukey’s multiple
comparison test using GraphPad Prism Software (Version 2.02, 2008, USA). Dierences between groups were
considered signicant at P < 0.05. Values in tables are given as means ± standard errors of the mean (SEM).
Results
The eects on growth performance of broiler chickens assessed from 0 to 35 days after
supplementing the basal diet with inorganic and organic zinc are summarized in Table 2.
The average daily feed intake was not inuenced by supplemental Zn. Intake of dietary Zn
sulphate resulted in improvement of the feed conversion ratio (P < 0.001) and increased
nal body weight (P < 0.01) compared to birds fed the diet enriched with Zn-Gly. The
average daily weight gain was signicantly higher in the Zn sulphate group than in the
unsupplemented control (P < 0.05) and Zn-Gly group (P < 0.001).
Antioxidant indicators of broilers supplemented with Zn from dierent sources are given
in Table 3. Activity of SOD measured in erythrocytes was signicantly elevated in the
Zn-Pro group compared to control birds (P < 0.05) and the Zn sulphate group (P < 0.05).
287
Neither the SOD activity nor the Cu/Zn SOD activity in liver and kidney were aected
by dietary Zn supplementation. Feed supplementation with Zn from Zn-Pro signicantly
increased the TBARS levels in plasma (P < 0.01) compared to all other groups. There was
a tendency for higher TBARS values in liver and kidney of chickens receiving Zn-Pro,
however, no signicant dierences between treatments were observed for this indicator.
The eects of various sources of dietary Zn on mineral concentration in plasma and tissues
are presented in Table 4. Addition of Zn-Pro to the diet signicantly increased Zn deposition
in liver (P < 0.05) compared to control birds fed only BD, but Zn concentration in plasma,
kidney and breast muscle was not aected by Zn supplementation. No dierences in
concentration of Mn and Cu in plasma and tissues were found between dietary treatments.
Discussion
After 35 days of feeding experimental diets, increased average daily weight gain was observed
in broilers from the Zn sulphate group, with an increase in the nal body weight compared to
288
Table 2. Eect of dierent zinc sources on growing performance of broilers during the feeding period of 5 weeks
(from 1 to 35 days of age).
Indicator BD Zn-sulphate Zn-Gly Zn-Pro
Feed intake
(g/bird/day)
79.60 ± 1.06 81.26 ± 0.97 80.03 ± 1.14 80.81 ± 0.93
Weight gain (g/bird/day) 48.63 ± 0.47
a
51.47 ± 0.87
b
47.12 ± 0.78
a
49.24 ± 0.32
ab
Feed conversion ratio (g/g) 1.64 ± 0.01
ab
1.58 ± 0.03
a
1.70 ± 0.01
b
1.64 ± 0.02
ab
Initial body weight
(g/bird)
46.47 ± 0.32 46.47 ± 0.41 46.57 ± 0.50 47.58 ± 0.72
Final body weight
(g/bird)
1733.7 ± 16.5
ab
1832.0 ± 30.3
b
1683.7 ± 27.5
a
1781.4 ± 36.1
ab
Mortality (%) 0.00 1.85 0.00 1.85
Results are presented as mean ± SEM,
a,b
-
values in a row with dierent letters in superscripts are signicantly
dierent (P < 0.05)
Table 3. Eect of dierent zinc sources on activity of SOD in erythrocytes and tissues, activity of Cu/Zn SOD in
tissues and TBARS concentration in plasma and tissues of 35-day-old broiler chickens.
Indicator BD Zn-sulphate Zn-Gly Zn-Pro
SOD
Erythrocytes (µkat/g Hb) 9.17 ± 1.04
a
9.16 ± 1.54
a
12.75 ± 0.67
ab
14.00 ± 1.30
b
Liver (µkat/mg protein) 0.83 ± 0.06 0.91 ± 0.06 0.88 ± 0.13 0.87 ± 0.04
Kidney (µkat/mg protein) 0.48 ± 0.02 0.48 ± 0.01 0.49 ± 0.01 0.50 ± 0.01
Cu/Zn SOD
Liver (µkat/mg protein) 0.67 ± 0.04 0.71 ± 0.06 0.71 ± 0.10 0.67 ± 0.03
Kidney (µkat/mg protein) 0.32 ± 0.02 0.32 ± 0.01 0.33 ± 0.01 0.33 ± 0.02
TBARS
Plasma (nmol/ml) 0.27 ± 0.02
a
0.25 ± 0.01
a
0.28 ± 0.01
a
0.37 ± 0.02
b
Liver (nmol/g protein) 151.5 ± 16.8 131.0 ±11.7 135.2 ± 12.3 163.8 ± 7.8
Kidney (nmol/g protein) 74.65 ± 2.19 71.84 ± 6.55 76.06 ± 7.32 89.74 ± 7.03
SOD – superoxide dismutase; Hb – haemoglobin; TBARS – thiobarbituric acid reactive substances; Results are presented
as mean ± SEM;
a,b
-
values in a row with dierent letters in superscripts are signicantly dierent (P < 0.05)
the Zn-Gly group. However, there was no dierence in feed intake between treatments, which
explains the improvement of feed conversion ratio in birds receiving inorganic Zn. These
ndings show that feed supplementation with Zn sulphate helps to improve the performance
of broiler chickens, although signicant dierence compared to unsupplemented control birds
was observed only in the average daily weight gain. Sahraei et al. (2013) recorded higher
weight gain in broilers fed similar dietary Zn concentrations from sulphate compared to control
birds from day 22 to 28, which is partially consistent with our results. They also reported
improvement of the feed conversion ratio in the birds supplemented with Zn sulphate and Zn-
Pro compared to the control group, and no signicant dierence in the nal body weight between
treatments, which was not conrmed in our experiment. Sunder et al. (2008) did not nd any
signicant eect of Zn sulphate supplemented to the BD at graded doses up to 320 mg Zn/kg for
3 weeks on broiler performance.
Several mechanisms by which Zn can exert its antioxidant action in a biological system
have been described (Powell 2000), but one of the most important functions of Zn is
related to its antioxidative eect mediated by Cu/Zn SOD. Elevated activity of SOD
in erythrocytes was observed in broilers fed dietary Zn-Pro compared to control birds
and the Zn sulphate group, and was associated with higher TBARS values in plasma
(Table 3). This nding could be explained by the ability of SOD to protect cells against
oxidative damage (Fridovich 1995) and may in this case be a reaction of antioxidant
defence mechanisms to a higher rate of lipid peroxidation. Zago and Oteiza (2001)
reported that Zn as an important component of the antioxidant defence network prevents
membrane damage from oxidation and can also partially inhibit formation of free radicals
and other potentially reactive substances.
No response to Zn supplementation in activities of total and Cu/Zn SOD in the liver
and kidney was observed in our experiment, which is in agreement with the results of
Liao et al. (2013). Nevertheless, other authors have reported an increase in Cu/Zn SOD
289
Table 4. Concentration of zinc, manganese and copper in plasma and tissues of broilers supplemented with zinc
from various sources.
Indicators BD Zn-sulphate Zn-Gly Zn-Pro
Plasma
Zn (mg/l) 1.88 ± 0.07 1.96 ± 0.12 1.85 ± 0.10 1.99 ± 0.12
Mn (µg/l) 4.03 ± 0.30 3.76 ± 0.32 4.12 ± 0.80 5.19 ± 0.87
Cu (mg/l) 0.18 ± 0.02 0.21 ± 0.02 0.19 ± 0.01 0.23 ± 0.01
Liver (mg/kg DM)
Zn 90.20 ± 2.09
a
99.74 ± 3.37
ab
96.67 ± 2.03
ab
101.00 ± 2.89
b
Mn 10.57 ± 0.77 10.65 ± 0.67 9.93 ± 0.50 11.14 ± 0.46
Cu 12.28 ± 0.32 13.47 ± 0.63 12.98 ± 0.65 12.22 ± 0.72
Kidney (mg/kg DM)
Zn 96.92 ± 0.71 96.21 ± 2.25 98.18 ± 1.07 96.40 ± 1.04
Mn 10.39 ± 0.22 10.30 ± 0.30 10.82 ± 0.25 10.25 ± 0.42
Cu 11.91 ± 0.13 12.49 ± 0.15 12.45 ± 0.25 12.48 ± 0.26
Breast muscle (mg/kg DM)
Zn 19.58 ± 0.46 19.45 ± 0.31 18.96 ± 0.30 20.65 ± 0.73
Mn 0.88 ± 0.09 0.93 ± 0.10 1.06 ± 0.07 0.93 ± 0.10
Cu 1.75 ± 0.23 1.81 ± 0.29 1.80 ± 0.25 1.83 ± 0.20
DM – dry matter; results are presented as mean ± SEM;
a,b
-
values in a row with dierent letters in superscripts
are signicantly dierent (P < 0.05)