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Received: 21 September 2016    Accepted: 28 April 2017

DOI: 10.1111/jpn.12753

ORIGINAL ARTICLE

Plantago ovata in broiler chicken nutrition: Performance,

carcass criteria, intestinal morphology, immunity, and intestinal
bacterial population

A. Divani | F. Bagherzadeh-Kasmani  | M. Mehri

Department of Animal Science, College of

Agriculture, University of Zabol, Zabol, Iran
Correspondence
F. Bagherzadeh-Kasmani, Department of
Animal Science, College of Agriculture,
University of Zabol, Zabol, Iran.
Email: fbkasmani@uoz.ac.ir
Summary
In this experiment, the effect of dietary Plantago ovata (PO) on performance, carcass
criteria, intestinal morphology, immunity, and intestinal bacterial population of broiler
chickens was evaluated. A total of 250 one-­day-­old male broiler chicks (Ross 308)
were randomly assigned to five treatments containing 0, 5, 10, 15, or 20 g/kg of PO
with five replicate pens and 10 birds in each replicate. Dietary PO increased body
weight gain and decreased feed conversion ratio in the finisher period, improving the
performance index (p < .05). Dietary treatments had no effects on carcass criteria, but
breast meat percentage showed an increasing trend with incremental levels of PO in
the diet (p = .069). The length of small intestine, especially jejunum section, as well as
the villus height, villus width, villus area, and goblet cell numbers were significantly
increased with supplemental PO (p < .05). Humoral and cellular immunity parameters,
and oxidation stability of meat were improved due to use of dietary PO (p < .05).
Dietary PO decreased the CFU of Escherichia coli, whereas the Lactobacilli population
was increased (p = .001). Broken-­line regression revealed that dietary PO at the rate of
10 g/kg may results in the best performance in broiler chickens. This study showed
that PO at the level of 10 g/kg could be considered as a beneficial feed additive in
broiler diet.

KEYWORDS
antibody titter, broiler chick, intestinal microbiota, oxidation stability, villus area

1 |  INTRODUCTION
Parallel to excluding antibiotics from the poultry diets, extensive
efforts are taking place to find safe alternatives for antibiotics around
the world. One of the most promising candidates are medicinal plants
as they contain bioactive substances with beneficial effects on ani-
mal health and metabolism (Abdel-­Wareth, Kehraus, Hippenstiel,
& Südekum, 2012; Abdel-­Wareth & Lohakare, 2014; Alimohamadi,
Taherpour, Ghasemi, & Fatahnia, 2014; Forouzanfar, Bazzaz, &
Hosseinzadeh, 2014; Ghasemi, Kasani, & Taherpour, 2014; Ghazaghi,
Mehri, & Bagherzadeh-­Kasmani, 2014; Lee, Everts, Kappert, Yeom,
& Beynen, 2003; Lee et al., 2003; Mehri, Sabaghi, & Bagherzadeh-­
Kasmani, 2015a,b; Mohammadi, Bagherzadeh-­Kasmani, Shojaian,
Mehri, & Karimi-­Torshizi, 2015; Rios & Recio, 2005; Sadeghi, Habibian,
Raei, Farhadi, & Khateri, 2015).
In general, the potential benefits of medicinal plants on bird
responses are attributed to their phenolic compounds (Windisch,
Schedle, Plitzner, & Kroismayr, 2008) that may stimulate the endog-
enous secretions of the gastrointestinal tract (Jang, Ko, Kang, & Lee,
2007; Lovkova, Buzuk, Sokolova, & Kliment’eva, 2001; Yang, Iji, &
Choct, 2009) and absorption capacity as well as antimicrobial and
antioxidant effects in the birds (Ghazaghi et al., 2014; Mehri et al.,
2015a,b).
Plantago ovata (PO) seeds come from plant of the Plantaginaceae
family, which mainly contain phenolic compounds (Nishib, Kodama,
Yukari Noguchi, & Han, 2001) and arabinoxylans (Guo, Cui, Wang,

J Anim Physiol Anim Nutr. 2017;1–11. © 2017 Blackwell Verlag GmbH |  1

wileyonlinelibrary.com/journal/jpn  
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2       DIVANI et al.
& Christopher Young, 2008) acting as antioxidants and pre-­biotics
respectively. Since ancient times, the mucilage of PO seed has been
used in home remedies and preparation of medicines. Recently, there
has been an upturn of interest in PO seeds because of its classification
as soluble dietary fibre, pre-­biotics, and antimicrobial agent with bene-
ficial effect on the gastrointestinal tract (BeMiller, Whistler, Barkalow,
& Chen, 1993; Rodrı́guez-­Cabezas et al., 2003).
Phenolic compounds such as plantaovaside (quercetin
3-­O-­rutinoside 3’-­O-­β-­apioside) and flavonoids along with acteoside
and forsythoside B phenylpropanoid glycosides have been isolated
from PO seeds (Nishib et al., 2001). Guo et al. (2008) found substan-
tial amounts of uronic acid (~15%) in the seed hull of PO, which is a
sugar acid.
Most grain phenolic compounds (~95%) are covalently bound to
cell wall arabinoxylans through ester bonds, while uronic acid is bound
to the arabinoxylans via the acid group. These complex structures
could be hydrolysed by specific intestinal bacteria possessing arab-
inoxylases, resulting in formation of arabinoxylan oligosaccharides
that may be elected as a new class of pre-­biotic candidates. Although
the composition of PO seed indicates that it could play as a natural
antioxidant and pre-­biotic with potential benefits to enhance poultry
health and performance, little information is currently available on
the pre-­biotic potency of arabinoxylan oligosaccharides in vivo(Ney-
rinck et al., 2012). Therefore, the present study aimed to evaluate the
dietary effects of PO on the performance, carcass criteria, intestinal
morphology, immunity, meat quality, and intestinal bacterial popula-
tion of broiler chickens.
2 |  MATERIALS AND METHODS
2.1 | Chemical composition of experimental diets
and PO
Moisture content of the diets was determined by drying in a forced-­air
oven at 135°C for 2 hr (method 930.15, AOAC, 2006). Gross energy
(GE) was determined using an adiabatic oxygen bomb calorimeter
(Model 6300, Parr Instruments, Moline, IL, USA). Experimental diets
and PO grains were analysed for CP (method 990.03, AOAC, 2006),
crude fibre (method 978.10, AOAC, 2006), crude fat (method 920.39,
AOAC, 2006), ash (method 942.05, AOAC, 2006), and amino acids
(method 982.30, AOAC, 2006). For amino acid analysis, all samples
were prepared using a 24-­hr hydrolysis in 6 N hydrochloric acid at
110°C under an atmosphere of nitrogen. For Met and Cys, perfor-
mic acid oxidation was performed before acid hydrolysis. Samples for
Trp analysis were hydrolysed using barium hydroxide (AOAC, 2006).
Chromatographic separations of amino acids were performed with a
Waters HPLC system (Waters, Milford, MA). It consisted of a 1525
Binary HPLC pump, a 2487 Dual λ absorbance detector operating
at 254 nm, Breeze chromatography software, and a Rheodyne 7725
injection valve (Cotati, CA, USA) which equipped with a 20-­μl sample
loop. The column was Pico tag (3.9 × 150 mm I.D.; particle size 5 μm).
Mucilage content of PO was measured according to Sharma and
Koul (1986). In brief, 10 ml of 0.1 N HCl was heated in a 100-­ml flask
until boiling, and then, it was taken off from the heater and 1 g of
dry seeds was added into the solution. After reheating the flask, the
dissociation process of seed husk was initiated and colour of seeds
was changed completely. Finally, the hot solution was filtered through
clean muslin cloth. To remove residual traces of mucilage, the seeds
were washed twice in 5 ml of hot water and filtered. Dissolved muci-
lage was mixed with 60 ml of 95% ethyl alcohol, stirred, and allowed to
stand for 5 hr. Finally, the supernatant liquid was discarded and resid-
ual precipitation was oven-­dried at 50°C.
The total sugar analysis of PO mucilage was performed by the
method of Guo et al. (2008). The sample was hydrolysed in 1 M H2SO4
at 100°C for 3 hr and diluted 10 times. The diluted samples were
passed through a 0.45-­μm filter and injected to a high-­performance
anion exchange chromatography with pulsed amperometric detection
(HPAEC-­PAD; Dionex-­5500, Dionex Corporation, Canada). Separations
were performed with isocratic eluent (100 mM NaOH) on a CarboPac
PA1 column (250.4 mm I.D., Dionex Corporation, Canada) and a guard
column (3 × 25 mm, Dionex Corporation, Canada) at a flow rate of 1 ml/
min. The column system was cleaned after each analysis with 300 mM
NaOH for 30 min. A post-­column delivery system of 600 mM NaOH
with a flow rate of 1 ml/min was added to the HPAEC-­PAD system. The
instrument was controlled, and data were processed using Dionex AI
450 software (Dionex Corporation, Canada). The amount of total sugar
was the sum of monosaccharides in PO mucilage.
2.2 | Animal housing and management
The Research Animal Ethic Committee of the University of Zabol
approved this experimental protocol. The birds in each pen replicate
had free access to feed and water during the experimental period.
The environmental conditions of experimental house were kept at
32 ± 1.5ºC with 60 ± 5% humidity at the first week, and then, the
temperature was gradually decreased by 3ºC each week.
2.3 | Experimental design and dietary treatments
A total of 250 one-­day-­old male broiler chicks (Ross 308) were ran-
domly assigned to five treatment diets containing 0, 5, 10, 15, or 20 g/kg
of PO with five replicates and 10 birds each. Before feed formula-
tion, all protein-­containing feed ingredients were analysed for CP and
essential amino acids, and feed formulation was performed based on
the nutrient analysis. As shown in Table 1, the experimental diets for
starter (0–10 day), grower (11–24 day), and finisher (25–39 day) were
formulated to meet or exceed all nutritional recommendations of
Aviagen (2014) for meat-­type broiler chickens. Control diets and four
PO diets (5, 10, 15, and 20 g PO/kg) were used in this experiment.
2.4 | Performance and carcass processing
During the experimental period (from 1 to 39 day of age), feed intake
(FI) and body weight gain (BWG) were measured at 10, 24, and
39 day of age, and then, feed conversion ratio (FCR) was calculated
accordingly. European production index (EPI), which is widely used to
T A B L E   1   Composition of experimental diets

Starter (1–10 day) Grower (11–24 day) Finisher (25–39 day)

DIVANI et al.

Plantago ovata (g/kg of diet) Plantago ovata (g/kg of diet) Plantago ovata (g/kg of diet)

Feed ingredients 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20

Corn, grain 460.95 462.63 464.31 465.99 467.66 489.84 491.65 492.53 493.35 494.16 536.18 537.99 539.79 541.59 543.40
Soybean meal 350.00 350.00 350.00 350.00 350.00 366.45 366.10 362.91 359.50 356.10 336.82 336.45 336.08 335.71 335.34
Corn gluten meal 86.98 85.15 83.32 81.49 79.66 24.35 22.79 23.74 24.87 26.00 6.20 4.67 3.13 1.59 0.05
Sunflower oil 31.75 31.83 31.92 32.00 32.09 58.00 58.00 58.00 58.00 58.00 62.00 62.00 62.00 62.00 62.00
Plantago ovata 0.00 5.00 10.00 15.00 20.00 0.00 5.00 10.00 15.00 20.00 0.00 5.00 10.0 15.00 20.0
Corn starch 20.00 15.00 10.00 5.00 0.00 20.00 15.00 10.00 5.00 0.00 20.00 15.00 10.0 5.00 0.0
DCP 17.14 17.15 17.16 17.17 17.18 14.87 14.88 14.92 14.94 14.97 13.71 13.72 13.73 13.74 13.76
Oyster shells 15.10 15.10 15.09 15.09 15.08 12.28 12.28 12.28 12.90 14.16 11.82 11.81 11.81 11.80 11.80
NaHCO3 4.39 4.40 4.41 4.42 4.42 2.94 2.96 3.13 3.31 3.49 3.47 3.50 3.53 3.55 3.58
L-­Lys ≅ HCl 4.24 4.26 4.27 4.29 4.30 2.11 2.14 2.23 2.33 2.43 0.83 0.85 0.88 0.90 0.92
DL-­Met 1.86 1.88 1.90 1.92 1.94 2.44 2.48 2.49 2.49 2.50 2.21 2.26 2.30 2.34 2.38
NaCl 0.62 0.61 0.61 0.60 0.60 1.34 1.33 2.38 2.88 2.75 1.75 1.74 1.74 1.73 1.72
L-­Thr 1.97 1.99 2.02 2.04 2.06 0.37 0.39 0.40 0.41 0.43 0.00 0.01 0.02 0.03 0.05
Mineral pre-­mixa 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50
b
Vitamin pre-­mix 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50 2.50
Nutrient specifications
ME (MJ/kg) 12.68 12.68 12.68 12.68 12.68 13.18 13.18 13.18 13.18 13.18 13.39 13.39 13.39 13.39 13.39
CP (g/kg) 244.70 244.70 244.70 244.70 244.70 220.00 220.00 220.00 220.00 220.00 200.00 200.00 200.00 200.00 200.00
Lys (g/kg) 14.50 14.50 14.50 14.50 14.50 12.80 12.80 12.80 12.80 12.80 11.00 11.00 11.00 11.00 11.00
Met (g/kg) 6.40 6.40 6.40 6.40 6.40 5.90 5.90 5.90 5.90 5.90 5.20 5.20 5.20 5.20 5.20
TSAA (g/kg) 10.80 10.80 10.80 10.80 10.80 9.80 9.80 9.80 9.80 9.80 8.80 8.80 8.80 8.80 8.80
Thr (g/kg) 9.90 9.90 9.90 9.90 9.90 8.50 8.50 8.50 8.50 8.50 7.40 7.40 7.40 7.40 7.40
Trp (g/kg) 2.60 2.60 2.60 2.60 2.60 2.50 2.50 2.50 2.50 2.50 2.30 2.30 2.30 2.30 2.30
Ca (g/kg) 10.50 10.50 10.50 10.50 10.50 9.00 9.00 9.00 9.00 9.00 8.50 8.50 8.50 8.50 8.50
Available P (g/kg) 5.00 5.00 5.00 5.00 5.00 4.50 4.50 4.50 4.50 4.50 4.20 4.20 4.20 4.20 4.20
DEB (mEq/kg)c 250.00 250.00 250.00 250.00 250.00 250.00 250.00 250.00 250.00 250.00 250.00 250.00 250.00 250.00 250.00
a
Mineral pre-­mix provided per kilogram of diet: Mn (MnSO4·H2O), 120 mg; Zn (ZnO), 110 mg; Fe (FeSO4·7H2O), 50 mg; Cu (CuSO4·5H2O), 16 mg; I [Ca (IO3)2·H2O], 1.80 mg; Se, 0.30 mg; Co (Co2O3), 0.20 mg;
and Mo, 0.16 mg.
b
Vitamin pre-­mix provided per kilogram of diet: vitamin A (vitamin A acetate), 12,000 IU; cholecalciferol, 5,000 IU; vitamin E (dl-­α-­tocopheryl acetate), 80 IU; vitamin B12, 0.60 mg; riboflavin, 8.8 mg; nicotin-
amide, 60 mg; calcium pantothenate, 35 mg; menadione (menadione dimethyl-­pyrimidinol), 1.50 mg; folic acid, 2.00 mg; thiamine, 3 mg; pyridoxine, 10 mg; biotin, 1 mg; choline chloride, 1,600 mg; and ethoxy-
quin, 125 mg.
|

c
DEB, dietary electrolyte balance (Na + K – Cl).
      3
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4       DIVANI et al.
describe the efficiency of broiler production, was calculated as [(body
weight (kg) × livability (%) ÷ rearing period (day) × FCR) ÷ 10] (Nollet,
Huyghebaert, & Spring, 2008). At the end of experiment, three birds
from each pen were euthanized by cervical dislocation and hot carcass
(without neck, giblets, and feet) was weighed immediately. The length
of small intestine and relative weight of different sections (e.g., breast
meat, thigh meat, heart, liver, proventriculus, gizzard, spleen, bursa of
Fabricius) to live body weight were measured accordingly.
2.5 | Intestinal morphology
As described by Awad, Ghareeb, and Böhm (2011), the ileal sections
of three birds in each replicate were fixed in 4% buffered formalin
for 48 hr. The processing consisted of serial dehydration, clearing
(Tissue Processor, Shandon Duplex, USA), and impregnation with
wax. Tissue sections with 5 μm thickness (three cross sections from
each of sample) were cut by a microtome and were fixed on slides.
The staining procedure was carried out using haematoxylin and
eosin. The slides were examined on an Olympus BX51 microscope
(Olympus Corporation, Tokyo, Japan) fitted with a digital video cam-
era (Olympus DP79, Japan). The images were analysed using ImageJ
analysis software (Version 1.47, Bethesda, MD, USA). A total of 15
intact well-­oriented, crypt–villus units were selected randomly for
each sample. The mean values attributed to individual birds were used
in the statistical analysis. Villus height was measured from the tip of
the villus to the villus–crypt junction, while crypt depth was defined as
the depth of the invagination between adjacent villi. The villus width
was measured at its middle. Area of villus was calculated by the fol-
lowing formula (Mehri et al., 2015a):
Villus area (cm2) = [(2π × villus width) ÷ 2] × villus length.
2.6 | Humoral immune responses
All birds were vaccinated against Newcastle disease virus (NDV)
and sheep red blood cells (SRBC) antigens. Blood sampling was per-
formed from four birds in each replicate at 39 day of age. Vaccination
against NDV was performed on day 21 using the lyophilized vaccine
(Live B1 strain; Vetrina; Zagreb, Croatia), and anti-­NDV titter was
assessed by a hemagglutination inhibition test on sera obtained on
day 39. Moreover, 0.1 ml of 5% SRBC in PBS was injected through
wing vein at 25 day of age. Antibody production against antigens was
assessed by a hemagglutination test in the serum samples according
to (Cheema, Qureshi, & Havenstein, 2003).
2.7 | Cellular immune responses
When birds were 28 days old, phytohemagglutinin-­P (PHA-­P) was
injected at dose of 0.1 ml of PHA-­P (1 mg of PHA-­P per 1 ml of ster-
ile phosphate buffer saline, PBS) in inter-­digital space between 3rd
and 4th toe of the right foot of the individual chick according to the
methodology described by Corrier and DeLoach (1990). The left foot
served as controls and received the same amount of sterile PBS. The
individual skin index for the right and left foot was calculated as the
difference between the skin thicknesses measured by micrometre
before and after 24 hr of injection and expressed as millimetres. Foot
pad index (FPI) was calculated as the difference in skin index of right
and left foot for individual bird.
2,4-­Dinitro 1-­chlorobenzene (DNCB; Merck; Darmstadt, Germany)
was dissolved in a mixture of acetone and olive oil (4:1 vol/vol) to a final
concentration of 1 mg/ml. On day 39, the skin of the birds was rubbed
with 0.1 ml of DNCB solution. An area of approximately 10 cm2 with-
out feathers on the left lateral abdomen was chosen for the challenge
with DNCB. The same area on the right side was treated with the sol-
vent alone. The skin thickness (on both sides) before and 24 hr after the
challenge was measured to assess reactions. Differences before and
after the challenge were calculated to determine the mean increase in
skin thickness in each bird (Verma, Johri, Swain, & Ameena, 2004).
2.8 | Malondialdehyde assay
At the end of experiment, two birds in each pen were killed by cervi-
cal dislocation and deboned meat of thigh portions was ground with
a blender and stored in −20°C for 30 day to determine the oxidation
stability in meat samples. To evaluate the extent of lipid peroxidation
in meat samples, third-­order derivative spectrophotometric method
developed by Botsoglou et al. (1994) with minor modifications was
adopted. In brief, one gram of grounded meat sample was weighed
and homogenized (Polytron homogenizer, PCU, Switzerland) with 4 ml
of 5% aqueous trichloroacetic acid (TCA) and 2.5 ml of 0.8% butylated
hydroxytoluene and then centrifuged at 3,000 × g for 3 min. The top
hexane layer was discarded, and the bottom layer was filtered and made
to 5 ml volume with 5% TCA, then placed into a screw-­capped tube con-
taining 3 ml of 0.8% aqueous 2-­thiobarbituric acid (TBA). Finally, tubes
were heated in 70°C water bath for 30 min, then immediately cooled
under tap water and submitted to spectrophotometry (UNIKON 933,
Kontron, Milan, Italy). The height of the third-­order derivative peak that
appeared at 521.5 nm was used for calculation of the malondialdehyde
(MDA) concentration, as secondary oxidation product, in the samples.
Tetraethoxypropane (1,1,3,3-­tetraethoxy propane, T9889, 97%, Sigma,
USA) was used as a MDA precursor in the standard curve. The concen-
tration of MDA was expressed as micrograms per gram of meat samples.
2.9 | Bacterial populations of ileal content
The ileal contents (10 cm anterior to the junction between the ceca,
ileum, and colon) of two birds in each pen were separately collected
into the sterile tubes for serial dilution as described by Ghazaghi et al.
(2014). In brief, 1 g of ileal digesta was added into the test tube con-
taining 9 ml of sterilized phosphate-­buffered saline, and buffered
solutions were transferred to the microbial laboratory of our insti-
tute. Bacterial populations were determined by serial dilution (10−4–
10−6) of ileal samples before inoculation onto Petri dishes. Plates for
Lactobacilli (grown on deMan, Rogosa, and Sharpe, MRS agar) and
Escherichia coli (grown on Mac Conkey agar) were incubated at 37°C
in anaerobic and aerobic media respectively. Plate count agar was
DIVANI et al. |
      5

T A B L E   2   Effect of Plantago ovata on the performance of broilers

Plantago ovata (g/kg of diet) Probabilities

Item 0 5 10 15 20 SEM Model Lin. Quad. Contrast

1–10 days
Body weight gain 19.3 18.0 20.1 19.2 18.0 0.43 .013 .338 .103 .378
(g/day)
Feed intake (g/day) 35.9 33.6 32.6 32.6 31.8 0.59 .001 .001 .075 <.001
Feed conversion 1.87 1.86 1.62 1.70 1.76 0.03 .001 .001 .001 .001
ratio
11–24 days
Body weight gain 52.1 50.3 52.6 51.3 47.6 0.82 .003 .006 .023 .094
(g/day)
Feed intake (g/day) 113 105 97.9 102 99.5 1.55 .001 .001 .001 <.001
Feed conversion 2.16 2.10 1.86 1.98 2.09 0.04 .001 .056 .001 .003
ratio
25–39 days
Body weight gain 80.7 91.3 103 96.1 99.6 2.74 .001 .001 .005 <.001
(g/day)
Feed intake (g/day) 213 221 212 211 221 5.00 .435 .714 .540 .562
Feed conversion 2.65 2.43 2.07 2.20 2.22 0.04 .001 .001 .001 <.001
ratio
1–39 days
Body weight gain 54.7 57.8 63.6 60.3 60.0 1.19 .001 .002 .002 <.001
(g/day)
Feed intake (g/day) 132 132 125 126 129 1.99 .096 .099 .091 .111
Feed conversion 2.41 2.28 1.97 2.09 2.15 0.02 .001 .001 .001 <.001
ratio

SEM, standard error of the means; Lin. Quad., linear and quadratic responses, respectively, to dietary inclusion levels; Contrast, contrast comparison per-
formed between control and PO groups.

used for total count of bacteria under aerobic (TAB) condition. Finally,
plates were counted between 24 and 48 hr after inoculation. All agar
media were obtained from the Merck Company, Germany. Colony-­
forming units (CFU) were defined as distinct colonies under magnifica-
tion using a binocular magnifier.
The orthogonal polynomial contrast test was performed to deter-
mine linear and quadratic effects of increasing inclusion level of PO
in diets on each measurement. Broken-­line regression analysis was
employed using NLIN procedure (Robbins, Saxton, & Southern, 2006)
to determine optimal inclusion rate of dietary PO [eq. 3]:

Y = L + U × (R − X) × (X < R) + V × (X − dR) × (X > R) (3)

2.10 | Statistical analysis
Growth performance data of each pen were analysed by the GLM pro-
cedure for completely randomized design [eq. 1], while carcass traits,
immunity responses, malondialdehyde assay, bacterial, and morpho-
logical measurements data were obtained on bird basis and analysed
by MIXED procedure [eq. 2], in which the effect of bird was included
as a random effect nested within fixed effect (SAS, 2002):
yij = μ + Ti + εij
yijk = μ + Ti + Bj (Ti ) + εijk
where yij = the variable of interest for the i treatment; μ  =  the overall
mean; Ti = treatment; εij = residual random error; yijk = the variable of
interest for the j bird nested in i treatment; μ  =  the overall mean;
Ti = treatment (fixed effect); Bj(Ti)  = the bird effect (random effect)
nested in i treatment; and εijk = residual random error.
where Y is the bird response; L is the asymptote for the first segment;
U and V are the slopes for the first and second lines, respectively, with
increasing or descending slope; and R is the break point that is consid-
ered as “optimal” point.
3 | RESULTS
(1)
Table 2 represents the FI, BWG, and FCR data for different stages
(2)
of growth. During 1–10 and 11–24 day of age, dietary PO signifi-
cantly affected the bird performance in which FI and FCR decreased
in birds that received 10 g/kg of dietary PO (p < .001). Contrast anal-
ysis showed that BWG was not affected by inclusion of PO in the
diet during the first 10 day of experiment (p = .378), while a nonlinear
reduction in BWG was observed from 11 to 24 day of age (p = .094).
6       | DIVANI et al.

400 (p < .0001; Figure 1). Broken-­line regression revealed that maximum

European production index

BWG and minimum FCR could be achieved by 10 g/kg of dietary PO
350
(Figure 2).
300 As shown in Table 3, dietary treatments did not affect the carcass
portions; however, breast meat showed an increasing trend with the
250
use of PO in the diet (p = .069). Inclusion of PO in the diet increased
200 the relative weight of proventriculus (p = .018).
Table 4 shows the effects of dietary treatments on morphologi-
150
cal characteristics of small intestine (SI). The length of SI was linearly
0

10

15

20
5
0.

Plantago ovata (g/kg) increased in birds that received PO (p = .001). Among three sections
of SI, the length of jejunum (p = .001) and ileum (p = .039) sections sig-
F I G U R E   1   Effect of dietary Plantago ovata on European
nificantly increased in birds fed supplemental PO; however, the length
production index of broiler chickens
of duodenum (p = .160) tends to be increased with supplemental PO.
During 25–39 and 1–39 day of age, BWG and FCR significantly Inclusion of PO in the diet increased villus height, villus width, villus
increased and decreased, respectively, in response to inclusion of area, and goblet numbers in the duodenum and jejunum in relation to
PO in the diet (p < .001). Dietary PO significantly improved the EPI the control group (p < .05).
Feed conversion ratio (g:g)

70 2.6
(a) (b)
Break point: 10.0
65 2.4
R 2: .95
Gain (g)

60 2.2

55 Break point: 10.0 2.0

R 2: .92
50 1.8
F I G U R E   2   Break points of maximum
0 5 10 15 20 25 0 5 10 15 20 25
gain and minimum feed conversion ratio in
Plantago ovata (g/kg) Plantago ovata (g/kg) response to dietary Plantago ovata

T A B L E   3   Effect of Plantago ovata on the carcass attributes and internal organs of broilersa

Plantago ovata (g/kg of diet) Probabilities

Item 0 5 10 15 20 SEM Fixed effect Random effect Lin. Quad. Contrast

Live weight (g) 2,273 2,396 2,578 2,372 2,462 90.8 .045 .305 .106 .081 .025
Carcass (g) 1,324 1,451 1,549 1,406 1,505 58.5 .013 .543 .022 .064 .003
Dressing (%) 58.4 60.6 60.1 59.2 61.1 1.27 .240 .129 .192 .766 .097
Breast meat (g) 526 582 621 550 570 30.8 .069 .496 .419 .035 .034
Breast meat 23.1 24.3 24.1 23.1 23.1 0.70 .284 .333 .463 .125 .384
yield (%)
Thigh meat (g) 417 423 446 424 444 16.4 .308 .471 .146 .697 .194
Thigh meat 18.4 17.7 17.3 17.9 18.1 0.60 .454 .554 .719 .092 .160
yield (%)
Heart (%) 0.62 0.59 0.57 0.64 0.62 0.02 .057 .026 .325 .130 .581
Liver (%) 2.12 2.03 2.13 2.27 2.16 0.13 .457 .984 .198 .989 .711
Proventriculus 0.49 0.48 0.45 0.53 0.51 0.0 .018 .700 .038 .102 .839
(%)
Bursa of 0.21 0.22 0.22 0.23 0.19 0.03 .705 .889 .881 .252 .725
Fabricius (%)
Spleen (%) 0.10 0.09 0.10 0.11 0.10 0.01 .614 .665 .367 .720 .713
Gizzard (%) 2.58 2.50 2.58 2.82 2.50 0.17 .257 .383 .546 .195 .701

SEM, standard error of the means; Lin. Quad., linear and quadratic responses, respectively, to dietary inclusion levels; Contrast, contrast comparison per-
formed between control and PO groups.
The percentage values are based on the relative weights of carcass parts or internal organs as the percentages of the live body weight.
DIVANI et al. |
      7

T A B L E   4   Effect of Plantago ovata on intestinal morphology of broilers

Plantago ovata (g/kg of diet) Probabilities

Fixed Random
Item 0 5 10 15 20 SEM effect effect Lin. Quad. Contrast

Small intestine (cm) 185 183 197 191 195 2.97 .001 .029 .002 .382 .024
Duodenum
Length (cm) 15.0 16.2 15.9 15.3 15.0 0.56 .160 .591 .451 .036 .187
Villus height (μm) 1,535 1,680 2,235 2,040 2,215 68.3 .001 .272 .001 .014 <.001
Villus width (μm) 145 150 165 175 145 9.55 .021 .630 .239 .006 .074
2
Villus area (cm ) 69.8 79.6 116 113 101 9.06 .001 .448 .001 .003 <.001
Crypt depth (μm) 147 150 166 161 171 3.98 .001 .059 .001 .576 <.001
Goblet number 22 22 25 23 25 0.80 .001 .467 .001 .925 .027
Jejunum
Length (cm) 81.9 81.6 88.2 86.5 91.8 1.36 .001 .286 .001 .440 <.001
Villus height (μm) 1,460 1,580 2,055 1,865 1,815 37.6 .001 .161 .001 .001 <.001
Villus width (μm) 135 120 145 160 120 9.55 .001 .630 .633 .023 .866
Villus area (cm2) 61.8 59.5 93.5 93.6 68.3 4.96 .001 .278 .001 .001 <.001
Crypt depth (μm) 173 171 197 173 168 16.3 .993 .904 .920 .616 .730
Goblet number 22 21 26 24 26 0.88 .001 .115 .001 .263 .002
Ileum
Length (cm) 87.6 85.4 92.6 89.5 88.3 2.08 .039 .024 .355 .241 .518
Villus height (μm) 1,205 1,215 1,300 1,270 1,280 33.9 .049 .021 .044 .320 .084
Villus width (μm) 120 155 170 160 155 10.7 .003 .691 .004 .001 <.001
Villus area (cm2) 45.5 59.2 69.5 63.9 62.3 5.23 .004 .852 .002 .002 <.001
Crypt depth (μm) 163 172 176 165 168 5.06 .120 .442 .820 .057 .083
Goblet number 22 23 27 25 26 2.27 .276 .631 .840 .398 .273

SEM, standard error of the means; Lin. Quad., linear and quadratic responses, respectively, to dietary inclusion levels; Contrast, contrast comparison per-
formed between control and PO groups.

All immunity parameters were affected by dietary treatments
(Table 5). Antibody production against SRBC (p = .054) and NDV
(p = .001) antigens increased in birds fed supplemental PO compared
with control. Moreover, FPI and skin thickness responses to PHA-­P
and DNCB challenge, respectively, increased in PO groups in relation
to the control group (p = .001). As shown in Table 5, a nonlinear reduc-
tion in MDA content of meat samples was observed due to use of
dietary PO (p = .001). Contrast analysis of control versus PO showed
that immunity parameters and oxidation stability of meat samples have
been improved due to use of PO in the diet (p = .001; Table 5). Broken-­
line regression revealed that maximum skin thickness responses to
PHA-­P and minimum MDA content of meat samples could be achieved
by 11.3 and 9.62 g/kg of dietary PO respectively (Figure 3).
As shown in Table 6, supplemental PO decreased the numbers of
TAB and E coli but increased Lactobacilli numbers (p = .001).
4 | DISCUSSION
Sugar analysis of PO mucilage showed that the main monosaccha-
rides were arabinose (52.33 per cent) and xylose (20.97 per cent)
presumably due to high content of arabinoxylan (Table 7), which
was parallel with previous studies (Edwards, Chaplin, Blackwood, &
Dettmar, 2003; Kennedy, Sandhu, & Southgate, 1979). The chemical
bonds of arabinoxylans, phenolic compounds, and uronic acid could
be hydrolysed by specific bacterial enzymes resulting in formation of
arabinoxylan oligosaccharides that could serve as pre-­biotics in the
intestine (Neyrinck et al., 2012). One of the most predominant mecha-
nisms of pre-­biotics is the increase in short-­chain fatty acids produc-
tion or modifying their relative concentrations (e.g., lactic acid, acetic
acid, propionic acid, and butyric acid) in the distal part of the gut dur-
ing the fermentation and thereby mild acidification of the intestinal
digesta and modulation of intestinal microflora. In addition, produc-
tion of butyric acid may cause high regeneration at epithelial levels
of intestinal cells through pro-­apoptotic potency. The pro-­apoptotic
reactions may increase the expression of binding proteins or active
carriers associated with mineral absorption, enhance immunity and
modulate the mucin production (Watson & Preedy, 2016).
The results of this study showed that 10 g/kg of dietary PO
increased BWG and EPI. This improvement in broiler performance was
associated with increasing breast mass and decreasing FCR, indicat-
ing that nutrient utilization might be more efficient in birds fed PO
in relation to control group. In fact, the use of PO in the diet signifi-
cantly increased the jejunal section of the SI providing better nutrient
 8       | DIVANI et al.

T A B L E   5   Effect of Plantago ovata on immune response to Newcastle disease virus, sheep red blood cells, phytohemagglutinin-­P, and
2,4-­dinitro 1-­chlorobenzene, and meat quality of growing broilers

Plantago ovata (g/kg of diet) Probabilities

Fixed Random
Item 0 5 10 15 20 SEM effect effect Lin. Quad. Contrast

Anti-­NDV titter (log2) 4.50 8.10 8.50 7.90 7.90 0.51 .001 .721 .001 .001 <.001
Anti-­SRBC titter (log2) 4.00 4.75 4.83 5.37 4.87 0.41 .054 .981 .001 .049 .004
Increase in foot pad index 0.19 0.44 0.46 0.45 0.34 0.04 .001 .335 .001 .001 <.001
to PHA-­P (mm)
Increase in mean skin 1.62 2.87 2.99 2.98 2.89 0.19 .001 .145 .001 .001 <.001
thickness to DNCB (mm)
MDA (μg/g) 0.16 0.14 0.11 0.14 0.15 0.003 .001 .998 .178 .001 <.001

NDV, newcastle disease virus; SRBC, sheep red blood cells; PHA-­P, phytohemagglutinin-­P; DNCB, dinitrochlorobenzene; MDA, malondialdehyde; SEM,
standard error of the means; Lin. Quad., linear and quadratic responses, respectively, to dietary inclusion levels; Contrast, contrast comparison performed
between control and PO groups.

0.6 0.18
Malondealdehyde (mg/kg)
response to PHA-P (mm)

(a) (b)
Break point: 9.62
0.16 R 2: .98
Skin thickness

0.4

0.14
Break point: 11.3
F I G U R E   3   Break points of
0.2 R 2: .83
0.12 maximum skin thickness response to
phytohemagglutinin-­P (PHA-­P) and
0.0 0.10
minimum amount of malondialdehyde
0 5 10 15 20 25 0 5 10 15 20 25 in meat samples in response to dietary
Plantago ovata (g/kg) Plantago ovata (g/kg) Plantago ovata

T A B L E   6   Effect of Plantago ovata on ileal total aerobic bacteria, Escherichia coli, and Lactobacilli populations in growing broilers

Plantago ovata (g/kg of diet) Probabilities

Microbial population Fixed Random

(Log10 CFU/g) 0 5 10 15 20 SEM effect effect Lin. Quad. Contrast

Total aerobic bacteria 9.40 9.14 9.05 7.81 6.53 0.07 .001 .157 .053 .042 <.001
E coli 7.11 6.98 6.87 6.77 6.46 0.04 .001 .721 .001 .001 <.001
Lactobacilli 8.74 9.05 9.07 9.08 9.20 0.03 .001 .981 .001 .002 <.001

CFU, colony-­forming unit; SEM, standard error of the means; Lin. Quad., Linear and quadratic responses, respectively, to dietary inclusion levels; Contrast,
contrast comparison performed between control and PO groups.

absorption in the gut. This study indicated that PO may increase the
development of the gastrointestinal tract that is evident with increas-
ing digestive and absorption capacities. Moreover, the proliferation
of intestinal cells possibly through enhancing mitotic phase of epi-
thelial cells resulted in longer villus length in birds receiving PO. As
indicated in this study, height and width of villus, crypt depth, and vil-
lus area significantly increased in birds that received dietary PO. The
higher villus height and crypt depth indicate that expression of brush
border enzymes and the rate tissue turnover to renewal villus have
been increased in PO groups. The shortest villi were found in control
treatments, which was concomitant with lowest CFU of Lactobacilli.
Mehri et al. (2015a) suggested that control birds had lower ileal
population of beneficial bacteria than those received dietary pepper-
mint. Apparently, Lactobacilli and Bifidobacteria have significant contri-
butions to villi height (Baurhoo, Phillip, & Ruiz-­Feria, 2007). In addition,
natural pre-­biotics in PO mucilage are feed components that cannot
be absorbed or hydrolysed by enzymes of the upper gastrointestinal
tract, but are fermented selectively by some types of bacteria in the
lower sections of intestine, thereby exerting a beneficial health effect
on their host and increasing villus area (Mehri et al., 2015a).
As shown by contrast analysis, the birds receiving PO had bet-
ter immunity responses and oxidation stability than those receiving
the control diet. A possible mechanism for increasing the immunity
response in birds fed medicinal plants is the protective effects of
DIVANI et al. |
      9

phenolic compounds in botanicals pertaining antioxidant capacity

82.08 ± 2.75
Mucilage (%)

19.45 ± 0.35

0.01
against oxidative damages in the cells (Khan et al., 2012). De la Fuente

Cys
(2002) showed that the antioxidant molecules preserve an adequate

Total
function of immune cells against homoeostatic disturbances caused by

0.02
oxidative stress. In addition to augmenting immunity, we showed that

Trp
dietary PO decreased the amounts of MDA in meat samples revealing
another antioxidant effects in the biological system (El Sheikh, 2014).

0.06
Lys
Plantago ovata could be considered as multiple source of phenolic
compounds such as flavonoids and propanoids with wide expectable
19.80 ± 0.45

0.06
impact on the bird responses (Haddadian, Haddadian, & Zahmatkash,

Met
1.17 ± 0.07
Mannose
CF (g/kg)

2014; Mehri et al., 2015b). The bioactive materials in medicinal plants

might also increase immune functions through the phagocytic capac-

0.07
ity of the immune cells (Karami, Lotfi, & Aminzade, 2013; Pourhossein
His et al., 2014). Moreover, the adaptive immune system is influenced by
0.14 bacterial colonization in the gut and any disturbances in this process
may result in malfunction of immunity (Round & Mazmanian, 2009).
Tyr

The present study showed that dietary PO changed the bacterial

community in the small intestine in favour of beneficial bacteria (e.g.,
1.44 ± 0.23
2.80 ± 0.05

Rhamnose

0.20
Asp

Lactobacilli). It has been documented that this relationship between

Ash (%)

modulation of gut microbiota and immunity system relies on the molec-

ular pathways governing the host–symbiont interactions that regulate
0.27
Phe

proper immune function (Round & Mazmanian, 2009). The CD4+ T cells
are a key component of the adaptive immune system, which are mostly
located in the lamina propria of the intestine. These cells have a pivotal
0.31

role in activating immune responses, and gut microbiota is the main

Ile
1.94 ± 0.06

factor in the development of CD4+ T cells (Wu & Wu, 2012).

4.35 ± 0.08

Glucose

Based on the current study, PO has shown the positive potential to

EE (%)

0.36
Ser

improve the microbial community of the gut. These beneficial effects

of PO may attribute to its fibre content with bifidogenic properties
(Elli, Cattivelli, Soldi, Bonatti, & Morelli, 2008). It has been reported
0.38
Thr

that PO fibre as a source of mucilage is usually listed among the

pre-­biotics (Bhatia & Ahuja, 2013). Pollet et al. (2012) showed that
arabinoxylans of PO could be used as pre-­biotics by human intesti-
17.54 ± 0.11

nal microbiota. Pre-­biotics have the ability to stimulate the growth of

4.23 ± 0.53
Galactose

0.39
Val

beneficial bacteria giving rise to production of unbranched short-­chain

CP (%)

fatty acids such as acetic, propionic, butyric, and lactic acids, thereby
lowering intestinal pH (Teitelbaum & Walker, 2002). Low pH of gut
0.41
Ala

medium inhibits the growth of potentially harmful bacteria.

T A B L E   7   Chemical composition of Plantago ovata (PO)

Monosaccharide analysis of Plantago ovata mucilage (%)

0.46
Pro

5 | CONCLUSION
Amino acid content of Plantago ovata seeds (%)
20.97 ± 0.88
GE (kcal/kg)

4618 ± 3.51

Arabinose

In conclusion, PO could be considered as a useful feed additive in

0.46
Arg

broiler production because of beneficial effects on broiler perfor-

mance, humoral and cellular immunities, and oxidation stability of
meat. The cfu of E  coli was decreased by PO, while the Lactobacilli
0.48
Leu

population as well as ileal absorption surface was increased. We spec-

ulated that the positive effects of PO may be mainly related to the
Proximate analysis

pre-­biotic properties of this herb.

0.59
52.33 ± 1.84

Gly
93.35 ± 0.13
DM (%)

Xylose

CO NFL I C T O F I NT ER ES T
1.35
Glu

There are no known conflicts of interest.

 |
10       DIVANI et al.
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How to cite this article: Divani A, Bagherzadeh-Kasmani F,
Mehri M. Plantago ovata in broiler chicken nutrition:
Performance, carcass criteria, intestinal morphology, immunity,
and intestinal bacterial population. J Anim Physiol Anim Nutr.
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