Food and Nutrition Sciences, 2013, 4, 1293-1300
Published Online December 2013 (http://www.scirp.org/journal/fns)
http://dx.doi.org/10.4236/fns.2013.412166
Open Access FNS
Bioavailability and Immunity Response in Broiler Breeders
on Organically Complexed Zinc Supplementation
Neeta Soni, Sumanta Kumar Mishra*, Rajakishore Swain, Abinash Das, Biswal Chichilichi,
Kamdev Sethy
Department of Animal Nutrition, College of Veterinary Science and Animal Husbandry, Orissa University of Agriculture and Tech-
nology, Bhubaneswar, India.
Received September 17th, 2013; revised October 17th, 2013; accepted October 24th, 2013
Copyright © 2013 Neeta Soni et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. In accor-
dance of the Creative Commons Attribution License all Copyrights © 2013 are reserved for SCIRP and the owner of the intellectual
property Neeta Soni et al. All Copyright © 2013 are guarded by law and by SCIRP as a guardian.
ABSTRACT
Two hundred and sixty four broiler breeder hens of 32 weeks of age were distributed randomly in four dietary treat-
ments. The dietary treatments were T0: Broiler breeder ration containing 40 ppm zinc (basal 29.8 ppm + 10.2 ppm inor-
ganic zinc), T1: T0 + organic zinc (zinc methionine) @ 20 ppm, T2: T0 + organic zinc @ 40 ppm and T3: T0 + organic
zinc @ 60 ppm. The experiment was continued from 32 to 48 weeks of age. At 48 weeks, the weight of lymphoid or-
gans, zinc levels in organs and immunity response were determined. The faecal zinc level was determined at monthly
interval. The weight lymphoid organs of different treatment groups (both organic and inorganic zinc fed groups) of the
broiler breeders did not differ significantly (P > 0.05). The cellular immune response of breeder birds to PHA-P was
significantly (P < 0.05) higher in group T3 than the rest of treated groups. The antibody titre to SRBC differed among
the treated groups. The zinc content of serum of broiler breeders of all the groups did not differ significantly (P > 0.05)
in all the periods of study. Zinc content in liver and tibia of broiler breeders in different dietary treatments of zinc dif-
fered significantly (P < 0.05) with higher levels were obtained on increasing zinc concentration in the diet. The zinc
level in the spleen and kidney of the broiler breeders in different dietary treatments did not differ significantly (P >
0.05). The average zinc content in the faeces of broiler breeder during 35 to 43 week of age did not differ significantly
(P > 0.05) among the treated groups. At 48 weeks of age, zinc content of the faeces of T3 was found to be significantly
(P < 0.05) higher than the rest of treated groups. Similarly, during the overall experimental period analysis, it was found
that zinc levels in the faeces of T2 and T3 were significantly (P < 0.05) higher than T1 and T0.
Keywords: Organic Zinc; Immunity; Bioavailability; Broiler Breeders
1. Introduction
Zinc impacts immunity in poultry [1]. Zinc deficiency
has been shown to decrease cellular immunity [2], thy-
mus [3] and spleen development [4]. Zinc is important
for proper disease resistance and its deficiency has re-
sulted in bacteremia [5], parasitic infections [6] and al-
teration in high-density lipoprotein cholesterol [7]. Zn-
methionine provides a source of zinc with greater bio-
logical availability than zinc from inorganic sources. Gill
(1997) [8] reported that chelated (organic) minerals are
more biologically available in animal digestive system
than inorganic minerals and that perhaps resulted in less
mineral excretion and pollution of the environment. To-
day, large scale commercial livestock production system
has given rise to many environmental concerns, since
excess mineral concentrations in the manure can lead to
mineral depositions that exceed crop nutrient require-
ment [9]. According to [10] and the opinion of Scientific
Committee for Animal Nutrition on use of zinc in feed-
ing stuffs, a clear indication of biological activity of zinc
is: the content of this element in liver, melethionine ac-
tivity, accumulation of zinc in the bones and levels of
zinc in blood serum are the method of estimation of
availability of this element in live animals. Considering
the higher bioavailability of organic zinc [11] the ex-
periment was planned to study the effect of organic zinc
*Corresponding author.

Bioavailability and Immunity Response in Broiler Breeders on Organically Complexed Zinc Supplementation 1294
supplementation in broiler breeder birds on bioavailabil-
ity and immune response.
2. Materials and Methods
2.1. Selection and Management of Experimental
Birds
Female broiler breeder birds of synthetic dam line,
maintained under All India Coordinated Research Net-
work Project on “Poultry Breeding” were selected for the
study. A total of two hundred and sixty four broiler
breeder hens of 32 weeks of age were distributed ran-
domly in four dietary treatments with three replicate per
treatment. The hens were selected on the basis of their
body weight and egg production. The hens were main-
tained in individual layer cages. Experimental diets were
offered @ 150 g/bird/day throughout the experimental
period. The hens were provided 24 hours free access to
clean drinking water. The experiment continued for a
period of sixteen weeks i.e. 32 to 48 weeks of age of the
hens. A basal diet was prepared to meet the nutrient re-
quirement of broiler breeders [12]. The ingredient com-
position and proximate composition of the basal diet is
given in Table 1. The basal diet was analyzed for proxi-
mate composition as per [13].
2.2. Dietary Treatments
The dietary treatments of the experiment were: T0:
Broiler breeder ration containing 40 ppm zinc (basal 29.8
ppm + 10.2 ppm inorganic zinc supplementation in form
of ZnSO4), T1: T0 + Supplementation of organic zinc
(zinc methionine) @ 20 ppm in the broiler breeder ration,
T2: T0 + Supplementation of organic zinc @ 40 ppm in
the broiler breeder ration and T3: T0 + Supplementation
of organic zinc @ 60 ppm in the broiler breeder ration.
2.3. Collection and Processing of Experimental
Samples
At the end of the experimental period (48 weeks of age),
three birds were randomly chosen from each replicate
and slaughtered for collection of liver, spleen and kidney.
The birds were kept off feed overnight before bleeding
and only water was provided. The live weight of the
birds were recorded as pre slaughter weight. The broiler
birds were bled by modified Kosher’s method [14]. Liver,
spleen, bursa of fabricus and kidney were weighed in a
top pan electric balance. Both the tibia bones were re-
moved from the slaughter birds. The tibia bones were
pressure cooked in deionised water for 15 minutes,
cleaned off all tissues and dried in an oven for 72 hours
until constant weight. Then the tibia bones were ex-
tracted in petroleum ether for 72 hours to remove fat and
dried for 24 hours in 105˚C. The collected liver, kidney
and spleen samples were oven dried at 100˚C for 24
hours and finely grounded. The zinc content in the liver,
kidney, bursa and spleen samples were determined by
Table 1. Ingredient and proximate composition of breeder basal diet.
Ingredients Percentage Proximate composition Percentage on DM basis
Maize 60.00 Moisture 9.17
Soyabean meal 19.50 Crude protein 16.08
De oiled rice bran 12.00 Ether extract 4.20
Mineral mixture# 3.00 Crude fibre 4.82
Oyster shell meal 5.00 Total ash 10.61
Common salt 0.30 Acid insoluble ash 2.54
L-lysine 0.03 Nitrogen free extract* 64.39
DL-methionine 0.05 Calcium 2.97
Feed additives used (g/kg of diet) Available phosphorus 0.42
Biocholine 0.50 Metabolisable energy* (Kcal/kg) 2751.25
Biobantox 0.50 Lysine* 0.84
Layvit 0.50 Methionine* 0.34
Livoline 0.25 Zinc 29.80 ppm
E-sel-powder 0.10
K-zyme 0.50
#
Mineral mixture—without zinc, *Calculated value.
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Bioavailability and Immunity Response in Broiler Breeders on Organically Complexed Zinc Supplementation 1295
digesting of 0.5 g samples at 120˚C using 5ml concentrate
HNO3 for 1 hour using KEL plus digestion system. The
digested samples were cooled and further digested with
30% H2O2 at 200˚C. The process continued until the
content appeared clear and colourless. At 35, 39, 43 and
48 weeks of experiment, 5 ml of blood was drawn from
the brachial vein of 12 birds per treatment (4 per replicate).
The serum was obtained by centrifugation of coagulated
blood at 700 × g for 5 min. The serum were frozen at
−20˚C until needed for analysis.
2.4. Measure of Immunity
2.4.1. Cellular Immunity
At 48 weeks of age, three birds from each replicate in
each dietary treatment were injected intra-dermally in the
right wattle with 100 micro gram of Phytohaemagluti-
nin-P (PHP-P) in 0.1 ml of normal saline to measure the
cellular immune response by Cutaneous Basophillc Hy-
per Sensitivity (CBH) test [15]. The thickness of wattle
was measured using digital calliper before inoculation
and 24 h post inoculation and CBH response was calcu-
lated using the formula:
Post-injection skin thickness
CBH response100
Pre-injection thickness

2.4.2. Humoral Immunity
The measure of humoral immunity was carried out as per
the method described by [16]. Sheep red blood cells
(SRBC) were used as test antigens to quantitatively ana-
lyse specific antibody response as measure of humoral
immunity. At 48 weeks of age, three birds from each
replicate in each dietary treatment were immunized in-
travenously via a wing vein with 0.07 ml packed RBC
mixed with 0.93 ml physiological saline (0.9% NaCl) for
measure primary response. Seven days following the
antigen challenge, blood samples were collected and se-
rum samples were used to measure humoral immunity.
Antibody production to SRBC was measured using micro
titration hemmagglutination technique with micro titer
plate U shape of 96 wells (8 rows × 12 column) accord-
ing [17,18]. All SRBC antibody titers were expressed as
log2 of the reciprocal of the highest serum dilution caus-
ing agglutination of SRBC.
2.5. Collection of Faeces
The faeces of the experimental broiler breeder birds were
collected at 35, 39, 43 and 48 weeks of age. Three birds
form each replicate of each group were taken for indi-
vidual collection of faeces. A polythene sheet was at-
tached under the cages of the birds and light was turn off
for 1 hour. The faeces were homogeneously mixed repli-
cate wise and representative samples of the faeces were
collected in moisture cup and were oven dried at 105˚C
for 24 h. For determination of zinc content in faeces, 2 gm
faecal samples were taken in a digestion tube and to it 12
ml of tri acid mixture (7 ml HNO3, 3 ml H2SO4 and 2 ml
Perchloric acid) were added and digested at 200˚C.
2.6. Statistical Analysis
Data retrieved from the experiment was subjected to sta-
tistical analysis wherever required. The statistical analy-
sis of the data was done according to [19].
3. Results and Discussion
3.1. Immunity
The effect of various levels of organic zinc on immune
response of broiler breeders is presented in Table 2. The
cellular immune response of breeder birds to PHA-P was
significantly higher in higher zinc fed groups with T3
recorded highest response. The influence on primary
antibody titer to SRBC was significantly (P < 0.05) lower
in T0 group (inorganic zinc) than that of other three or-
ganic zinc fed groups. [20] reported that immune re-
sponse to PHA-P injection was enhanced when dietary
zinc supplementation was solely from ZnAA. They re-
ported that PHA-P (mm) was 0.97 in ZnSO4 group and
1.12 in ZnAA group and they observed significant dif-
ference between these two groups. [21] reported that zinc
as zinc-methionine supplementation (100 mg/kg zinc to a
basal diet containing 36.8 mg/kg zinc) had better effect
on primary immune response to SRBC relative to control.
Researcher has demonstrated supplementing broiler
breeder hen diets with zinc-methionine rather than inor-
ganic zinc sources increased cellular immune response of
progeny to PHA-P [22]. [23] concluded that supplemen-
tation of 20 ppm zinc significantly improved immune
response and impact was more prominent with ZnAA
(organic zinc) compared to ZnSO4. The broiler breeder
hens provided diets supplemented with zinc from zinc
amino acid which might have increased thymulin activity;
therefore, enhancing immune response through increased
maturation of T-lymphocyte and activation of B lym-
Table 2. Effect on immune response of broiler breeders in different dietary treatments of zinc.
Parameters T0 T
1 T 2 T 3 P value
CBH 165.89c ± 10.98 192.66c ± 6.023 243.96b ± 7.76 291.48a ± 12.49 <0.01
SRBC 4.33b ± 0.40 5.88a ± 0.30 6.22a ± 0.52 6.44a ± 0.68 0.02
Means with different superscripts in a row differ significantly (P < 0.05).
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Bioavailability and Immunity Response in Broiler Breeders on Organically Complexed Zinc Supplementation 1296
phocytes by T-helper cells [20]. Moreover, zinc has been
shown to directly influence the immune system [24].
This element is required for normal immune function
[25].
In contrast to this finding, [26] reported that zinc sup-
plementation (80 mg/kg zinc from ZnSO4) had lower
effect on primary immune response to SRBC relative to
control. Similarly, [1] reported that on feeding of zinc
both from inorganic and organic sources did not have any
significant effect on either cellular or humoral immunity.
Also, [27] reported that supplementation of zinc me-
thionine partially or completely in place of inorganic
sources had no much influence on primary antibody titer
to SRBC
The weight of lymphoid organs (spleen and bursa) and
liver of female broiler breeder of different dietary treat-
ment has been presented in Table 3. The Weight lym-
phoid organs of different treatment groups (both organic
and inorganic zinc fed groups) of the broiler breeders did
not differ significantly (P > 0.05). [27] reported that die-
tary addition of organic zinc in place of inorganic source
in broiler diet increased the relative weight of thymus as
a proportion of live body weight but no significant effect
on the weight of spleen and bursa of fabricius. [28] in
their experiments in broiler reported that all the immune
organs (bursa of fabricius, spleen and thymus) were sig-
nificantly affected by the level of zinc-glycine in the diet
at 21 day of age but at 42 day, no significant improve-
ment in these organs were observed except for thymus.
In contrast to this finding, [29] observed significant
higher weight of spleen in zinc-proteanate group than the
control group. Supplementation of zinc did not improve
the weight of the lymphoid organs as more nutrients be-
ing reprationed to develop body weight and production
and immune system need a small amount of nutrient in
relation to what is needed for growth and production [30].
The weight of the liver (percentage of body weight) in-
creased from T0 to T3 with significant lower liver weight
was recorded in T0 group. This might be due to numeri-
cal higher body weight of birds in T3 than that of T0
group.
3.2. Bioavailability
Measuring the deposition or storage of minerals in se-
lected tissues (tibia or plasma zinc, liver copper and tibia
manganese) is most common output in trace mineral
relative bioavailability experiments [31]. Tissue mineral
concentrations are indicators of body storage and mineral
status and have been used as biomarker in requirement
and bioavailability study [32]. According to some au-
thors [33] and the opinion of Scientific Committee for
Animal Nutrition on use of zinc of feeding stuffs, a clear
indication of biological activity of zinc are: the content of
this element in liver, melethionine activity, accumulation
of zinc in the bones and levels of zinc in blood serum are
the method of estimation of availability of this element in
live animals.
The zinc content in the serum of broiler breeder in dif-
ferent dietary treatment during the experimental period is
presented in Table 4. The zinc content of serum of
broiler breeders of all the groups did not differed signifi-
cantly in all the periods of study. No increasing or de-
creasing trend was observed even on feeding higher le-
Table 3. Weight lymphoid organs and liver (percentage of live weight) of broiler breeders in different dietary treatments of
zinc.
Immune organ T0 T
1 T 2 T 3 P value
Spleen 1.73 ± 0.15 1.82 ± 0.12 1.98 ± 0.13 2.15 ± 0.17 0.21
Liver 2.42c ± 0.14 2.82cb ± 0.18 2.97b ± 0.22 3.60a ± 0.19 <0.01
Bursa 0.14 ± 0.04 0.12 ± 0.02 0.14 ± 0.03 0.17 ± 0.04 0.72
Means with different superscripts in a row differ significantly (P < 0.05).
Table 4. Zinc content (ppm) of serum of broiler breeders in different dietary treatments of zinc.
Weeks T0 T
1 T 2 T 3 P value
35 week 11.06 ± 0.45 10.58 ± 0.31 11.29 ± 0.25 11.27 ± 0.30 0.42
39 weeks 11.15 ± 0.36 11.26 ± 0.51 11.81 ± 0.52 11.66 ± 0.40 0.70
43 weeks 12.04 ± 0.47 11.26 ± 0.35 11.76 ± 0.54 12.24 ± 0.50 0.50
48 week 11.92 ± 0.38 12.10 ± 0.34 11.81 ± 0.45 12.58 ± 0.44 0.56
Average 11.54 ± 0.21 11.30 ± 0.20 11.67 ± 0.22 11.94 ± 0.21 0.21
M
eans with different superscripts in a row differ significantly (P < 0.05).
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Bioavailability and Immunity Response in Broiler Breeders on Organically Complexed Zinc Supplementation 1297
vels of zinc in organic form or replacement of inorganic
zinc with organic zinc in the diet of broiler breeders. [34]
in their experiment on broilers reported that zinc content
of serum of both organic and inorganic fed groups did
not differ significantly which is in agreement with our
finding. In contrast to this, [29] reported significantly
higher level of serum zinc in zinc-proteanate group than
control. Similarly, [35] reported that broiler chicken re-
ceived diet containing zinc-methionine had increased
level serum zinc compared to the content of this element
in birds received zinc sulphate. The non-significant level
of serum zinc in different periods of study might be due
to mineral homeostasis which is precisely maintained in
the body and is predominantly achieved by balancing
tissue storage and excretion [36].
Zinc content in liver, tibia, kidney and spleen of
broiler breeders in different dietary treatments is pre-
sented in Table 5. Zinc content in liver and tibia of
broiler breeders in different dietary treatments of zinc
differed significantly (P < 0.05) with higher levels ob-
tained on increasing zinc concentration in the diet. The
increased level of zinc in tibia as observed in the present
study is in agreement with the findings of [29,34,35,
37,38]. In contrast to this finding, [39] reported dietary
treatment by replacing 50% of the inorganic Zn, Cu, Mn
in the control ration with chelated minerals at 14 and 52
day, there was no significant difference between the con-
trol and treatment group for tibia zinc content. This
might be due to lower duration of feeding. [26] reported
that feeding broiler chicken with feed containing either
zinc-methionine or ZnO had no significant effect on the
presence of microelements in the bone. Moreover, based
on tibia zinc content, [11] reported that zinc from zinc
methionine, is more bio-available than zinc from ZnSO4
or ZnO. Similarly, [9] reported the impact of different die-
tary concentrations of minerals on their deposition rate in
tibia. Their results showed that with feeding different
diets containing same concentration of zinc, manganese
and copper, birds were able to deposit organic form of
minerals more efficient than their inorganic form.
[37] reported that the increased dietary addition of zinc
from both organic and inorganic sources increased the
liver zinc content. [40] reported that on supplementation
of 100 ppm zn-methionine chelate over the NRC level in
control group exhibited higher zinc content in the liver
(104.53 ppm) of zinc-methionine supplemented group
than control (99.50 ppm) in spite of any significant dif-
ference between the two groups. In support to our finding,
[26] reported that feeding broiler chicken with feed con-
taining either zinc-methionine or ZnO had no significant
effect on the presence of microelements in the liver and
pancreas at the same level of feeding but they observed
significant difference in zinc levels of those organs at
different levels of feeding.
The zinc level in the spleen and kidney of the broiler
breeders in different dietary treatments did not differ
significantly. No such reported work was available on
this study. Probably, the weight of immune organ have
significance in birds on feeding different source and level
of zinc rather than the level of zinc in those organs.
3.3. Faecal Excretion
The zinc content in the faeces of broiler breeder in dif-
ferent dietary treatment during the experimental period
has been presented in Table 6. The zinc content in the
faeces (excretion) of broiler breeders in different dietary
treatments of zinc did not differ significantly among all
the treatments during the periods of study viz. 32 - 35
weeks, 36 - 39 weeks and 40 - 43 weeks of age. Even the
zinc level was higher in T3 and T2 but the zinc excretion
was not significant which implied better bioavailability
of organic zinc. [41] reported that one of the benefits of
feeding chelated minerals to broilers was to improve
mineral bioavailability, allowing reduction in dietary
mineral supplementation and thus reduced excretion.
Organically complexed trace minerals provide alternative
pathways for absorption, thus leading to a reduction in
the excretion of minerals [42]. But during 44 - 48 weeks
of age, the zinc excretion in faeces was significantly
higher in T3 than that of T2, T1 and T0 groups. The over-
all zinc excretion in faeces of T3 did not differ signifi-
cantly with T2 which implied higher retention of zinc in
T3 as it contained 20 ppm which is more organic zinc
Table 5. Zinc content (ppm) in organs and chicks of broiler breeders in different dietary treatments of zinc.
Organs T0 T
1 T 2 T 3 P value
Liver 75.76c ± 2.28 83.37bc ± 3.29 90.92b ± 3.27 106.12a ± 3.065 <0.01
Kidney 91.65 ± 1.184 89.85 ± 0.82 90.51 ± 0.89 92.73 ± 0.74 0.15
Spleen 85.62 ± 1.43 82.33 ± 1.68 84.89 ± 2.51 85.69 ± 1.72 0.56
Tibia 203.0c ± 9.54 320.81b ± 16.71 375.22a ± 12.98 381.84a ± 13.99 <0.01
chick 55.25d ± 1.51 59.38c ± 1.37 65.6b ± 1.43 71.81a ± 0.97 <0.01
Means with different superscripts in a row differ significantly (P < 0.05).
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Bioavailability and Immunity Response in Broiler Breeders on Organically Complexed Zinc Supplementation 1298
Table 6. Zinc content (ppm) in the faeces (excretion) of broiler breeders in different dietary treatments of zinc.
Weeks T0 T
1 T 2 T 3 P value
35 Week 119.24 ± 2.98 118.55 ± 2.43 122.88 ± 3.079 127.81 ± 2.24 0.08
39 weeks 124.74 ± 2.57 126.33 ± 2.76 133.58 ± 2.002 129.36 ± 2.99 0.10
43 weeks 119.81 ± 2.42 124.88 ± 2.47 126.85 ± 2.41 125.80 ± 1.45 0.14
48 week 120.01c ± 2.16 122.25c ± 1.91 128.47b ± 1.37 137.79a ± 1.32 <0.01
Average 120.95b ± 1.27 123.008b ± 1.26 127.95a ± 1.27 130.19a ± 1.27 <0.01
Means with different superscripts in a row differ significantly (P < 0.05).
than that of T2. This implied that in the 20 ppm organic
zinc group where the diet contained 60 ppm of total zinc
as compared to 40 ppm in control group, the bioavail-
ability was significantly improved as the faecal loss in
both groups were comparable. [43] fed broilers with
basal diet contained 30 mg/ kg zinc in control and added
inorganic zinc at the levels of 20, 40, 80 mg/kg. Similarly
organic zinc was added to other groups at the levels of 20,
40, 80 mg/kg. They reported that neither in ZnSO4 or
organic alone supplemented groups, there was a reduc-
tion in zinc excretion. But they observed significantly
lower zinc excretion in both organic and inorganic sup-
plemented group. They also reported that the cumulative
zinc excretion of organic zinc fed groups were signifi-
cantly lower than inorganic zinc fed groups. [44] re-
ported that the total amount of inorganic minerals in a
broiler premix could be totally replaced by 20% organic
minerals without affecting growth performance and at the
same time reducing environmental pollution. [45] re-
ported that organic minerals that are chelated to small
peptides have much greater bioavailability through in-
creased selective transport of peptide at gut level. En-
hanced bioavailability of mineral source can potentially
reduce the amount of a mineral that is added to a diet to
meet nutritional requirement, leading to reduced amount
of mineral excreted by birds [46].
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