Livestock Science 191 (2016) 8085

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Livestock Science
journal homepage: www.elsevier.com/locate/livsci

Effects of supplementation of manganese with or without phytase on

growth performance, carcass traits, muscle and tibia composition, and
immunity in broiler chickens
A. Ghosh, G.P. Mandal, A. Roy, A.K. Patra n
Department of Animal Nutrition, West Bengal University of Animal and Fishery Sciences, Belgachia, Kolkata, India

art ic l e i nf o

a b s t r a c t

Article history:
Received 17 December 2015
Received in revised form
21 June 2016
Accepted 19 July 2016

An experiment was conducted to study the effect of Mn and phytase supplementation on performance,
carcass traits, and immunity in broiler chickens. A total of 350 one-day-old unsexed broiler chickens were
randomly allotted to 7 dietary treatments (5 replicate pens of 10 chicks per pen) in a 13  2 factorial
arrangement with 3 concentrations of supplemental Mn and with or without phytase addition. The
control diet represented a basal diet with no supplemental Mn and phytase. The basal diet was supplemented with Mn at 50, 75, and 100 mg/kg diet and microbial phytase was added at a dose of 500 FTU/
kg. Average daily gain, feed intake, and feed conversion ratio were similar among the treatments. During
the starter, grower, and nisher periods, 5, 7.5, and 17.5% of the broiler chickens, respectively, developed
leg abnormalities in the control diet, which were not observed in the treatment groups. The serum
concentrations of glucose, total cholesterol, Ca, and P were not affected by supplemental Mn and phytase
or their interaction. Antibody titer against Newcastle virus disease vaccine on 16 d and 32 d of the
feeding trial (10 d after vaccination) were increased (Po 0.05) by Mn or phytase supplementation, but
the antibody titer was not affected by the Mn and phytase interaction. Carcass traits such as hot carcass
weight, eviscerated carcass weight, dressing percentage, breast, leg, frame, and giblet were not altered by
Mn or phytase or both supplementation. Abdominal fat content was decreased (Po 0.05) by high concentrations of Mn supplementation (75 and 100 mg/kg), but not by phytase addition. Chemical composition (moisture, protein, ash, and fat) in thigh and breast muscle, and total ash, Ca, and P concentrations in tibia at 42 d were similar among the dietary treatments. Thus, supplementation of Mn at
75100 mg/kg to a basal corn-soybean diet could be benecial for improving immunity and decreasing
fat deposition, and phytase supplementation could also improve immunity measures in broiler chickens.
& 2016 Elsevier B.V. All rights reserved.

Keywords:
Manganese
Phytate
Broiler chicken
Growth
Carcass traits
Immunity

1. Introduction
Mn is an essential trace mineral and Mn requirements in avian
species are higher than that of mammals. This is because chicks
absorb Mn less efciently than do mammals (Halpin et al., 1986;
Turk et al., 1982). The absorption efcacy of Mn from inorganic
Mn-sulphate was estimated to be 1.71% for corn-soybean diets to
2.40% for pure casein-dextrose diets fed to chicks (Halpin et al.,
1986), and the bioavailability of Mn is highly variable depending
upon the Mn sources (Li et al., 2004). The NRC (1994) recommends
60 mg Mn/kg diet for broiler chickens. This requirement is based
on research conducted at least 20 years ago with broiler chickens
of genetic characteristics that differ from existing genetic traits
today. The evaluation of the dietary Mn requirement is based on
n

Corresponding author.
E-mail address: patra_amlan@yahoo.com (A.K. Patra).

http://dx.doi.org/10.1016/j.livsci.2016.07.014
1871-1413/& 2016 Elsevier B.V. All rights reserved.

some specic performance indices such as average daily gain

(ADG) and leg abnormalities (Lu et al., 2007). However, other indices for Mn requirements may be more representative, such as
immune function. Mn is required for Mn-superoxide dismutase
(MnSOD) enzyme function, which has been shown to contribute to
the integrity of macrophases and heterophils, virulence to pathogenic microorganisms, and antibody production (Cook-Mills and
Fraker, 1993; Cox et al., 2003; Lynch and Kuramitsu, 2000). Reevaluation of the Mn requirement in broiler chickens using more
specic and sensitive indices such as MnSOD activity has been
advocated (Li et al., 2004; Lu et al., 2007; Suttle, 2010). Based on
ADG and incidences of leg abnormality along with heart MnSOD
activity, and tissue Mn concentrations, Luo et al. (1991) recommended 120 mg Mn/kg corn-soybean meal basal diets in
broiler chickens.
Poultry industry faces environmental challenges such excess
nutrient and mineral excretion, and gas emission. Commercial
practice of feeding Mn above the recommended requirement (e.g.,

A. Ghosh et al. / Livestock Science 191 (2016) 8085

NRC, 1994) results in excretion of high level of this element in

excreta (Inal et al., 2001; Wang et al., 2008), which may cause
environmental pollution (Powers and Angel, 2008). In addition,
supplementation of high concentration of a mineral affects the
bioavalibility of other minerals masking the advantages of the
mineral supplementation (Collins and Moran, 1999). Reports show
that high concentrations of Mn in diets may depress Cu concentrations in tibia and liver (Gajula et al., 2011) and decrease
immunity by unbalancing trace mineral concentrations in lymphoid organs of broiler chickens (Liu et al., 2012). The strategy for
reducing Mn excretion is to supplement microbial phytase to diets,
which hydrolyses phytic acid to inositol and improved the absorption of Mn (Mohanna and Nya, 1999). Indeed, phytase supplementation increased tibia Mn concentration of broiler chickens
fed on a corn-soybean based diet (Singh et al., 2003; Sunder et al.,
2006).
Another major concern for the modern day poultry industry as
well as consumers of chicken meat is excessive fat deposition in
chicken meat (Choct et al., 2000; Fouad and El-Senousey, 2014). In
response to concern of health-conscious consumers, current research aims at reducing excessive fat deposition in broiler chickens
without affecting growth performance by dietary means. Supplementation of Mn decreased abdominal fat deposition in broilers
(Lu et al., 2007; Sands and Smith, 1999) by decreasing the activity
of lipoprotein lipase of abdominal fat (Mersmann, 1998).
Efcacy of phytase supplementation in maize-soybean based
diets on P and Ca utilization has been studied thoroughly. There
are several studies showing signicant interaction effects between
phytase and other minerals such as Ca, P, and Zn on intake, growth
and mineral content in tibia and plasma (e.g., Paiva et al., 2014;
Qian et al., 1996; Sebastian et al., 1996). However, the interaction
effects of phytase and Mn on growth performance, carcass traits,
and immunity in broiler chickens are limited. It was hypothesized
that phytase and Mn addition in a basal diet could affect some
response variables associated with Mn in broiler chickens. Therefore, the objective of this study was to determine the effects of
graded doses of Mn supplementation in a diet based on maizesoybean with or without addition of phytase on growth performance, immune response, carcass traits, and meat composition in
broiler chickens.

2. Materials and methods

2.1. Broiler chicken management and diet
The experiment was conducted following the ethical guidelines
of animal care and use of this institute. A total of 350 one-day-old
unsexed broiler chickens (Vencobb 400 breed, Venkys, Pune, Maharashtra) were purchased from local hatchery and brought to the
poultry shed 6 h post-hatch. Immediately after arrival, they were
weighed and randomly allotted to 17 treatments (5 replicate pens
of 10 chicks per pen). Broiler chickens were maintained in a deep
litter system made up of husks and sawdust. The treatment diets
were offered from d 2. Continuous illumination was given for rst
15 d. A dark period of 1 and 3 h during night was provided from d
1625 and d 2635, respectively, to reduce the activity of broiler
chickens. The broiler chickens were vaccinated against Newcastle
(ND) disease at d 6 and 22, and infectious bursal disease at d 12.
The broiler chickens were allowed ad libitum access to the
experimental diets in mash form and water containing no detectable amount of Mn. The starter (d 010), grower (d 1022) and
nisher (d 2242) diet (Table 1) were formulated using maize
grain and soyabean meal. Seven dietary treatments were designed
in a 3  2 factorial arrangement with 3 concentrations of supplemental Mn and with or without phytase addition plus one control

81

Table 1
Ingredients and chemical composition of diets.1
Item

Starter
(0 to 10 d)

Ingredient (%)
Maize
57.3
Soyabean meal
38.0
Rice bran oil
0.8
Dicalcium phosphate
2.0
Limestone powder
1.0
Common salt
0.22
L-Lys.HCl
0.08
DL-Met
0.14
Sodium bicarbonate
0.20
Toxin binder
0.05
Bacitracin methylene
0.04
disalicylate
Cocciodistate
0.05
Trace mineralsa
0.03
Vitaminsb
0.05
Choline chloride
0.04
Energy and nutrient composition
Metabolizable energy
2.91
(Mcal/kg)c
22.4
Crude protein (%)d
Ether extract (%)d
3.23
Crude ber (%)d
3.75
c
Lys (%)
1.31
c
Met (%)
0.51
d
Ca (%)
1.06
Available P (%)c
0.50
Mn (mg/kg)d
26.4

Grower
(10 to 22 d)

Finisher
(22 to 42 d)

61.1
33.0
2.0
1.9
1.0
0.22
0.11
0.16
0.20
0.05
0.04

62.8
31.0
2.5
1.8
1.0
0.22
0.08
0.13
0.20
0.05
0.04

0.05
0.05
0.05
0.04

0.05
0.05
0.05
0.04

3.02

3.06

20.6
4.34
3.41
1.20
0.50
0.96
0.48
24.7

20.0
5.03
3.33
1.12
0.46
0.92
0.45
23.3

1
For phytase diets, a small increase of 0.4 to 0.5% of maize grain with a decrease of 0.4 to 0.5% of dicalcium phosphate, limestone powder, soybean meal, and
rice bran oil was made with similar calculated concentrations of crude protein and
metabolizable energy. These slight changes in ingredient composition would result
in minor changes in other nutrient composition.
a
Supplied per kilogram of diet: Cu, 4.0 mg; Zn, 75 mg; Fe, 20 mg; and I, 0.35
mg.
b
Supplied per kilogram of diet: vitamin A, 13,000 IU; vitamin D3, 2,500 IU;
vitamin E, 30 IU; vitamin K, 3 mg; vitamin B1, 4 mg; vitamin B2, 9 mg; vitamin B6, 4
mg; vitamin B12, 20 g; biotin, 0.15 mg; foilc acid, 2 mg; niacin, 45 mg; and pantothenic acid, 15 mg.
c
Calculated value.
d
Analyzed value.

diet. The control diet represented a corn-soybean meal-based

basal diet with no supplemental Mn or phytase. The control diet
contained approximately 25 mg Mn/kg diet, and was supplemented with Mn at 50, 75, and 100 mg/kg diet using MnSO4, and a
commercial microbial phytase was added at a dose of 500 FTU/kg.
When phytase was added to the diets, the amounts of incorporation of limestone powder and dicalcium phosphate were adjusted
accounting 0.16 g available P and 0.2 total Ca per 100 FTU phytase/
kg (Kornegay, 2001). Thus, the phytase diet contained 0.1% less Ca
and 0.08% less available P compared with non-phytase diet. For
phytase diets, a small increase of 0.40.5% of maize grain with a
decrease of 0.40.5% of dicalcium phosphate, limestone powder,
soybean meal, and rice bran oil was made with similar calculated
concentrations of crude protein and metabolizable energy in diets.
These slight changes in ingredient composition would result in
insignicant differences in other nutrient composition.
2.2. Performance traits and incidence of leg abnormalities
Initial body weights of the broiler chickens were recorded at
the start, and replicate-wise body weight and feed intake was
recorded at weekly intervals. Average daily gain (ADG), average
daily feed intake (ADFI), and feed conversion ratio (FCR) were
calculated from body weights and feed intake data. Leg

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A. Ghosh et al. / Livestock Science 191 (2016) 8085

abnormalities were recorded on every broiler chicken by examining the tibiotarsal joint for deformity (Luo et al., 2007) at the
end of each week. A broiler chicken that walked normally with no
visual detectable abnormality at the tibiotarsal joint considered as
normal gait, and any deviations from the normal gait were considered as leg abnormalities.
2.3. Carcass traits and collection of tissue and blood samples
After termination of experiment on d 42, 4 broiler chickens
from each replicate (2 males and 2 females) were selected according to the average weight within the pen after 12 h of fasting,
weighed individually, euthanized by cutting the carotid arteries,
and then were immediately bled. Head and feet were removed
from these broiler chickens, and the eviscerated carcass was
weighed to determine the dressing percentage. Breast, legs, giblets, muscle, and abdominal fat were removed and weighed to
determine the content of these parts and chemical composition of
muscles. Breast and thigh muscles, and legs were stored individually in plastic bags at  20 C for determination of tibial ash
and chemical composition of muscles. On d 42, 2.0 mL blood was
collected from 4 broiler chickens per replicate after 12 h of fasting,
from wing vein in the morning. To obtain serum sample 1.5 mL
blood was placed into clean and dry test tubes and kept in slanting
position to clot before being centrifuged at 3000  g for 15 min at
room temperature . Another 0.5 mL of blood samples were taken
in another clean and dry test tubes containing anticoagulant (heparin), and was centrifuged at 3000  g at room temperature to
separate the plasma from the red blood cells. Both serum and
plasma sample were collected into small plastic vials, and stored at
20 C for further analysis.
2.4. Humoral immune response
All the experimental chickens were vaccinated against F and
Lasota virus strains of Newcastle disease (ND) on 6 and 22 d of age,
respectively. Blood samples were collected from 3 chickens per
replicate (total 15 chickens per treatment) at 16 and 32 d of age
(10 d after each vaccination). Following collection, blood samples
were allowed to clot, centrifuged at 3000  g at room temperature
to obtain serum samples. Antibody titer against ND virus was
detected by ND virus antibody test (Bangalore Genei, Bengaluru,
India).

using General Linear Model (GLM) procedure of SPSS (1997). The

model included the main effects of Mn, phytase and their interaction and the two-way 3  2 treatment structure was nested
within plus 1 factor following the suggestion of Piepho et al.
(2006). Statistical analysis on ADG, ADFI, and FCR was performed
using pen as the experimental unit whereas individual broiler
chicken data were used for other variables. For the analyses of
ADG, ADFI, and FCR, period and treatment period interaction were
included in the model, but were dropped in subsequent analyses
as the interaction between period and treatment was not signicant. The differences among the mean were tested by Tukey's
test and P o0.05 was considered to be statistically signicant.

3. Results
Body weights, ADG, ADFI, and FCR were similar among the
dietary treatments in spite of the addition of Mn in presence or
absence of phytase (Table 2). The results suggest that Mn concentration in control diet was adequate for optimum body weight
gain up to 42 d of age in chickens. During starter, grower, and
nisher periods, 5%, 7.5%, and 17.5% of the broiler chickens, respectively, developed leg abnormalities in control group, which
were not observed in treatment groups.
The serum concentrations of glucose, total cholesterol, Ca, and
P were not affected by supplemental Mn or phytase level or their
interaction (Table 3). Humoral immune response measured by
determining the antibody titer against ND vaccine on 16 and 32 d
of the trial (10 d after vaccination) revealed that both Mn and
phytase supplementation increased (Po0.05) the antibody titer.
Carcass traits such as hot carcass weight, eviscerated carcass
weight, dressing percentage, breast, leg, frame, and giblet were not
altered by Mn or phytase or the interaction (Table 4). Abdominal
fat concentrations were decreased (Po0.05) by high doses of Mn
supplementation (75 and 100 mg/kg); however this variable did
not change with phytase supplementation. Chemical composition
(moisture, protein, ash, and fat) of thigh and breast muscle of
chickens at 42 d was similar among the dietary treatments
(Table 5). There was also no signicant effect of these treatments
on concentrations of total ash, Ca, and P concentration in tibia.

2.5. Chemical analysis

Samples of feeds were analyzed for crude protein (method
988.05), Ca (method 927.02), ether extract (method 2003.05), and
P (method 965.17) following AOAC (2002) methods. Concentration
of Mn (method 968.08; AOAC, 2002) in the feeds was estimated in
an air-acetylene ame on atomic absorption spectrophotometer
(Perkin Elmer A Analyst 100, Wellesley, MA, USA). Composite
muscle samples from breast and leg in equal proportion were
analyzed for moisture (method 934.01), protein (method 928.08),
and fat (method 960.39) of AOAC (2002). Ash (method 923.03;
AOAC, 2002) in muscle and tibia was analyzed by igniting in quartz
crucible at 550 C for 6 h, and concentrations of Ca and P in tibia
were determined using AOAC (2002) methods (927.02 and 965.17,
respectively). Concentration of plasma glucose and serum total
cholesterol, Ca, and P was determined by using diagnostic kit
(Span Diagonostics Ltd., Surat, India).
2.6. Statistical analysis
The data were analyzed as a 1 (control) 3 (Mn concentration)  2 (with or without phytase) factorial arrangement

Table 2
Effects of Mn and phytase supplementation on average daily gain (ADG), average daily
feed intake (ADFI), and feed conversion ratio (FCR) in broiler chickens (0 to 42 d of age).
Item

ADG (g)

Phytase
(U/kg):
Mn (mg/
kg)

d 0 to 14
d 14 to 28
d 28 to 42
d 0 to 42
ADFI (g) d 0 to 14
d 14 to 28
d 28 to 42
d 0 to 42
FCR
d 14
d 28
d 42

SEM Effecta

500

50

75

100

50

75

100

23.8
51.1
64.5
46.5
30.1
86.9
136
84.4
1.27
1.56
1.81

23.4
50.1
65.2
46.2
30.0
84.6
133
82.6
1.28
1.56
1.79

23.4
50.1
65.2
46.2
30.0
84.6
133
82.6
1.28
1.56
1.79

23.6
50.9
64.2
46.2
31.1
86.4
133
83.6
1.32
1.58
1.81

23.7
52.6
62.2
46.2
30.4
88.9
132
83.9
1.28
1.56
1.82

24.6
53.2
62.4
46.7
31.4
87.1
136
84.8
1.27
1.52
1.81

24.6
49.3
63.5
45.8
30.9
86.5
135
84.0
1.25
1.59
1.84

0.2
0.4
0.5
0.3
0.2
0.3
0.4
0.3
0.01
0.01
0.01

NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS

SEM, standard error of mean; NS, no effect (P4 0.05) of M and phytase, and their
interaction.
a

The effect of Mn, phytase, and their interaction.

A. Ghosh et al. / Livestock Science 191 (2016) 8085

83

Table 3
Effects of Mn and phytase supplementation on tibial mineral content, blood glucose, cholesterol, Ca, and P concentrations, and antibody titer in broiler chickens.
Item

SEM

Effecta

47.4
35.8
18.1

1.2
0.4
0.3

NS
NS
NS

251
165
10.4
5.92

256
179
10.3
5.62

25
21
0.7
0.38

NS
NS
NS
NS

7,616
7,669

7,626
7,731

40
31

** *

Phytase (U/kg):

Mn (mg/kg)

50

75

100

50

75

100

46.6
36.3
18.4

48.5
36.8
18.6

48.3
36.5
18.8

48.2
36.7
18.4

47.6
35.5
17.5

47.6
36.2
17.8

246
176
11.0
5.48

249
172
11.5
6.03

243
181
10.9
6.26

245
167
11.3
5.83

241
161
11.1
5.77

7,128
7,343

7,320
7,470

7,518
7,614

7,589
7,709

7,422
7,638

Tibia
Ash (%)
Ca (%)
P (%)
Blood
Glucose (mg/dL)
Total cholesterol (mg/dL)
Ca (mg/dL)
P (mg/dL)
Antibody titre
d 16
d 32

500

,
,

** *

SEM, standard error of mean; NS, no effect (P40.05) of Mn and phytase, and their interaction.
a

The effect of Mn, phytase, and their interaction.

Mn effect, P o 0.01.
Phytase effect, P o 0.05.

Table 4
Effects of Mn and phytase supplementation on carcass characteristics in broiler
chickens at 42 d of age.
SEM Effecta

Item Phytase
(U/kg):
Mn
(mg/kg)

Hot carcass (g)

Eviscerated
carcass (g)
Breast (g)
Legs (g)
Frame (g)
Giblet (g)
Breast (%)
Legs (%)
Frame (%)
Abdominal fat
(%)
Dressing %

1,713 1,673 1,718 1,707 1,690 1,714 1,669 13

1,313 1,324 1,343 1,305 1,304 1,312 1,303 10

NS
NS

413
434
273
127
32.3
34.0
21.4
2.48

472
424
289
119
35.6
32.1
21.8
1.92

459
430
300
129
34.2
32.0
22.4
1.51

468
425
280
119
35.9
32.6
21.5
1.07

455
427
280
132
34.9
32.7
21.5
1.80

458
437
280
124
34.9
33.4
21.4
1.49

457
418
289
117
35.2
32.2
22.1
1.15

6
4
5
2
0.4
0.3
0.3
0.09

NS
NS
NS
NS
NS
NS
NS

66.8

68.6

68.3

67.7

67.8

67.1

68.2

0.5

NS

500
50

75

100

50

75

100

**

SEM, standard error of mean; NS, no effect (P 40.05) of Mn and phytase, and their
interaction.
a

The effect of Mn, phytase, and their interaction.

Mn effect, P o 0.01.

Table 5
Effects of Mn and phytase supplementation on chemical composition (% as fresh
basis) of breast and thigh muscles of broiler chickens at 42 d of age.
Item Phytase (U/kg): 0
Mn (mg/kg)
Breast muscle
Moisture
Protein
Ash
Fat
Thigh muscle
Moisture
Protein
Ash
Fat

SEM Effecta

500

50

75

100

50

75

100

72.0
21.1
4.34
2.32

72.5
20.9
4.95
2.06

72.4
21.1
4.79
1.93

72.1
20.9
4.73
1.93

72.7
21.4
4.58
2.15

71.8
21.3
4.88
1.96

71.9
21.0
4.69
1.97

1.7
0.5
0.33
0.25

NS
NS
NS
NS

72.6
17.5
3.45
4.72

72.8
17.6
3.48
4.52

72.2
18.0
3.67
4.43

73.8
18.2
3.82
4.39

72.3
17.8
3.81
4.49

73.5
18.2
3.95
4.37

73.2
18.3
3.96
4.37

1.7
0.5
0.23
0.30

NS
NS
NS
NS

SEM, standard error of mean; NS, no effect (P 40.05) of Mn and phytase, and their
interaction.
a

The effect of Mn, phytase, and their interaction.

4. Discussion
Mn has high afnity for phytic acid, and forms an insoluble
complex in weak acidic to neutral pH of the gastrointestinal tract
of chickens reducing the availability of Mn (Mohanna and Nya,
1999). Thus, it was hypothesized that phytase addition in the diet
could affect some response variables linked to Mn in broiler
chickens. There is limited information to study the interaction
effect between phytase and Mn supplementation. In a study of
Attia et al. (2010), phytase supplementation reduced Mn requirement in dual purpose laying hens. In the present study, Mn and
phytase interactions were not signicant for any response variables, which were in agreement with the ndings of Mohanna and
Nya (1999) with respect to growth performance, feed intake, and
tibia ash content. The interaction effect was not noted probably
due to high concentration of Mn in the basal diet and low amount
of Mn-phytate complexes in these diets.
4.1. Growth performance and incidence of leg abnormalities
The growth performance of the broiler chickens was not affected by Mn supplementation. The NRC (1994) recommendations
for poultry birds are a safe allowance for growth performance and
leg abnormalities to cope with poorly available sources and extreme variations in Ca and P intake (Underwood and Suttle, 2001).
Similar to our ndings, Berta et al. (2004) did not observe any
signicant effect of Mn supplementation (0, 30, 60, and 240 mg/
kg) on ADG, feed intake, and FCR in broiler chickens fed on a basal
corn-soyabean meal diet containing Mn at 23 mg/kg, which indicated that the basal diet contained adequate level of Mn for
optimum growth of broiler chickens. Similarly, Mn supplementation (100500 mg/kg) to a basal diet containing Mn concentrations
at 1923 mg/kg did not improve ADG, feed intake or FCR in broiler
chickens (Lu et al., 2006). Results of growth performance in the
current study are consistent with other studies (Lu et al., 2007;
Sunder et al., 2006), indicating that the basal corn-soyabean meal
diet containing 2326 mg Mn/kg was adequate for optimum
growth performance. In contrary to our ndings, a previous study
reported improvement in growth performance of broiler chickens
due to supplementation of Mn (Luo et al., 1991).
Skeletal deformities generally occur when Mn intake is inadequate (60 mg/kg feed; Sauveur, 1984). The need for Mn for
normal skeletal development appears to be related to its role in

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A. Ghosh et al. / Livestock Science 191 (2016) 8085

proteoglycan biosynthesis for the development of organic matrix

in bone (Sauveur, 1984). Mn deciency is one of the major factors
inducing leg abnormalities in broiler chickens (Lu et al., 2007). In
the studies of Lu et al. (2006, 2007), incidences of leg abnormalities decreased or tended to decrease in broiler chickens due to
supplementation of Mn at 100500 mg/kg as MnSO4 to a basal
corn-soybean diet containing 1923 mg Mn/kg diet, but was not
completely prevented. These studies suggested that a greater
dietary Mn level might be required to minimize leg abnormalities
(Sunder et al., 2006). In the study of Sunder et al. (2006) also,
supplementation of Mn at 100 mg/kg DM to a basal diet containing 37 mg/kg DM alleviated leg abnormality scores and increased
Mn concentrations in bone ash, though total ash content in tibia
was similar. Structural components of cartilage might have affected by deciency of Mn in the diets (Sunder et al., 2006). Our
results are in partial agreement with the previous reports. Unlike
in this study, broiler chickens in the previous experiments (Lu
et al., 2006, 2007) were housed in cages, which may be one of the
contributory factors for development of leg abnormalities in Mn
supplemented groups given that factors other than Mn might be
involved in leg abnormality development (Luo et al., 1991).
4.2. Tibia ash, Ca, and P concentration
The Mn and phytase supplementation did not affect ash, Ca,
and P concentrations in tibia. The Ca and P concentrations in Mn
supplemented groups without phytase were as per recommendation of NRC (1994); however, Ca and P concentrations
in phytase supplemented groups were lowered by 0.1 and 0.08%,
respectively. Therefore, the present investigation revealed that
addition of phytase enzyme improved bioavailability of Ca and P.
Addition of microbial phytase in poultry diet is a tool to improve
the availability of P for plant derived feed ingredients. More sensitive and reliable indicator for determining the phytate P utilization is the bone ash concentration (Koch and Mahan, 1985).
Improvement of tibia ash percentage by addition of phytase in
corn-soyabean meal diets containing low available P was reported
in previous studies (Cabahug et al., 1999; Picn-Rubio et al., 2009).
Perney et al. (1993) reported improvement of tibia and toe ash
percentage due to the addition of dietary phytase. This suggests an
improvement in bone mineralization as a result of increased P and
Ca utilization, which was caused by the liberation of inorganic P
and Ca molecules by the phytase enzyme. Similarly, Cortes et al.
(2007) reported that the addition of 750 FTU/kg of phytase could
allow for reducing the inorganic P concentration in broiler starter
diets by 0.1% while improving the growth performances. The results are consistent with previous ndings and indicate that addition of phytase could allow for reduced available P level in diets
of chickens without affecting the tibia ash, Ca, and P concentration.
4.3. Blood Ca, P, glucose, and cholesterol concentrations
The serum Ca and P concentrations were not affected by supplemental Mn and phytase or their interaction. These results
suggest that amount of Ca and P absorption and excretion was not
affected by any diets. Serum glucose and total cholesterol concentrations across different experimental diets did not differ owing to Mn and phytase supplementation. These ndings were in
corroboration with the ndings of Sands and Smith (2002) who
observed that plasma glucose and total cholesterol concentrations
were not affected by Mn supplementation (60 or 120 mg Mn/kg)
to a diet fed to chickens. However, Luo et al. (1991) reported that
plasma glucose concentration was increased and serum total
cholesterol concentration was unaltered because of Mn supplementation in the diet of chickens. Huff et al. (1998) observed also
no difference in serum cholesterol concentration in serum of

chickens supplemented with phytase.

4.4. Carcass characteristics and chemical composition of meat
In agreement with our results, previous reports indicated that
Mn supplementation did not affect the dressing percentage or
percentage of breast muscle or leg muscle (Collins and Moran,
1999; Lu et al., 2006, 2007). Sands and Smith (1999) observed that
addition of 240 mg/kg of Mn as Mn proteinate to the diets of
chickens decreased abdominal fat deposition compared to unsupplemented control (containing 19 mg Mn/kg in the starter diet
and 28 mg/kg in the grower diet). Lu et al. (2006) supplemented
maize-soybean meal diets (containing 22.7 mg Mn/kg in the
starter diet and 18.9 mg of Mn/kg in grower diet) with 100, 200,
300, 400, and 500 mg/kg of Mn, and observed that abdominal fat
content decreased quadratically as dietary Mn concentration increased. Similarly, Lu et al. (2007) also observed a decreased in
abdominal fat percentage in chickens fed supplemented Mn at 100
or 200 mg/kg. Decreased abdominal fat percentage in this study
owing to Mn addition agrees with the aforementioned studies,
indicating the Mn plays an important role in reducing abdominal
fat percentage of broiler chickens. Supplementation of Mn may
decrease abdominal fat deposition by decreasing activity of lipoprotein lipase and malate dehydrogenase activities or increasing
hormone-sensitive lipase activity in abdominal adipose tissue (Lu
et al., 2007; Mersmann, 1998).
4.5. Humural immune response
Supplementation of Mn and phytase increased the antibody
titers against ND vaccine. It was also observed that improvement
of antibody titer was not observed beyond 75 mg Mn/kg irrespective of phytase addition. Moreover, similar antibody titers
were found at 75 mg Mn/kg without phytase and 50 mg Mn with
phytase. Consequently from the results of this study, it can be
deduced that Mn supplementation at 75 mg/kg diet without
phytase or at 50 mg/kg diet with phytase could increase the antibody titers against ND virus, but Mn supplementation beyond
75 mg/kg may not show further benecial response in improving
immunity.
Manganese plays a role in immunological function possibly by
interacting with neutrophils and macrophages through plasma
membrane of cells and producing antibodies in many species
(Fletcher et al., 1988). Manganese contributes to superoxide dismutase activity (Luo et al., 2006), which is vital for macrophage
and heterophil integrity (Cook-Mills and Fraker, 1993). Sunder
et al. (2006) reported no signicant inuence on antibody titers up
to 800 or 1,600 mg Mn/kg. The current study indicated that for
enhanced humoral immune response, Mn supplementation
(75 mg/kg) in the diets of chickens should be higher than NRC
(1994) recommendation. However, Luo et al. (1991) supplemented
a basal diet (18 mg Mn/kg) of broiler chickens with graded level of
Mn from MnSO4 (50, 90, 130, 280, and 2,840 mg/kg) and reported
that Mn supplementation did not inuence the antibody titer levels. The excess exposure to Mn may result in Mn accumulations
in immune organs and disturb the balance of other trace minerals
in immune organs inducing immune suppression in molecular
levels (Liu et al., 2012).

5. Conclusions
Supplementation of Mn to a basal diet containing 25 mg Mn/kg
diet had no benecial effects on growth performance and carcass
traits. However, Mn supplementation increased antibody titer
against New Castle disease at 75 mg/kg diet and decreased

A. Ghosh et al. / Livestock Science 191 (2016) 8085

abdominal fat at 100 mg/kg diet, and incidences of leg abnormalities compared with the control basal diet. There are no signicant interaction effects between Mn and phytase supplementation. Thus, this study conrmed that Mn supplementation at
concentrations of 75100 mg/kg to a basal corn-soybean diet could
be benecial for improving immunity, reducing incidences of leg
deformity, and decreasing fat deposition in broiler chickens.

Conict of interest
Authors declare that they do not have any conict of interest
related to this article.

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