Journal of Applied Animal Research

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Estimation of dietary threonine requirement for

growth and immune responses of broilers

Mehdi Taghinejad-Roudbaneh, Mohammad Javad Babaee, Mohammad

Afrooziyeh & Bahram Alizadeh

To cite this article: Mehdi Taghinejad-Roudbaneh, Mohammad Javad Babaee, Mohammad

Afrooziyeh & Bahram Alizadeh (2013) Estimation of dietary threonine requirement for growth
and immune responses of broilers, Journal of Applied Animal Research, 41:4, 474-483, DOI:
10.1080/09712119.2013.795896

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Published online: 19 Jun 2013.

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Journal of Applied Animal Research, 2013
Vol. 41, No. 4, 474
483, http://dx.doi.org/10.1080/09712119.2013.795896

Estimation of dietary threonine requirement for growth and immune responses of broilers
Mehdi Taghinejad-Roudbaneha*, Mohammad Javad Babaeea, Mohammad Afrooziyeha and Bahram Alizadehb
a
Department of Animal Science, Islamic Azad University, Tabriz, Iran; bDepartment of Animal Science, Tabriz University,
Tabriz, Iran
(Received 30 January 2012; final version received 13 March 2013)

This study was conducted to determine the effects of supplementing threonine (Thr) at different levels in the diets
on growth performance and immune responses of broiler chickens challenged with infectious bursal disease
(IBD) and also to estimate Thr requirement based on different response criterion. A total of 300 one-day-old
male broiler chicks were assigned to one of the six dietary treatments. Chickens were fed eight graded levels of
Thr: 0.60, 0.67, 0.74, 0.81, 0.88 or 0.95% from day 21 to 42 of age. On day 28, all birds were challenged with a
commercial live-IBDV vaccine. Body weight gain (BWG), feed conversion ratio (FCR) and carcass char-
acterisation (breast, thigh and fat pad percentage) were significantly influenced by Thr levels. Increasing advisable
level up to 0.81% of the diet resulted in significant improvement in the FCR and BWG. Thr supplementation had
great effects on the antibody titer against IBD; the broilers receiving the Thr levels of higher than National
Research Council (NRC) recommendation have a higher IBD antibody titer than those that received lower levels
of Thr. The highest (0.822490.0211) and the lowest (0.724090.0140) were estimated with straight broken-line
analysis for immune response (14 days post-challenge) and breast weight, respectively. The results obtained in the
present study indicated that Thr requirements of broiler based on the recommendation of NRC are not sufficient
to meet the requirement of the new commercial broiler companies under stress and non-hygienic conditions. The
best level of Thr based on the current study was 0.81% for support growth performance and immune function.
Keywords: broiler; immune system; performance; threonine; serology

1. Introduction environmental conditions. The Lys and sulphur

Dietary amino acid concentration should closely meet
maintenance and tissue accretion needs of commer-
cial broilers, especially towards the middle and end
of the growing period. Inadequate formulation of
dietary amino acids impairs protein utilisation and
increases total nitrogen excretion. Threonine (Thr)
may be considered as the third limiting amino acid in
poultry diets based on corn, wheat and soybean meal,
following Met and Lys (Dozier et al. 2000; Rosa &
Pesti 2001). Kidd (2000) reported that Thr deficiency
resulted in decreasing the utilisation of total sulphur
AA and Lys. Optimal levels of these amino acids are
needed to support optimum growth and health of
broilers (Rehman et al. 2012). Nutrient requirement
standards have been reported by the National Re-
search Council (NRC 1994) and through more than
28 years due to genetic selection, management prac-
tices and feed formulation changes birds have more
rapid growth and better performance than previous
years (Williams et al. 2000). On the other hand, NRC
recommendations are usually based on the needs of
healthy birds under ideal conditions, but birds in
commercial systems are normally exposed to different
kinds of stresses, diseases and also combinations of
amino acid requirements have been evaluated exten-
sively, but less information is available on the Thr
requirements of chicks (Webel et al. 1996). Evaluated
Thr requirement is based on the performance for
exam. Rosa and Pesti (2001) suggested 0.69% Thr for
growth and 0.68% for feed conversion ratio (FCR) of
growing chickens (classic strains) in the starter period,
whereas, in poultry production, it is very important to
improve immunity so as to prevent infectious dis-
eases. Minimising immunosuppression and its impact
is an important strategy for success in the broiler
industry. However, strategies to control immunosup-
pression are largely based on vaccination pro-
grammes for broiler breeders and broiler progeny
and management to minimise stress during rearing
(Fussell 1998). Utilisation of immunostimulants is
one solution to improve the immunity of animals and
to decrease their susceptibility to infectious diseases.
There are extremely important interactions, syner-
gisms and antagonisms between nutrition and im-
munity that markedly affect productivity of poultry.
Experiments in chickens and mammalian species have
shown that protein or amino acid deficiencies may
reduce the circulating antibodies available to challenge

*Corresponding author. Email: mtaghinejad@iaut.ac.ir

# 2013 Taylor & Francis

 Journal of Applied Animal Research 475

organisms. Minimum requirements for a given nu- Dietary amino acid content was determined by ion-
trient for maximum production are fully established exchange chromatography on an autoanalyzer. The
(NRC 1994). On the other hand, the National analysed values of protein and amino acid contents
Research Council (NRC) recommendation are usually were agree with calculated values (Table 1).
based on the needs of healthy birds under ideal Treatments were achieved by the addition of
management, but birds in commercial systems are crystalline L-Thr (98.5% Thr) at the expense of inert
normally exposed to different kinds of stresses that it filler in the test diet containing 0.6% Thr (Table 1) to
may be necessary for new research to conduct multiple give 0.60, 0.67, 0.74, 0.81, 0.88 or 0.95% Thr from
focus and attention to the actual broiler requirements. day 21
42 of age. No antimicrobial, anticoccidial
However, in many cases, it is not known whether the drugs or feed enzymes were included in the basal
requirement values that maximise productivity in diets. The chicks were vaccinated against Newcastle
healthy, unchallenged birds are optimal for immuno-
competence and disease resistance. There is some Table 1. Ingredients and nutrient composition of starter
evidence that essential amino acids levels in the feed and grower diets.
higher than NRC specifications are needed to achieve Ingredientsa Starter 1
21 Grower 21
42
optimal growth performance, immunocompetence
Corn and grain 31.53 31.44
and disease resistance (Kidd et al. 2001; Quentin et al. Wheat 20.00 30.00
2005; Taghinejad-Roudbaneh et al. 2011). Therefore, Corn and gluten meal 2.72 11.96
it may be necessary for new research to conduct Wheat bran 0 10.00
multiple focus and attention to the actual broiler Soybean meal 35.3 8.24
requirements. Therefore, the present experiment was Canola oil 5.96 3.68
Dicalcium phosphate 1.99 1.37
conducted to determine Thr requirement based on Oyster-shell 1.42 1.82
immune responses of broiler chickens challenged with DL-Methionine 0.17 0.00
infectious bursal disease (IBD). L-Lys Hcl 0.00 0.80
L-Thr 0.00 0.00
Common salt 0.4 0.28
Vitamin premixb 0.25 0.25
2. Materials and methods Mineral premixc 0.25 0.25
Calculated composition
A total of 500 day-old male broiler chicks (Ross 308) ME (kcal/g) 3100 3100
were obtained from a local hatchery. Chicks were CP (%) 22.30 19.40
raised from 1 to 21 d of age before commencement of Lys (%) 1.07 1.00
the trial. During this period, the birds were submitted Methionine 0.49 0.38
Methionine cystine (%) 0.88 0.73
to conventional broiler chicken management and Thr (%) 0.78 0.60
housed in floor pens (with 1.5 m2 area per each pen) Tryptophan (%) 0.19 0.17
in an environmentally controlled broiler house with Ca 0.97 0.9
litter floors with wood shavings as litter material. The Available phosphorus 0.48 0.35
relative humidity was between 75
85%. Feed and Na 0.19 0.15
Analysed composition
water were provided ad libitium, and birds were CP (%) 22.38 19.50
maintained on 23 light:1 dark schedule lighting. They Lys (%) 1.1 0.99
received a commercial broiler starter diet, formulated Methionine (%) 0.49 0.41
to meet or exceed the nutritional requirements of Methionine cystine (%) 0.89 0.76
Tryptophan (%) 0.21 0.17
broilers, as recommended by the NRC (1994). After an
Thr (%) 0.79 0.61
overnight fast, at 21-d of age, 300 chicks of similar
a
weight were randomly assigned to 30 clean pens in the Based on NRC (1994) feed composition tables.
b
Vitamin premix provided the following per kilogramme of diet.
same broiler house for the starter period. Average vitamin A, 5000 IU (as all trans-retinyl acetate); cholecalciferol,
body weights (means9standard error) at d 21 were 1000 IU; vitamin E, 10 IU (as all-rac-a-tocopherol acetate);
730930 g. The ambient temperature was gradually riboflavin, 4.4 mg; Ca pantothenate, 12 mg; nicotinic acid, 44
mg; choline Cl, 220 mg; vitamin B12, 6.6 mg;vitamin B6, 2.2 mg;
decreased from 328C when the birds were 1 d old to menadione, 1.1 mg (as menadione sodium bisulfitecomplex); folic
228C when 28 d old. The protein and AA levels of the acid, 0.55 mg; d-biotin, 0.11 mg; thiamine, 1.1 mg (asthiamine
corn, soybean meal and corn gluten meal were mononitrate); ethoxyquin, 125 mg.
c
Provided per kilogramme of diet. Mn (from MnSO4_H2O),
analysed before diets formulation. Experimental diet 100 mg; Zn (from ZnSO4_7H2O), 100 mg; Fe, (from FeSO4_
was formulated to meet or exceed the NRC (1994) 7H2O), 50 mg; Cu (from CuSO4_5H2O), 10 mg; I (from Ca (IO3)2_
nutrition recommendations from 21 to 42 d of age for H2O), 1 mg.
Thr was added to the basal diet at the expense of a 2:1 mixture of
all nutrients except Thr (Table 1). Dietary nitrogen glutamic acid and glycine to maintain equal nitrogen concen-
(N6.25) was measured by the Kjeldahl procedure. trations among treatments.
476 M. Taghinejad-Roudbaneh et al.
disease (Animal Health, Fort Dodge, Iowa, USA) on
day 7 (Intraocular) and on day 21(intranasal).
The chickens were weighed individually weekly.
Feed intake was recorded weekly and FCR was
calculated. Mortality was recorded daily in each
subgroup. On d 42, three randomly chosen chickens
from each pen were wing-banded and moved to
separate floor pens for an overnight fast (approxi-
mately 12 h); water was provided ad libitum. The
following morning, chickens were randomly crated
and transported to the processing room where each
was weighed, euthanised by cervical dislocation and
then scalded, defeathered and eviscerated. Abdominal
fat pads and livers were collected from the birds
after they were partially eviscerated. The weights of
thymus, spleen and bursa of Fabricius were recorded.
Organ weights were expressed on a relative body
weight basis. Carcasses were weighed and chilled on
ice in a walk-in cooler at 58C overnight. The
following day, breast (without skin) and thigh were
excised, and weights were recorded. The abdominal
fat pad percentage was calculated based on chilled
carcass weight.
2.1. Challenge protocol
On day 28, all birds were challenged by oral route
with a commercial live-IBD vaccine. The strain was
characterised as an intermediate virulent classical
strain. The content of the 1000 dose of IBD vaccine
vial was reconstituted in 100 mL distilled water, and
each bird was inoculated with 1 mL IBD virus into
the lumen of the crop by oral gavage, and finally each
bird received a dose ten times greater than the
standard IBD vaccine.
2.2. Serology and immune responses
On the 28th, 35th and 42nd day ten birds from each
group were chosen at random, and blood samples
were collected from the brachial vein. Serum was
separated by centrifugation (3000 g, 15 min), and
antibody titre against IBD was performed using
commercially available ELISA kits (IDEXX, Labs
Inc., Westbrook, Maine, USA) according to the manu-
facturer’s instructions. ELISA absorbency was mea-
sured at 650 nm using an ELISA reader (Bio-Tek
Instruments Inc. ELX 800; Winooski, Vermont) by
standard procedures. Serum albumin, globulin, alkaline
phosphatase (ALP), alanine aminotransferase (ALT),
aspartate aminotransferase (AST) and lactate dehy-
drogenase (LDH) were measured by specific com-
mercial kits (Roche Diagnostica, Basel, Switzerland)
using an autoanalyzer (HITACHI 902 automatic
autoanalyzer).
2.3. Statistical analysis
The experimental design was a completely rando-
mised design. The experimental unit was the pen
mean. All data were analysed by two-way ANOVA,
using the GLM procedure of SAS (SAS Institute
2004). The requirement based on BWG, FCR, carcass
breast, thigh weight and immune response to IBD
challenge was determined applying a non-linear
procedure of SAS. The iterative procedure makes
repeated estimates for coefficients and minimises
residual error until the best-fit lines are achieved:
one line with a slope of zero and the other with a
marked slope. The two lines are fitted to the values
using the following equations:
For linear broken line : y ¼ L þ UðR  XÞ  I
For quadratic broken line : L þ UðR  XÞ
 ðR  XÞ  I
where X independent variable, R requirement,
ydependent variable, L  theoretical maximum,
I1 (if X BR) or I0 (if X R), and U rate
constant. The coefficient of determination (R2 value)
was determined as follows: R2 1 (residual sum of
squares/corrected total sum of squares).
3. Results
Responses to dietary Thr level were significant
(P B0.05) for BWG, FCR, feed intake, percentage
of fat pad carcass efficiency body, breast and thigh
weight (Table 2). The BWG, BW, carcass efficiency,
breast and thigh weight and feed intake increased
with increasing Thr level in diet. The highest BWG
(1756.6 g), BW (2484.7 g) and carcass efficiency
(68.6%) were in diet containing 0.88% Thr, and the
highest breast (427 g) and feed intake (3500 g) were in
diet containing 0.95% Thr, and the highest thigh
weight (433.0 g) was in diet containing 0.81% Thr.
FCR and percentage of fat pad decreased as Thr
increased in the diet, and lowest level of FCR was in
diet containing 0.81% Thr and the lowest percentage
of fat pad was in diet containing 0.74% Thr.
However, spleen percentage was not affected by Thr
level in diet. The BWG, BW and FCR in diet
containing 0.81% Thr did not significantly differ
with higher level of Thr in diet but significantly
(P B0.05) differ with diet containing lower levels of
Thr. The feed intake, breast and thigh weight in diet
containing 0.74% Thr did not significantly differ
(P B0.05) with higher level of Thr in diet but
significantly differ with diet containing lower levels
of Thr (Table 2).
 Journal of Applied Animal Research 477

Table 2. The effects of dietary Thr on BWG, feed intake, FCR, breast meat, thigh weight, carcass efficiency and abdominal fat
pat percentage of male broiler chicks (21
42 d).

Thr (%) n BWG (g) Feed intake (g) FCR (g/g) BW (g) Spleen (%) Thigh(g) Breast (g) CE (%) Fat pad (%)
d b a d
0.6 5 1441.8 3272.4 2.27 2164.5 0.094 392.6c 360.8c 60.1c 2.87a
0.67 5 1548.8c 3304.4b 2.13b 2276.1c 0.096 406.6b 397.3b 62.2bc 2. 19bc
0.74 5 1686.4b 3481.5a 2.06c 2424.8b 0.114 428.2a 422.9a 64.0abc 2.06c
0.81 5 11754.3a 3467.1a 1.98d 2479.9a 0.116 433.0a 425.5a 68.2a 2.33bc
0.88 5 1756.6a 3497.2a 1.99d 2484.7a 0.124 430.9a 426.0a 68.6a 2.60ab
0.95 5 1752.2a 3500.4a 2.00d 2483.8a 0.134 431.7a 427.0a 66.6ab 2.52abc
SEM 5 8.66 28.52 0.015 9.78 0.016 2.61 3.83 1.58 0.166
ANOVA df Probabilities
Thr 5 B0.0001 B0.0001 B0.0001 B0.0001 0.47 B0.0001 B0.0001 0.005 0.02
Error 24
R2 0.97 0.73 0.91 0.97 0.16 0.89 0.90 0.46 0.39
REGRESSION
Thr 1 B0.0001 0.0038 B0.0001 0.009 B0.0001 B0.0001 B0.0001 0.05 0.0018
Thr 1 B0.0001 0.0145 B0.0001 0.009 B0.0001 B0.0001 B0.0001 0.081 0.0138
Thr
Error 27
R2 0.96 0.62 0.89 0.83 0.77 0.87 0.92 0.09 0.77
a, b, c, d
BWG, Body weight gain; Weigth gain. BW, body weight. Means in the column with different letters differ (p B0.05).

The effect of Thr level on liver function enzymes,
blood parameters and serum antibody titer were
significant (PB0.05) (Table 3). The highest AST,
LDH and ALP, were observed in the chicken fed with
diet containing 0.81% Thr. However, the highest ALT
was found in the group with 0.88% Thr supplementa-
tion, and the chickens with 0.74% Thr supplemented
diet had the highest serum total protein. Immune
response to live-IBDV vaccine significantly increased
with increasing Thr in the diets (PB0.001). The diet
containing 0.81% Thr had the highest serum antibody
titer (log2) against IBDV in a pre-challenge stage (3.00),
7 days post-challenge (3.11) and 14 days post-challenge
(3.80). On the other hand, the lowest level of antibody
in these stages were found in a group with the diet
containing 0.6% Thr (81% of NRC recommendation).
Regression analyses showed significant linear
effects (P B0.05) due to dietary Thr concentrations
for all response parameters except for ALP and serum
globulin. A quadratic effect (P B0.05) due to dietary
Thr concentration was noted for all parameters
except for carcass efficiency, ALT, LDH, ALP and
serum globulin (Tables 2 and 3).
The Thr requirement was determined as a percen-
tage of the diets using linear and quadratic broken-line
analysis with different response criterion (Table 4 and

Table 3. The effects of dietary Thr on liver function enzymes, blood parameters and serum antibody titer (log2) of broiler
chickens challenged with IBD.

Thr (%) N AST ALT LDH ALP Albumin TP Globulin A/G Titr1 Titr2 Titr3
d c c b b b b b c e
0.6 5 314.5 11.5 3536.4 1384.75 1.426 4.526 3 0.47 2.08 2.13 2.96d
0.67 5 338.5c 12.076bc 3578c 1416.00b 1.726a 4.8ab 3.1ab 0.56a 2.33b 2.50d 3.05c
0.74 5 355.5b 13abc 3648.3c 1463.25b 1.45a 7.9a 3.15ab 0.56a 2.91a 2.70c 3.42b
0.81 5 379.5a 13.6ab 4536.8a 1641.25a 1.75a 5.11a 3.32a 0.54a 3.00a 3.11a 3.80a
0.88 5 376.25a 14.126 a
4333.5ab 1345.00b 1.79a 5.02a 3.3a 0.53ab 2.91a 2.91b 3.80a
0.95 5 371.0ab 13.25ab 4102.3b 1455.25b 1.8a 4.95a 3.2ab 0.56a 2.90a 2.75bc 3.79a
SEM 8.66 28.52 0.015 9.78 0.016 2.61 3.83 1.58 0.072 0.064 0.074
ANOVA df Probabilities
Thr 5 B.0001 0.014 B.0001 0.0018 0.0001 0.006 0.117 0.034 B.0001 B.0001 B.0001
Error 24
R2 0.81 0.42 0.78 0.53 0.63 0.47 0.29 0.37 0.85 0.85 0.85
REGRESSION
Thr 1 B.0001 0.03 0.04 0.06 0.0019 0.0047 0.0768 0.0908 B.0001 B.0001 B.0001
Thr Thr 1 0.0001 0.06 0.07 0.07 0.0039 0.0084 0.1048 0.11481 B.0001 B.0001 B.0001
Error 27
R2 0.77 0.39 0.49 0.12 0.49 0.40 0.19 0.13 0.84 0.85 0.78
AST, Aspartate Aminotransferase ((U/L)); ALT, Alanine Aminotransferase ((U/L)); LDH, Lactate Dehydrogenase ((U/L)); ALP, Alcalin
phosphatase((U/L)); Albumin, Globulin and TP, Albumin Globulin and total protein ((mmol /L)). a, b, c, dMeans in the column with different
letters differ (p B0.05).
478 M. Taghinejad-Roudbaneh et al.

Figure 1. Fitted broken-line plot of body weight gain (BWG) as a function of dietary Threonine level (% of diet). The BWG
data points are replications of 10 chicks during a 21 d feeding trial. The break point occurred at 0.781890.00681 and 0.87109
0.0180 as a percentage of diet with Linear and Quadratic broken line, respectively.

Figure 2. Fitted broken-line plot of feed conversion ratio (FCR) as a function of dietary Threonine (% of diet). The FCR data
points are replications of 10 chicks during a 21 d feeding trial. The break point occurred at 0.733290.0216 and 0.85489
0.0286 as a percentage of diet with Linear and Quadratic broken line, respectively.

Figure 3. Fitted linear broken-line plot of serum antibody titer (log2) as a function of dietary Threonine level (% of diet). The
serum antibody titer data points are replications of 10 chicks during a 21 d feeding trial. The break point occurred 0.75319
0.0147, 0.787490.0280 and 0.822490.0211 as a percentage of diet at 28, 35 and 42 days old.

Figures 1
4). The quadratic broken-line model esti- 0.0140). Thr requirements with straight broken-line
mated higher requirements than the straight broken- analysis were estimated for immune response (14
line model for all response criterions (Table 4). The days post-challenge) and breast weight, respectively.
highest (0.822490.0211) and the lowest (0.72409 And the highest (0.963690.0742) and the lowest
 Journal of Applied Animal Research 479

Figure 4. Fitted quadratic broken-line plot of serum antibody titer (log2) as a function of dietary Threonine level (% of diet).
The serum antibody titer data points are replications of 10 chicks during a 21 d feeding trial. The break point occurred
0.833690.0413, 0.834990.0487 and 0.963690.0742 as a percentage of diet at 28, 35 and 42 days old.

Table 4. Dietary Thr requirement based on broken-line model analyses.

Estimated requirement

Linear broken-line analysis1

Response criterion Intercept (L) Slope (U) Break point (R) P-value R2
BWG (g) 1754.494.9317 1747.9986.2865 0.781890.00681 B0.0001 0.97
FCR (g/g) 2.007590.00982 1.971490.3967 0.733290.0216 B0.0001 0.85
FI (g) 3488.2917.1123 1493.29299.4 0.760790.0244 B0.0001 0.67
Breast (g) 425.491.8315 520.6974.0039 0.724090.0140 B0.0001 0.90
Tight 431.991.4737 254.4925.783 0.759590.0122 B0.0001 0.88
Body weight (g) 2482.895.6023 1859.3998.020 0.774590.00696 B0.0001 0.97
Titr1 2.938790.0431 5.957190.7547 0.753190.0147 B0.0001 0.82
Titr2 2.927390.0460 4.100090.8054 0.787490.0280 B0.0001 0.74
Titr3 3.795090.0539 4.145790.4870 0.822490.0211 B0.0001 0.82
Quadratic broken-line analysis2
BWG (g) 1758.997.1792 4254.19535.8 0.878090.0171 B0.0001 0.96
FCR (g/g) 1.989890.0106 4.318590.9925 0.854890.0286 B0.0001 0.90
FI (g) 3500.1922.9189 2829.991433.6 0.894790.0746 B0.0001 0.65
Breast (g) 426.592.0852 1715.99394.2 0.796490.0216 B0.0001 0.90
Tight 432.391.7863 709.49194.6 0.841790.0323 B0.0001 0.87
Body weight (g) 2488.097.7755 4557.39619.1 0.871090.0180 B0.0001 0.96
Titr1 2.947190.0513 16.943196.1675 0.833690.0413 B0.0001 0.79
Titr2 2.906890.0517 14.357696.1157 0.834990.0487 B0.0001 0.73
Titr3 3.836790.0870 7.339792.7856 0.963690.0742 B0.0001 0.78
1. The linear broken-line model is y LU(R x), where L is the ordinate, R is the abscissa of the break point, and the value of R is zero at
values of x R. Values are reported as9SEM.
2. The quadratic broken-line model is yLU (R x) (Rx) where L is the ordinate, R is the abscissa of the break point, and the value
R is zero at values of xR. Values are reported as9SEM.

(0.796490.0216). Thr requirements with quadratic 4. Discussion

broken-line analysis were estimated for immune
response 14 day post-challenge and breast weight,
respectively. All estimated requirements except esti-
mated based on FCR and breast weight were higher
than NRC (1998) recommendation.
Increasing Thr level in the diet improved breast meat
weight (from 360.8 to 427 g), BWG (from 1441.8 to
1756.6 g), thigh weight (from 392.6 to 433 g) and
decreased FCR (from 2.27 to 1.98). Similarly Mack
et al. (1999) and Dozier et al. (2000, 2001) indicated
480 M. Taghinejad-Roudbaneh et al.
that supplemental Thr improved breast percentage. In
contrast to our results, Dozier et al. (2000) reported
that Thr supplementation had no significant effect on
the proportion of drumsticks and thighs. Also Dozier
et al. (2000, 2001) indicated that total carcass meat
yield was not affected by Thr concentration. Lemme
(2003) reported that FCR of male broilers optimised
as dietary Thr was increased from 0.55 to 1.05%.
Tugay et al. (2009) showed that the highest body
weight gain was at 0.75%, and better feed efficiency
was in 0.85% Thr for broilers occurred. In the current
study, increasing the Thr concentration of the diet
from 0.6 to 0.81% improved the growth rate, body
weight and FCR of broilers. In addition, increasing
the Thr concentration of the diet from 0.6 to 0.74%
improved the feed intake, carcass efficiency and
breast and thigh weight of broilers.
Thr supplementation did not significantly alter
the abdominal fat proportion, as reported in earlier
studies (Dozier et al. 2001; Rosa & Pesti 2001). The
effect of dietary Thr on absolute and relative abdom-
inal fat pad weight in broilers has typically been
inconsistent (Kidd & Kerr 1997; Corzo et al. 2003;
Kidd et al. 2004). No dietary Thr effect was observed
for relative weights of spleen (Table 2). Results are in
agreement with those presented by Kidd et al. (2001).
The estimated Thr requirement based on BWG
(0.781890.00681) was higher than that of breast
meat (0.724090.0140), FCR (0.733290.0216), feed
intake (0.760790.0244), thigh (0.759590.0122) and
body weight (0.774590.00696). Dietary Thr needs
observed for live performance measurements are in
agreement with those previously published for birds
during the same age period (Webel et al. 1996; Penz
et al. 1997; Mack et al. 1999). Webel et al. (1996) fed
graduations of Thr to Ross Hubbard broilers from
d 21 to 42 in a corn
peanut meal-based diet and
reported that optimal feed conversion occurred when
dietary Thr was 0.70%. In addition, Kidd and Kerr
(1997) fed graduations of Thr to Ross Ross 308
male broilers in a corn
peanut meal-based diet and
determined that the live performance needs during 30
to 42 d are satisfied with 0.70% total Thr. These live
performance needs for Thr (Kidd & Kerr 1997) are in
agreement with the former estimates (Webel et al.
1996; Penz et al. 1997), since they were obtained in the
last half of the 21- to 42-d period. Similar to the result
of the current study, Kidd and Kerr (1997) evaluated
carcass traits and found that a higher level of Thr
(0.78% total of diet) was needed to yield good breast
meat accretion. Amino acid requirements in growing
animals depend on several factors like genotype, age
and sex (Samadi & Liebert 2007; Muhl & Liebert
2008; Rehman et al. 2012). The NRC (1994) suggest
that 21- to 42-d-old broilers fed diets containing 3200
kcal/kg should receive a total dietary Thr level of
0.74% of diet. However, Ojano-Dirain and Waldroup
(2002) suggested that the modern rapidly growing
broiler may have Thr requirements greater (0.78%)
than those generally recommended (0.74%) by NRC
(1994). Lemme (2001) reported that depending on the
performance criteria chosen, the optimum dietary Thr
levels for 20- to 42-d-old broilers to achieve 95% of
the asymptotic response for BWG, FCR and breast
meat yield were 0.66, 0.68 and 0.70%, respectively,
compared to the 0.74% suggested by NRC (1994).
The method of statistical evaluation can influence
the estimation of an amino acid and requirement
(Taghinejad-Roudbaneh et al. 2011). The most com-
mon statistical models used to determine amino acid
requirements are the broken-line (one-slope), two-
slope, quadratic, and ascending quadratic with pla-
teau models (Vedenov & Pesti 2008). Estimated Thr
requirement values with quadratic broken-line model
in all response criterions in this experiment were
higher than estimated values with straight broken-line
model (Table 4). The result of this experiment closed
with the results of Barbour et al. (1993) and
Taghinejad-Roudbaneh et al. (2011) that found
statistical evaluation influences the estimate of an
amino acid requirement and the quadratic procedure
estimates were higher for all protein sources when
compared with straight broken-line model. In addi-
tion, Vazquez and Pesti (1997) concluded that ‘as-
cending quadratic with plateau’ model may
theoretically be more realistic and adequate than the
‘ascending line with plateau’ model for predicting the
amino acid requirement for broiler chicks.
Additionally, environmental conditions and
graded stimulation of the immune system may also
affect requirement studies (Kidd et al. 2003).Thr
participates in protein synthesis and its catabolism
generates many products important in metabolism
(i.e., glycine, acetyl-Coa and pyruvate). Thr serves as a
component of body protein and a precursor of glysine
and serine, is involved in immune responses and
needed in gastrointestinal mucin production (Lemme
2003). Thr is a major component of intestinal mucin
and plasma a-globulin in animals (Kim et al. 2007).
By way of protein synthesis and cellular signalling
mechanisms, Peng et al. (2007) show that addition of
2 mM-Thr to the culture medium prevented apopto-
sis, stimulated cell growth and promoted antibody
production in lymphocytes. Bhargava et al. (1971)
found that Thr need in chickens for antibody produc-
tion to Newcastle disease virus was higher than that
for growth. Thr is the most abundant essential amino
acid in immunoglobulin protein, and Defa et al.
(1999) show that growing pigs fed with Thr deficient
diets have a significant lower plasma concentration of
 Journal of Applied Animal Research
total or specific IgG titre following bovine serum
albumin injection and swine fever vaccination. Wang
et al. (2006) demonstrated that the requirement to
optimize immunity was higher than to maximize
weight gain and FCR in pig, as the intake of true
ileal digestible Thr increased from 4.1 to 6.6g per day,
serum IgG concentrations increased in response to
increased intake of true ileal digestible Thr. Thr is a
major component of plasma g-globulin in poultry
(Tenenhouse & Deutsch 1966). Bhargava et al. (1971)
reported that antibody titers of chicks increased as
dietary Thr increased. With increasing Thr level in
diet, immune responses against IBVD were improved
(Table 3). NRC requirements for amino acids and
protein are designed to support maximum growth
and production under ideal condition and good
maintenance. The estimated Thr requirements vary
with response criterion, and when requirement was
estimated based on immune response 14 days post-
challenge with IBVD, this value was higher than
other response criterions (Table 3 and Figures 3
and 4). The control of immunosuppressive diseases
is of prime importance for the nascent poultry
industry in developing countries. In this regard, the
improvement of feed formulation and enhancement
of genetic resistance to economically important dis-
eases should be considered (Bumstead et al. 1991).
The resistance against infectious challenges requires
an intense response orchestrated by the immune
system. From the nutritional standpoint, feed sub-
strates (amino acids, energy and enzymes) are needed
to activate such a response (Rubin et al. 2007).
Minimum requirements for a given nutrient for
maximum production are fully established (NRC
1994). As a result of the current study, nutrition
recommended by NRC are adequate for healthy birds
under ideal management, but the combination of
environment condition and disease increase the
requirement for Thr ( 0.73% vs. 0.82%).
From the nutritional standpoint, amino acids are
needed for proliferation of lymphocytes, establish-
ment of germinative centres in the bursa of Fabricius
to refine immunoglobulin affinity, recruitment of new
bone marrow monocytes and heterocytes and synth-
esis of effector molecules (immunoglobulins, nitric
oxide, lysozyme and complement) and communica-
tion molecules (cytokines and eicosanoids, for in-
stance). Methionine plays an important role in
humoral and cellular immune responses (Swain &
Johri 2000; Shini et al. 2005). Additionally, there is
evidence that Thr modulates immune function in
livestock (Li et al. 2007), and immune system is
sensitive to dietary Thr intake (Li et al. 1999). It has
been reported that Thr is a major component of
intestinal mucin and plasma g-globulin in animals
481
(Kim et al. 2007). Indeed, serum antibody titers
increased with increasing dietary intake of Thr in
chickens infected with the Newcastle disease virus
(Bhargava et al. 1971). Also, dietary supplementation
with Thr increased serum levels of IgG in sows
(Cuaron et al. 1984). Further, increasing dietary Thr
intake increased antibody production, serum IgG
levels and jejunal mucosal concentrations of IgG
and IgA (Wang et al. 2006). Furthermore, it should
be notified that dietary methionine can influence
the metabolism of other amino acids (Moritoki &
Yoshida 1970; Sanchez et al. 1972).
It has been demonstrated that the activities of
many liver enzymes involved in the catabolism of
amino acids decrease when a low protein diet is fed,
but increase with high protein intakes (Muramatsu
et al. 1971). In our experiment, liver enzymes were
affected by Thr level of diet, whereas in the group
receiving 0.81% Thr, we found a significant raise in
the concentration of ALT, AST and LDH. The
findings may also explain that the current NRC
recommendations for thr in broiler are probably not
appropriate for broiler health under stress and dis-
eases condition. Although, it should be noted that the
concentration of ALT, AST and LDH in the group
receiving 0.6% Thr also was in the normal range
(Marjanovic et al. 1975; Chattopadhyay et al. 2006).
5. Conclusion
In conclusion, the results obtained on the present
study indicated that Thr requirements of broiler
based on recommendation of the NRC (1994) are
sufficient to meet the requirements of the new
commercial poultry and commercial broiler compa-
nies under hygienic condition. However, it may
reduce profitability under conditions of disease or if
the broilers encounter the stress conditions. There-
fore, in order to overcome this problem, Thr level
during non-hygienic should be increased. The best
level of Thr based on current study is 0.81% for
support growth performance and immune function.
Acknowledgement
This work is supported by the Tabriz Branch, Islamic Azad
University, and by an endowment from the Animal Science
Department of agricultural faculty. The author would like
to thank Mr. Mehdi Alizade for his collaborations.
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