Efficacy of ajwain (Trachyspermum ammi L.) seed at graded levels of dietary threonine on growth performance, serum metabolites, intestinal ...
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Received: 13 November 2019 | Revised: 8 February 2020 | Accepted: 21 February 2020
DOI: 10.1111/jpn.13357
ORIGINAL ARTICLE
Efficacy of ajwain (Trachyspermum ammi L.) seed at graded
levels of dietary threonine on growth performance, serum
metabolites, intestinal morphology and microbial population in
broiler chickens
Alireza Kolbadinejad | Vahid Rezaeipour
Department of Animal Science, Qaemshahr
Branch, Islamic Azad University, Qaemshahr, Abstract
Iran The present study investigated the influence of dietary threonine (Thr) levels and
Correspondence ajwain seed (AS) on the growth performance, immunity, blood metabolites, ileal mi-
Vahid Rezaeipour, Department of Animal croflora and jejunum morphology of broiler chickens. A total of 600-day-old male
Science, Qaemshahr Branch, Islamic Azad
University, Qaemshahr, Iran. broiler chickens (Ross 308) were randomly allocated among five replicates of eight
Emails: vrezaeipour@gmail.com, dietary treatments according to a 4 × 2 factorial arrangement of treatments, includ-
vrezaeipour@qaemiau.ac.ir
ing four different levels of Thr (100, 105, 110 and 115% of requirements) and two
Funding information inclusion rates of AS (0 or 10 g/kg of the diet). Body weight gain and FCR improved
Islamic Azad University, Qaemshahr Branch
in broiler chickens fed 105% Thr supplement during the starter, finisher and whole
periods of the experiment (p < .05). The broiler chickens fed AS supplemented diet
had a greater weight gain (25–42 days) and better FCR (1–10 and 25–42 days) than
those without AS (p < .05). A significant interaction was observed between Thr and
AS supplementation for lymphocyte, heterophil/lymphocyte (H/L), bursa and spleen
(p < .05). The liver enzyme activity of aspartate aminotransferase (AST) was higher in
the chicks that received diet without AS supplement than those fed AS diet (p < .05).
Inclusion of 105% dietary Thr and 10 g/kg AS increased the viable cell counts of
Lactobacilli and decreased the population of the Escherichia coli in broilers (p < .05).
In jejunum morphology, the broiler chickens fed 105% Thr diet had a greater villus
length, width and crypt depth (p < .05). Also, the villus length was longer in broilers
that received AS supplemented diet (p < .05). It is concluded that the dietary 105%
of Thr and AS supplement individually improved growth performance, ileal microbial
population and jejunum morphology of broiler chickens.
KEYWORDS
amino acid, broilers, medicinal plant, microbiota
1 | I NTRO D U C TI O N synthesis, especially at the beginning of the growth period and
increasing nitrogen excretion (Taghinejad-Roudbaneh, Babaee,
Providing sufficient amino acids in poultry diets is one of the most Afrooziyeh, & Alizadeh, 2013). Threonine (Thr) may be considered
important factors influencing poultry production. Inadequate the third most limiting amino acid in corn–soya bean meal based
formulation of amino acids in the broiler diets reduces protein diets (Rezaeipour, Fononi, & Irani, 2012). In chicks, Thr serves as a
J Anim Physiol Anim Nutr. 2020;00:1–10. wileyonlinelibrary.com/journal/jpn © 2020 Blackwell Verlag GmbH | 12 | KOLBADINEJAD and REZAEIPOUR
precursor for Glycine to synthesis of uric acid (Kidd, Virden, Corzo, the aim of this study was to investigate the effects of four levels
Dozier, & Burnham, 2005). On the other hand, poultry are not able of dietary Thr and 2 levels of AS in broiler chicken diets on the
of synthesizing Thr via de novo mechanism, which makes it a neces- growth performance, immunity, serum metabolites, intestinal
sary amino acid in the diet (Eftekhari, Rezaeipour, & Abdullahpour, morphology and microbial population.
2015). It is well documented that Thr has an important role in the
structure and function of the gastrointestinal tract, which Thr is an
integral component (nearly 40%) of the intestinal mucin glycopro- 2 | M ATE R I A L S A N D M E TH O DS
teins (Corzo, Kidd, Dozier, Pharr, & Koutsos, 2007). The mucin is
synthesized by the goblet cells distributed along the villi (Azzam, 2.1 | Broiler chickens and dietary treatments
Dong, Xie, & Zou, 2012). It is reported that much of this mucin is ex-
creted through faeces and the associated Thr cannot be recovered A total of 600 one-day-old male broiler chickens of the Ross strain
(Eftekhari et al., 2015). Therefore, supply of Thr in the broiler diets were purchased from a commercial hatchery (Gorgan, Iran), and ran-
by adding L-Thr or animal and plant protein materials such as soya domly distributed to eight experimental groups with five replicate
bean meal and meat meal may be suitable strategies to compensate pens of 15 birds per pen. The broiler chickens were kept in floor pens
for Thr deficiency. Therefore, several trials have been conducted on (1.1 × 1.6 m) for the trial period of 42 days. All pens were equipped
the Thr requirements in broiler chickens (Corzo et al., 2007; Kidd, with a nipple drinker and feeder. The lighting programme was 24 hr
Kerr, & Anthony, 1997; Kidd et al., 2005; Rosa, Pesti, Edwards, & a day for the first week and then reduced to 16 hr of light and 8 hr
Bakalli, 2001). of dark during 7–42 days. The room temperature was set at 34°C
Due to the ban of the use of in-feed antibiotics in European during the first week and gradually reduced to 3ºC each week until it
Union, there is a clear demand for safe alternatives feed addi- reached 24ºC. Feed and water were provided ad libitum throughout
tives in poultry industry. Several studies have been conducted the experiment.
on the ability of medicinal plants and their essential oil or ex- The broiler chickens were fed commercial corn–soya bean
tract to act as growth promoters in broiler chickens and Japanese meal broiler diets (as mash) that were formulated to meet or
quails (Akbari, Torki, & Kaviani, 2016; Ghazaghi, Mehri, & exceed the nutrients requirements (except for Thr) of the Ross
Bagherzadeh-Kasmani, 2014; Khosravinia, 2015; Mehri, Sabaghi, strain (2009). The ingredients, composition and nutritional value
& Bagherzadeh-Kasmani, 2015b; Torki, Akbari, & Kaviani, 2015). of the starter (1–10 days of age), grower (11–24 days of age)
One such plants ajwain (Trachyspermum ammi), is a long known and finisher (25–42 days of age) diets are presented in Table 1.
herb with various medical properties widely cultivated in the Fresh ajwain seeds (AS) were obtained from a local market in
west and northwest of Iran (Hajiaghapour & Rezaeipour, 2018). June, and then were dried under room temperature for 6 days.
The ajwain seeds (AS) possess several medicinal attribute, includ- The air-dried seeds were finely ground to powder and stored
ing antibacterial, antioxidants and antifungal properties (Vitali at 4ºC until used. The experimental diets were arranged as a
et al., 2016). Also, anticoccidial and antioxidant impacts of ajwain 4 × 2 factorial, including dietary Thr (100, 105, 110 or 115% of
powder have been documented in broiler chickens (Abbas et al., the requirements) and supplementation of AS (0 or 10 g/kg of
2019). It has been reported that the most active compounds of AS the diet).
include thymol and carvacrol, which are important pharmacolog-
ically active substances (Kheiri, Faghani, & Landy, 2017). Besides,
it is found that the major components of essential oils derived 2.2 | Growth performance and carcass attributes
from ajwain seeds are γ-terpinene (31.02%), thymol (29.20%),
p-cymene (22.87%) and β-pinene (4.13%) (Hazrati, Rezaeipour, The broiler chickens were weighed by pen at the end of the starter
& Asadzadeh, 2019). Our previous studies well documented (10 days of age), grower (24 days of age) and finisher (42 days of age)
that essential oils from ajwain seeds have beneficial effects on periods. Feed consumption was also measured on day 10, 24 and 42
the gut health and productive performance of Japanese quails and feed conversion ratio was calculated by dividing feed intake by
(Hajiaghapour & Rezaeipour, 2018; Hazrati et al., 2019). On the the body weight gain at the same times.
other hand, the previous researches have focused on the ef- At the end of the study (42 days of age), five broiler chickens
fect of dietary Thr in combination with Saccharomyces cerrviciae per treatment were randomly selected and euthanized for the
(Rezaeipour et al., 2012), feed form and particle size (Rezaeipour assessment of carcass attributes. The viscera for each bird were
& Gazani, 2014) and organic acids (Eftekhari et al., 2015) in the removed manually, and carcass compartments, including the rel-
broiler chickens. Due to the beneficial and common effects of Thr ative weight of the breast, thigh, liver, gallbladder, heart, gizzard
and medicinal plants such as AS on the gut health and morphol- and proventriculus were calculated. In order to determination of
ogy, it can be hypothesized that concurrent use of these two di- the lymphoid organs weight, the weight of bursa and spleen was
etary supplements may improve broiler performance. Therefore, also measured.KOLBADINEJAD and REZAEIPOUR | 3
2.3 | Immune response and serum biochemistry concentrations by spectrophotometric method using commercially
available kites (Parsazmun).
Blood samples were collected from five broiler chickens per treat- In order to measure the antibody titre to Newcastle disease
ment into the non-heparinized tubes through the wing vein on virus (NDV), the broiler chickens were vaccinated against NDV
35 days of age. The blood sample of each sterile test tube was cen- at 7 days of age. The non-heparinized blood samples (five broiler
trifuged at 5,000 g for 5 min to isolate serum. Then, sera were ana- chickens per treatment) were withdrawn from the wing vein
lysed by the using commercial kits (Parsazmun) for liver enzymes 10 days after vaccination. The samples were centrifuged (3,000 g
activity, including aspartate aminotransferase (AST) and alanine for 15 min) at room temperature to separate serum. Serum sam-
aminotransferase (ALT). The remaining part of the serum sample ples were used for the hemagglutination inhibition (HI) test to mea-
was used to measure the serum biochemical metabolites, includ- sure antibody response against NDV (Marquardt, Synder, Savage,
ing glucose, cholesterol, triglyceride and total protein and albumin Kadavil, & Yancey, 1984).
At the end of the trial (day 42), five broiler chickens per treat-
ment were bled and their blood was into tubes containing heparin
TA B L E 1 Composition of basal diets (as-fed basis) as an anticoagulant. The blood smears were provided using May-
Starter Grower Finisher Grunwald-Giemsa stain and the counts of heterophil (H) and lym-
phocyte (L) were conducted.
Item day 0–10 day 10–24 day 24–42
Ingredient (%)
Corn grain 56.00 58.01 62.10 2.4 | Jejunum morphology and microbial
Soya bean meal (440 g 37.42 35.00 30.31 enumeration
CP/kg)
Soya bean oil 1.70 2.90 3.90 To study the jejunum morphology and microbial population, a total
Oyster shell 1.38 1.12 1.00 of five randomly chosen broiler chickens per treatment were eu-
Dicalcium phosphate 1.83 1.63 1.49 thanized on day 42. The gastrointestinal tract was removed, and
Common salt 0.15 0.20 0.15 a segment of the proximal jejunum (approximately 3 cm in length)
Sodium bicarbonate 0.35 0.27 0.25
was excised for morphometric indices. The sample was flushed with
a physiological saline to remove the contents, and then fixed in 10%
Vitamin premix 0.25 0.25 0.25
formalin solution (10%) for 72 hr. The segment was processed, and
Mineral premixb 0.25 0.25 0.25
paraffin embedded after dehydration.Then, a 5-mm section was
DL-Methionine 0.32 0.25 0.21
placed on the glass slide and stained with eosin blue. The 10 long-
L-Lysine-HCl 0.20 0.10 0.08
est and straightest villi and associated crypts were measured in each
Choline chloride 0.05 - -
segment by an automated upright microscope system with LED
L-Threoninec 0.10 0.02 0.01 Illumination (Eftekhari et al., 2015).
Chemical composition Fresh digesta was collected from ileum of five broiler chickens
Metabolizable energy 12.3 12.7 13.1 per treatment to evaluate the number of colony-forming units (cfu)
(MJ/kg) of Lactobacilli, Escherichia coli and total bacteria on day 42. Briefly,
Crude protein (%) 21.9 20.9 19.1 One gram per each sample was serially diluted from 10–1 to 10–7 in
Calcium (%) 1.03 0.88 0.80 sterilized physiological saline solution (NaCl 85%). From these suspen-
Available phosphorous 0.49 0.45 0.41 sions, 0.1 ml was plated onto the following media. E. coli was cultured
(%) on eosin methylene blue agar (Merck) at 37°C for 24 hr. Lactobacilli
Sodium (%) 0.20 0.18 0.16 bacteria were enumerated on de Man, Rogosa, Sharpe agar (Merck)
Lysine (%) 1.40 1.26 1.12 after incubation for 48–72 hr at 37°C. The standard plate count agar
Methionine + Cysteine 1.04 0.94 0.85 (Merck) was used to determination of the enumeration of the total
(%) count bacteria.
Threonine (%) 0.95 0.84 0.76
a
Provides per kilogram of diet: 9,000 IU vitamin A; 2,000 IU vitamin
D3; 18 IU vitamin E; 2 mg menadione; 1.8 mg thiamine; 6.6 mg 2.5 | Statistical analysis
riboflavin; 30 mg niacin; 3 mg pyridoxine; 15 µg vitamin B12; 100 mg
D-pantothenic acid; 1 mg folic acid; 0.1 mg biotin; 500 mg choline
Data were subjected to general linear model (GLM procedure) of SAS
chloride; and 100 mg antioxidant.
b (SAS, 1999) to evaluate the effects of dietary treatments on broiler
Provides per kilogram of diet: 100 mg Mn; 84.7 mg Zn; 50 mg Fe;
10 mg Cu; 1 mg I; and 0.2 mg Se. chickens. The following model was assumed in the analysis of all stud-
c
The dose titrations were achieved by addition of crystalline L-threonine ied parameters: Yijk = µ + Ai + Bj + ABij + eijk, where Yijk = observed
to the basal diets. value for a particular character, µ = overall mean, Ai = effect of Thr4 | KOLBADINEJAD and REZAEIPOUR
levels, Bj = effect of AS, ABij = interaction effect between Thr and AS the present trial, feed intake was not affected by the dietary
and eijk = random error associated with the ijkth recording. The orthog- treatments.
onal polynomial contrasts were used to assess linear and quadratic ef- The dietary treatments had no influence on the relative weight of
fect of dietary Thr. Statements of statistical significance are based on the breast, thigh, liver, heart, gallbladder, gizzard and proventriculus
a probability of p < .05. in broiler chickens at day 42 (Table 3).
3 | R E S U LT S 3.2 | Immunity and lymphoid organs
3.1 | Growth performance and carcass attributes According to Table 4, a significant interaction was observed between
Thr and AS supplementation for lymphocyte, H/L, bursa and spleen
According to Table 2, the diets supplemented with 105% Thr en- (p < .05). Also, enhanced relative weight of spleen was observed for
hanced the body weight gain of broiler chickens during the starter broiler chickens supplemented with 105 and 115% Thr compared
(1–10 days), finisher (25–42 days) and total (1–42 days) periods of with 100 and 110% Thr (p < .05).
the trial (p < .05). Like body weight gain, the addition of 105% Thr
to the diet resulted in an improvement in FCR during starter, fin-
isher and the whole experimental periods (p < .05). The results of 3.3 | Serum metabolites
Table 2 also indicated that the broiler chickens fed AS supplemented
diet had a greater body weight gain and better FCR than those All of the serum metabolites, Except for plasma AST activity, were
without AS during the finisher (25–42 days) phase (p < .05). In not affected by the dietary treatments (Table 5). The liver enzyme
TA B L E 2 Effects of treatments on feed intake, live weight gain and feed conversion ratio (FCR) of broiler chickens
Feed intake (g/days) Live weight gain (g/days) FCR (g/g)
Thr (%) AS (g/kg) 1–10 11–24 25–42 1–42 1–10 11–24 25–42 1–42 1–10 11–24 25–42 1–42
100 0 28.7 88.3 169 95.4 20.7 52.7 63.3 45.6 1.38 1.68 2.72 2.10
105 29.6 88.0 169 95.6 20.1 52.9 70.6 47.8 1.48 1.67 2.41 2.00
110 28.7 88.1 167 94.7 20.9 54.1 75.6 50.2 1.37 1.63 2.22 1.89
115 29.5 87.8 171 96.4 21.6 49.5 87.3 52.8 1.37 1.78 1.97 1.83
100 10 28.8 88.0 168 95.1 19.8 50.0 72.9 59.4 1.46 1.78 2.32 2.01
105 29.3 88.2 171 96.2 19.9 51.6 74.9 48.8 1.48 1.71 2.30 1.97
110 28.5 88.0 166 94.4 20.5 52.9 75.6 47.5 1.40 1.67 2.44 1.99
115 27.9 87.3 164 93.1 18.2 48.4 74.5 47.0 1.54 1.84 2.23 1.98
SEM 0.42 0.32 2.16 0.78 0.57 1.79 3.36 1.15 0.04 0.06 0.12 0.05
Main effects
100 29.2 88.2 169 95.5 20.4ab 52.8 66.9b 46.7b 1.43 1.67 2.56a 2.05a
105 29.1 87.9 169 95.6 21.2a 51.8 81.4a 51.5a 1.37 1.70 2.10 b 1.85b
ab ab b ab
110 29.1 88.1 170 95.7 19.9 50.7 73.9 48.2 1.46 1.74 2.31 1.99a
115 28.2 87.7 165 93.7 19.3b 50.6 71.8b 47.2b 1.47 1.75 2.33ab 1.97ab
SEM 0.30 0.23 1.52 0.55 0.40 1.25 2.37 0.81 0.02 0.04 0.08 0.03
b b a
0 28.7 88.1 168 94.9 20.5 52.4 70.2 47.8 1.40 1.69 2.42 1.99
10 29.1 87.8 169 95.3 19.9 50.6 76.8a 49.1 1.46a 1.75 2.23b 1.94
SEM 0.21 0.16 1.08 0.39 0.28 0.88 1.67 0.57 0.01 0.03 0.06 0.02
p-value
Thr .09 .46 .14 .06 .01 .61KOLBADINEJAD and REZAEIPOUR | 5
TA B L E 3 Effects of treatments on
Thr (%) AS (g/kg) Breast Thigh Liver Heart Gizzard Proventriculus
carcass characteristics and internal organs
of broiler chickens (g/100 g body weight 100 0 22.7 18.5 2.28 0.50 3.14 0.47
of bird) 105 22.1 19.0 2.42 0.49 3.22 0.50
110 22.5 18.6 1.87 0.55 3.55 0.44
115 22.0 19.1 2.30 0.47 3.03 0.44
100 10 23.1 20.1 2.19 0.63 3.42 0.50
105 23.4 18.2 2.20 0.46 3.22 0.48
110 22.0 17.7 2.13 0.56 3.17 0.47
115 24.0 18.3 2.32 0.58 3.02 0.48
SEM 0.93 0.61 0.17 0.05 0.27 0.03
Main effects
100 22.4 19.3 2.35 0.50 3.18 0.48
105 22.6 18.6 2.08 0.51 3.29 0.44
110 24.7 18.1 2.19 0.54 3.32 0.49
115 23.5 18.7 2.23 0.57 3.10 0.47
SEM 0.66 0.43 0.12 0.03 0.19 0.02
0 22.6b 18.8 2.12 0.56 3.32 0.47
10 24.0a 18.6 2.31 0.50 3.12 0.47
SEM 0.46 0.30 0.08 0.02 0.13 0.01
p-value
Thr .07 .31 .50 .53 .83 .36
AS .01 .12 .12 .11 .30 .86
Thr × AS .11 .13 .66 .28 .74 .84
Linear .10 .31 .38 .40 .61 .87
Quadratic .23 .61 .21 .91 .86 .08
Abbreviations: AS, ajwain seed; Thr, threonine.
Means in the same column with different letters differ significantly (p < .05).
activity of AST was higher in the chicks that received diet without contrast with these findings, the different inclusion rates of Thr sup-
AS supplement than those fed AS diet (p < .05). plementation had no influence on the growth performance of broiler
chickens (Eftekhari et al., 2015; Kidd et al., 2005). In agreement
with our results, (Khan, Nawaz, & Zahoor, 2006) and (Rezaeipour
3.4 | Jejunum morphology and microbial population et al., 2012) observed an improvement in the growth performance
of broiler chickens fed different levels of dietary Thr. Similarly, an
The morphological results (Table 6) revealed an increase in villus increase in growth performance was found in broiler chickens as
length, width and crypt depth of the jejunum in 105% Thr group dietary Thr increased (Taghinejad-Roudbaneh et al., 2013). It is re-
(p < .05). Furthermore, the AS supplemented diet increased the jeju- ported that the extensive involvement of Thr in the gut mucin may
nal villus length in broiler chickens (p < .05). be associated with its effect on the broiler performance (Dozier,
In ileal microbial composition (Table 6), the number of Lactobacilli Moran, & Kidd, 2001).
bacteria tended to be higher in broiler chickens receiving 105% Thr The present results showed that the growth performance was
and AS diet (p < .05). In addition, the viable cell count of E. coli was improved in broiler chickens that received AS diet than those
reduced in ileal digesta collected from the broilers receiving 105% without AS supplement. The result was supported by the find-
Thr or AS diets (p < .05). In this experiment, none of the experimental ings of (Valiollahi, Gholami, Namjoo, Rahimian, & Rafiee, 2014)
diets influenced total bacteria in broilers. who indicated that the growth performance was improved in
broiler chickens fed diet supplemented with ajwain powder. This
response may be due to the active substances in the medicinal
4 | D I S CU S S I O N plants with highly odours and pungent taste (Mehri, Sabaghi, &
Bagherzadeh-Kasmani, 2015a). In a study with Japanese quails
In the present experiment, it is observed that 105% Thr supplemen- and in contrast with our results, it is observed that the effect of
tation improved the body weight gain and FCR in broiler chickens. In ajwain powder on the performance criteria was negligible (Kheiri6 | KOLBADINEJAD and REZAEIPOUR
TA B L E 4 Effects of treatments on immune response, antibody titre against Newcastle disease virus (NDV) and lymphoid organs (g/100g
body weight of bird)
Cell-mediated immunity NDV titre (log2) Lymphoid organs
Thr (%) AS (g/kg) Heterophil (%) Lymphocyte (%) H/L 10 days* Bursa Spleen
b ab b
100 0 19.0 41.0 0.46 3.48 0.05 0.07b
105 18.2 41.8a 0.43b 3.38 0.06ab 0.08ab
b a a
110 19.8 40.6 0.49 3.78 0.08 0.10a
115 18.6 41.4ab 0.45ab 3.34 0.05b 0.10a
b a a
100 10 19.8 40.2 0.49 3.32 0.07 0.11a
105 18.0 42.0a 0.43b 2.78 0.07a 0.07b
a b ab
110 17.8 42.2 0.42 3.36 0.06 0.10a
115 19.6 40.5 0.51a 3.74 0.07a 0.09ab
SEM 0.71 0.69 0.02 0.23 0.009 0.006
Main effects
100 18.6 41.4 0.45 3.43 0.05 0.08b
105 19.2 40.8 0.47 3.56 0.06 0.10a
110 18.9 41.2 0.46 3.05 0.07 0.09ab
115 18.7 41.1 0.46 3.55 0.06 0.10a
SEM 0.50 0.48 0.01 0.15 0.006 0.004
0 19.1 40.9 0.47 3.48 0.06 0.10
10 18.6 41.3 0.45 3.31 0.06 0.09
SEM 0.35 0.34 0.01 0.11 0.004 0.003
p-value
Thr .84 .94 .88 .10 .49 .01
AS .32 .54 .47 .28 .93 .11
Thr × AS .07 .04 .03 .19 .02 .03
Linear .67 .66 .65 .10 .12 .08
Quadratic .47 .67 .53 .11 .94 .04
Abbreviations: AS, ajwain seed; Thr, threonine.
*Days post-inoculation.
Means in the same column with different letters differ significantly (p < .05).
et al., 2017). Although the underlying mechanisms by which phy- dietary Thr in different levels did not influence the cell blood counts
togenics affect bird performance is not yet clear, it is documented (CBC) of broiler chickens (Rezaeipour et al., 2012). On the contrary,
that the variation in poultry responses to the aromatic plant seeds (Eftekhari et al., 2015) reported that the H/L ratio was higher in
may be related to the chemical composition of the various seeds, broiler chickens that received different levels of dietary Thr in their
environmental parameters, bird type and diet form (Behnamifar, diet. In addition, an improvement in NDV titre was observed in broil-
Rahimi, Karimi Torshizi, Hasanpor, & Mohamadzade, 2015). This ers with Thr supplementation (Maroufyan et al.., 2010). According to
study has been conducted in June and July months. Therefore, a (Taghinejad-Roudbaneh et al., 2013), Thr is one of the most import-
part of the decline in the rate of FCR in these broilers compared ant components of the plasma γ-globulin and antibody titre against
with commercial conditions may be attributed to the high ambient NDV may be enhanced with increasing of Thr intake in broiler chick-
temperature. ens. In spleen relative weight, the findings of (Eftekhari et al., 2015)
The experimental treatments had no effect on the cell-mediated did not support our result. According to the authors, the dietary Thr
immunity (H, L and H/L ratio), antibody titre against NDV and the did not affect the relative weight of spleen in broiler chickens. There
weight of the bursa in broilers. However, increasing Thr inclusion is no data regarding the mode of action of Thr on the lymphoid or-
rates (105 and 115%) in the diet enhanced the relative weight of the gans status in broilers and further study is needed to evaluate this
spleen in broilers. In agreement with these results, antibody titre mechanism in broiler chickens.
against NDV (Abbasi, Mahdavi, Samie, & Jahanian, 2014) and H/L The plasma AST activity was lower in the broiler chickens that
ratio (Corzo et al., 2007) did not alter in response to enhanced Thr in received AS supplemented diet. However, the other serum metabo-
broiler chickens. Similarly, in our previous study, it is found that the lites were not influenced by the dietary treatments. Liver as a mainKOLBADINEJAD and REZAEIPOUR | 7
TA B L E 5 Effects of treatments on serum metabolites (g/dL) and plasma enzymes activity (IU/L) of broiler chickens
Thr (%) AS (g/kg) Glucose Triglyceride Cholesterol Total protein albumin ALT AST
100 0 175 50.0 108 4.03 2.04 3.04 236
105 212 58.4 119 4.10 2.34 3.22 226
110 213 49.9 112 3.68 1.93 2.96 237
115 225 52.3 105 3.72 2.21 3.21 232
100 10 200 44.8 112 3.82 2.25 3.24 232
105 207 48.2 98.1 3.85 1.92 3.08 229
110 224 41.7 106 3.73 2.10 2.76 229
115 209 48.5 96.1 3.96 1.85 2.86 221
SEM 14.0 4.00 9.01 0.22 0.21 0.28 3.91
Main effects
100 193 54.2 114 4.06 2.19 3.13 231
105 219 51.1 108 3.70 2.07 3.08 234
110 203 46.5 105 3.83 2.09 3.16 231
115 216 45.1 101 3.85 1.97 2.80 225
SEM 9.92 2.8 6.36 0.15 0.15 0.20 2.76
0 203 46.6 109 3.82 2.08 3.00 233a
10 213 51.9 104 3.91 2.07 3.08 227b
SEM 7.01 1.99 4.49 0.11 0.10 0.14 1.95
p-value
Thr .24 .10 .53 .43 .78 .57 .12
AS .33 .07 .42 .56 .99 .67 .04
Thr × AS .32 .85 .53 .96 .29 .90 .82
Linear .47 .06 .32 .30 .62 .91 .89
Quadratic .10 .82 .92 .21 .70 .79 .32
Abbreviations: ALT, alanine aminotransferase; AS, ajwain seed; AST,aaspartate aminotransferase; Thr, threonine.
Means in the same column with different letters differ significantly (p < .05).
organ in metabolism is so sensitive to nutritional status in poultry (Eftekhari et al., 2015). On the other hand, it is known that the func-
and serum activity of AST and ALT are usually considered as more tional development and integrity of the gut mucosa is related to
specific indicators to liver health status. It is reported that the plasma morphometric development in broilers (Chee, Iji, Choct, Mikkelsen,
activity of liver enzymes such as AST and ALT decreased when the & Kocher, 2010). According to the results of (Abbasi et al., 2014),
liver function is normal (Rezaeipour, Barsalani, & Abdullahpour, a large amount of Thr (30 to 50%) as well as some amino acids are
2016). Therefore, it is concluded that the addition of AS to the broil- directly utilized by the gut and are not available for extra-intesti-
ers diet may have a beneficial effect on the liver function following a nal tissues. Furthermore, the negative influence of inadequate Thr
decreasing in AST activity. Along with the potent antioxidant activ- on the intestinal villus length was reported by (Hamard, Sève, & Le
ity, the ajwain methanolic substances revealed to exhibit a hepato- Floc’h, N., 2007).
protective activity (Zarshenas, Moein, Samani, & Petramfar, 2013). In the present experiment, the villus length was greater in broil-
The results of the present trial showed that the villus length, ers that received AS supplementation than those fed diet without
width and crypt depth were greater in broiler chickens that received AS additive. Several reports are available on the beneficial or no im-
105% Thr diet. In parallel with these results, it has been observed pact of phytogenic feed additives on the intestinal villi length and
that diets supplemented with different levels of Thr improve the in- crypt depth of broiler chickens (Demir, Sarica, Ozcan, & Suicmez,
testinal morphometric indices in broiler chickens (Abbasi et al., 2014; 2005; Jamroz, Wiliczkiewicz, Wertelecki, Orda, & Skorupińska,
Rezaeipour et al., 2012; Zaefarian, Zaghari, & Shivazad, 2008). In 2005). Therefore, the results of different studies do not provide a
contrast, no differences were observed in the intestinal morphology consistent view. However, improved digestive capacity in the gas-
of broilers fed different levels of Thr (Kazempour, Shargh, Jahanian, trointestinal tract may be considered an indirect side influence of
& Hassani, 2017). It is well documented that the dietary Thr has an phytogenic stabilizing the microbial eubiosis in the gut (Hashemi &
important role in high demand for the synthesis of intestinal mucin Davoodi, 2011). This conclusion is agreement with the present study
glycoproteins and intestinal mucosal integrity in broiler chickens regarding the effect of AS on the intestinal microbial population8 | KOLBADINEJAD and REZAEIPOUR
TA B L E 6 Effects of treatments on jejunum morphology and viable cell counts of microflora in ileo-caecal of broiler chickens
Jejunum morphology (µm) Microbial population (log10 cfu/g)
Thr (%) AS (g/kg) VL VW CD VL/CD Lactobacillus E. coli Total bacteria
100 0 1,148 126 182 6.29 6.85 6.61 8.58
105 1,158 127 181 6.39 7.20 6.32 9.05
110 1,170 132 187 6.24 7.72 5.40 9.03
115 1,186 133 184 6.43 8.56 5.13 9.01
100 10 1,146 126 174 6.56 6.69 6.71 9.02
105 1,159 126 181 6.38 7.21 5.94 9.06
110 1,146 124 181 6.32 7.22 6.62 8.64
115 1,155 131 178 6.47 7.44 6.39 9.25
SEM 7.27 1.49 2.56 0.08 0.19 0.22 0.25
Main effects
100 1,153b 126b 181ab 6.34 7.03b 6.46a 8.81
a a a a b
105 1,178 132 186 6.33 8.14 5.27 9.02
110 1,152b 126b 178b 6.47 6.95b 6.33b 9.04
b b ab b b
115 1,150 127 180 6.40 7.32 7.50 8.94
SEM 5.13 1.05 1.81 0.05 0.13 0.16 0.17
b b a
0 1,152 127 181 6.35 7.12 6.33 8.82
10 1,164a 129 181 6.42 7.60a 5.94b 9.09
SEM 3.63 0.75 1.27 0.04 0.09 0.11 0.12
p-value
Thr .02 .04 .03 .34 .04 .01 .81
AS .04 .07 .97 .26 .01 .04 .13
Thr × AS .96 .06 .18 .12 .43 .61 .51
Linear .97 .98 .16 .12 .68 .56 .38
Quadratic .02 .02 .03 .34 .02 .03 .69
Abbreviations: AS, ajwain seed; CD, crypt depth; Thr, threonine; VL, villus length: VW, villus width.
Means in the same column with different letters differ significantly (p < .05).
in broiler chickens. Nevertheless, the limited data are available at The results showed that the addition of AS in the diet decreased
the effect of AS on the intestinal morphometric indices in animals. CFU of E. coli and increased beneficial Lactobacillus enumeration.
Therefore, the direct comparison cannot be made. Antibacterial activity and immune enhancement probably are the
The dietary Thr (at level of 105%) increased the colony number of two major mechanisms by which herbal plants exert positive ef-
Lactobacilli, while the viable cell count of E. coli decreased. The result fects on the growth performance and health of animals (Yang, Iji,
of the previous study did not support the present finding (Eftekhari & Choct, 2009). (Valiollahi et al., 2014) came to the same result
et al., 2015). In agreement with our results, Thr supplementation de- in broilers where dietary inclusion of ajwain powder decreased
clined E. coli and Salmonella colonies and increased the Lactobacillus the E. coli population and increased the cfu of Lactobacilli. It is re-
colonies in the caecal contents (Chen et al., 2017). In addition, (Chee ported that the positive effect of medicinal plants, such as ajwain
et al., 2010) observed that excess dietary Thr increased the cfu of on the growth performance of broiler chickens, may be due to the
Lactobacilli, whereas declined the E. coli population in broiler chick- antibacterial and antioxidant properties via photogenic agents
ens. In a study with laying lens, it has been showed that dietary Thr (Kheiri et al., 2017). These phytogenic agents may exert either in-
supplementation increased the abundance of potential beneficial hibitory or stimulatory effects on the harmful bacteria and endog-
bacteria in low crude protein diets (Dong, Azzam, & Zou, 2017). The enous secretion of the gut, respectively, through the functional
beneficial effect of dietary Thr on the intestinal microbial compo- substances including alkaloids, phenolic compounds, terpenoids
sition was possibly associated with increased mucin synthesis and and triterpene saponins (Ghazaghi et al., 2014). AS contains an
immunoglobulins secretions resulting from Thr supplementation essential oil containing about 50%–55% thymol which is a strong
(Chen et al., 2017). It is reported that mucins cannot be digested in antibacterial and fungicide (Chung, Khanh, Lee, & Ahmad, 2007).
the small intestine and thereby can reach the caecum, acting as a In an in vitro study, the antimicrobial activity of the ajwain seed
substrate for beneficial bacteria (Dong et al., 2017). essential oil was investigated (Vitali et al., 2016). According to theKOLBADINEJAD and REZAEIPOUR | 9
authors, among gram-negative species of bacteria, E. coli were in- Chen, Y. P., Cheng, Y. F., Li, X. H., Yang, W. L., Wen, C., Zhuang, S., &
Zhou, Y. M. (2017). Effects of threonine supplementation on the
hibited by the addition of ajwain seed essential oil.
growth performance, immunity, oxidative status, intestinal integrity,
The present study indicated that the inclusion rate of dietary and barrier function of broilers at the early age. Poultry Science, 96,
Thr (105% of requirement) and AS supplement (10 g/kg of diet) in 405–413. https://doi.org/10.3382/ps/pew240
the diet individually improved growth performance, intestinal mor- Chung, I.-M., Khanh, T. D., Lee, O.-K., & Ahmad, A. (2007). Chemical con-
stitutents from ajwain seeds (Trachyspermum ammi) and inhibitory
phology and microbial population of broiler chickens. On the other
activity of thymol, lupeol and fatty acids on barnyardgrass and radish
hand, except for the immunity parameters, no interaction effects seeds. Asian Journal of Chemistry, 19, 1524.
were observed between dietary Thr and AS in the other traits. Corzo, A., Kidd, M. T., Dozier, W. A., Pharr, G. T., & Koutsos, E. A. (2007).
Dietary threonine needs for growth and immunity of broilers raised
AC K N OW L E D G E M E N T S under different litter conditions. The Journal of Applied Poultry
Research, 16, 574–582. https://doi.org/10.3382/japr.2007-00046
The authors gratefully thank the financial support by Islamic Azad
Demir, E., Sarica, S., Ozcan, M., & Suicmez, M. (2005). The use of natu-
University, Qaemshahr Branch. They would also like to thank ral feed additives as alternatives to an antibiotic growth promoter in
Rohullah Abdollahpour (Qaemshahr Branch, Islamic Azad University) broiler diets. Archiv Fur Geflugelkunde, 69, 110–116.
for his statistical guidance. Dong, X. Y., Azzam, M. M. M., & Zou, X. T. (2017). Effects of dietary
threonine supplementation on intestinal barrier function and gut mi-
crobiota of laying hens. Poultry Science, 96, 3654–3663. https://doi.
C O N FL I C T O F I N T E R E S T S org/10.3382/ps/pex185
The authors hereby certify that they have no conflict of interest. Dozier, W., Moran, E. Jr, & Kidd, M. (2001). Comparisons of male and
female broiler responses to dietary threonine from 42 to 56 days
of age. Journal of Applied Poultry Research, 10, 53–59. https://doi.
A N I M A L W E L FA R E S TAT E M E N T
org/10.1093/japr/10.1.53
All broiler chickens were handled in accordance with guidelines Eftekhari, A., Rezaeipour, V., & Abdullahpour, R. (2015). Effects of acid-
described by the Department of Animal Science, Islamic Azad ified drinking water on performance, carcass, immune response, je-
University (Qaemshahr Branch, Qaemshahr, Iran). junum morphology, and microbiota activity of broiler chickens fed
diets containing graded levels of threonine. Livestock Science, 180,
158–163. https://doi.org/10.1016/j.livsci.2015.07.010
ORCID Ghazaghi, M., Mehri, M., & Bagherzadeh-Kasmani, F. (2014). Effects of di-
Vahid Rezaeipour https://orcid.org/0000-0003-3651-2852 etary Mentha spicata on performance, blood metabolites, meat qual-
ity and microbial ecosystem of small intestine in growing Japanese
quail. Animal Feed Science and Technology, 194, 89–98. https://doi.
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L-threonine and Saccharomyces cerevisiae on performance, intestinal metabolites, intestinal morphology and microbial population
morphology and immune response of broiler chickens. South African in broiler chickens. J Anim Physiol Anim Nutr. 2020;00:1–10.
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