Effects of a Lactobacillus salivarius mixture on performance, intestinal health and serum lipids of broiler chickens
Effects of a Lactobacillus salivarius mixture on performance, intestinal health and serum lipids of broiler chickens
Parisa Shokryazdan 0 1 2
Mohammad Faseleh Jahromi 0 1 2
Juan Boo Liang 0 1 2
Kalavathy Ramasamy 0 2
Chin Chin Sieo 0 2
Yin Wan Ho 0 2
0 a Current address: Agriculture Biotechnology Research Institute of Iran (ABRII) , East and North-East Branch, Mashhad , Iran ¤b Current address: Faculty of Biotechnology and Biomolecular Sciences , Universiti Putra Malaysia, UPM, Serdang, Selangor , Malaysia
1 Institute of Tropical Agriculture , Universiti Putra Malaysia, UPM, Serdang, Selangor , Malaysia , 2 Faculty of Pharmacy , Universiti Teknologi MARA, Puncak Alam, Selangor , Malaysia , 3 Institute of Bioscience , Universiti Putra Malaysia, UPM, Serdang, Selangor , Malaysia
2 Editor: Francesco Cappello, University of Palermo , ITALY
The ban or severe restriction on the use of antibiotics in poultry feeds to promote growth has led to considerable interest to find alternative approaches. Probiotics have been considered as such alternatives. In the present study, the effects of a Lactobacillus mixture composed from three previously isolated Lactobacillus salivarius strains (CI1, CI2 and CI3) from chicken intestines on performance, intestinal health status and serum lipids of broiler chickens has been evaluated. Supplementation of the mixture at a concentration of 0.5 or 1 g kg-1 of diet to broilers for 42 days improved body weight, body weight gain and FCR, reduced total cholesterol, LDL-cholesterol and triglycerides, increased populations of beneficial bacteria such as lactobacilli and bifidobacteria, decreased harmful bacteria such as E. coli and total aerobes, reduced harmful cecal bacterial enzymes such as β-glucosidase and β-glucuronidase, and improved intestinal histomorphology of broilers. Because of its remarkable efficacy on broiler chickens, the L. salivarius mixture could be considered as a good potential probiotic for chickens, and its benefits should be further evaluated on a commercial scale.
Data Availability Statement: All relevant data are
within the paper.
Funding: This study was supported by the LRGS
Fasa 1/2012 (Universiti Putra Malaysia) provided
by the Ministry of Education, Malaysia.
Competing interests: The authors have declared
that no competing interests exist.
The use of probiotics as an alternative to antibiotic growth promoters has attracted
considerable interest due to its beneficial impacts on the health, performance and productivity of
]. Probiotics or direct-fed microbials are `live microbial supplements which
beneficially affect the health of the host animal by improving its intestinal microbial balance'
Lactic acid bacteria, particularly Lactobacillus strains, are frequently used as probiotics [
Lactobacillus strains have a high ability to attach to the intestinal epithelium and are able to
establish in the chicken intestine within a day after hatching [
], so they are considered to be
normal bacterial flora of the gastrointestinal tract (GIT) of chickens [
]. Bacterial strains used
as probiotics for animals should be isolated from the natural GIT microflora of the same type
of animal in order to have more specific application [
Although different probiotics may be developed for different purposes, a potential probiotic
strain intended for chickens mostly is developed towards improving the performance, general
health and productivity of chickens, which are usually achieved by affecting intestinal
microbial populations, serum lipids and intestinal morphology [
]. It has been reported
that probiotic strains can help to maintain the microbial balance in the GIT as well as make
changes in the composition of the intestinal microflora by increasing beneficial bacteria and
decreasing harmful pathogens . This could be due to competitive exclusion by competing
for nutrients and attachment sites on the intestinal epithelial wall, or production of
antimicrobial substances by probiotic strains or a synergy of both actions [
In terms of cholesterol lowering effects of probiotics, several mechanisms have been
proposed, which are based on reduction of cholesterol synthesis or increase in degradation and
excretion of cholesterol [
]. It has been also reported that some probiotic strains with BSH
activity are able to reduce serum cholesterol through deconjugation of bile salts [
addition, some probiotic cultures have been reported to be able of improving the morphology of
chicken intestine toward increasing nutrient absorption and endogenous digestive enzymes
secretion surface [
On the other hand, in terms of safety aspects, potential probiotic strains must not produce
harmful toxic enzymes such as β-glucosidase and β-glucuronidase, which can cause toxic
compounds being released in the colon.
In an earlier study [
], we have isolated several Lactobacillus strains from the intestines of
chickens, identified the strains and assessed (in vitro) their ability to survive and colonize the
GIT. Three strains (L. salivarius CI1, CI2 and CI3), which exhibited good probiotic properties
such as tolerance to acid, bile and pancreatic enzymes, and a strong ability to adhere to the
intestinal epithelial cells were selected as potential probiotics for chickens [
]. In the present
study, the in vivo effects of these three L. salivarius strains (as a mixture) on the growth
performance, cecal microbial populations, serum lipids, organ weights, intestinal villus and crypt
lengths and harmful cecal bacterial enzyme activities (β-glucosidase and β-glucuronidase) of
broiler chickens were investigated to confirm their potential as an effective probiotic mixture
Materials and methods
Preparation of Lactobacillus cultures
The three L. salivarius strains (CI1, CI2 and CI3) were cultured separately in MRS broth
medium (Merck, Darmstadt, Germany) and incubated for 24 h at 37ÊC in anaerobic jars
(Oxoid, Basingstoke, UK) containing gaspack (AnaeroGen, Oxoid, UK). After incubation, the
cultures were centrifuged at 5000 × g for 10 min at 4ÊC. Supernatants were discarded and cell
pellets were washed three times with deionized water. The cell pellet of each L. salivarius strain
was freeze-dried separately, and then mixed together in the ratio of 1:1:1 (w:w:w at 1×109 CFU
g-1). The mixture of Lactobacillus cultures was stored at -20ÊC and used daily as a dietary
supplement for broiler chickens.
Chickens and diets
Two hundred and seventy one-d-old male broiler chicks (Cobb 500), obtained from a local
commercial hatchery, were used in this experiment. The chicks were housed in stainless steel,
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three-tiered battery cages (0.9 m length, 0.6 m width and 0.6 m height) with raised wire netted
floors in an open house under natural tropical conditions. From d 1 to 14, the wire netted
floors of the cages were lined with papers, which were changed daily. After that, sliding
stainless steel trays were placed under the cages to collect feces, which were removed daily. For the
first 14 d, chicks were brooded with a 100 W bulb.
The chicks were weighed on per cage basis and randomly allocated to three dietary
treatment groups. Each dietary treatment consisted of six replicate cages of 15 chicks per cage. The
dietary treatments were: (i) basal diet (control) (ii) basal diet + 1 g kg-1 of mixture of three L.
salivarius strains (LC) and (iii) basal diet + 0.5 g kg-1 LC. The basal diet was an antibiotic-free,
corn-soybean meal diet (Table 1) formulated to meet the nutrient requirements for starter (1
to 21 d) and grower (22 to 42 d) periods [
]. The feed was in a mash form, and was fed to
chickens twice daily at 09:00 h and 17:00 h, in the way that chickens had ad libitum access to
the feed. The mixture of L. salivarius strains (LC) was mixed in the feed daily using a feed
mixer machine. The viability of the Lactobacillus cells was checked biweekly using
conventional spread plate method. The experimental period was 42 d. The study was approved by the
Ethics Committee of the Universiti Putra Malaysia, and the care and management of chickens
and sampling procedures were in compliance with the guidelines of the Federation of Animal
Science Societies [
]. Besides, the animals' health and welfare were monitored by a qualified
poultry veterinarian who is a member of the research team.
3 / 20
Sample collection and analysis
Feed residual was collected once a day before morning feeding and feed consumption on
percage basis was recorded daily. Body weight was recorded weekly, and body weight gain
was calculated based on that; feed conversion ratio (FCR) was calculated as feed intake per
weight gain unit. Mortality was recorded as it occurred, and the dead birds were immediately
removed from the cages. At d 21 and 42, 18 chickens per treatment (three chickens per
replicate cage) were randomly selected, weighed and euthanized by severing the jugular vein. Blood
was collected in non-heparinized blood collection tubes to obtain the serum. The carcasses
were opened immediately, and organs such as the heart, liver, spleen, bursa, and pancreas were
removed and weighed. Small intestine was collected for villus and crypt length measurements,
and cecal contents were collected for analyses of cecal microbial populations and
determination of harmful cecal bacterial enzyme (β-glucosidase and β-glucuronidase) activities.
Microbiological analyses of cecal contents
Cecal contents were analyzed for microbial populations using a conventional method (spread
plate method) and a molecular technique (real-time PCR assay). For the conventional method,
the cecal contents were used immediately after collection, however, for the molecular
technique the cecal contents were preserved in -20ÊC until the day of assessment.
Conventional spread plate method. For the conventional spread plate method, 1 g of
cecal content was suspended in 9 ml phosphate buffer saline (PBS) (8 g NaCl, 0.2 g KCl, 1.44 g
Na2HPO4, 0.24 g KH2PO4 in 1 l distilled water, pH 7.2) and vortexed for 1 min. Samples were
serially diluted in sterile diluents (0.5 g kg-1 peptone water in distilled water) and 100 μl of 10−4
to 10−6 dilutions were streaked on appropriate selective media for enumeration of different
groups of bacteria. de Man, Rogosa and Sharpe (MRS) agar medium was used for enumeration
of lactobacilli, Bifidus Selective agar for bifidobacteria, Brain-Heart Infusion agar for total
aerobes, Brilliant Green agar for Salmonella and Eosin Methylene Blue agar for E. coli (all media
from Sigma, Saint Louis, USA, except MRS from Merck). After incubation in appropriate
conditions for each group of bacteria (72 h at 37ÊC in anaerobic condition for lactobacilli and
bifidobacteria, and 48 h at 39ÊC in aerobic condition for Salmonella, E. coli and total aerobes),
colonies on the plates were counted and microbial population was expressed as log10 CFU g-1
Real-time PCR assay. For quantitative real-time PCR assay, total DNA was extracted
from cecal samples using the QIAamp DNA Stool Mini Kit (Qiagen Inc., USA). Quantification
carried out based on the standard curve method in real-time PCR. The standard curves were
constructed using number of copies of the 16S rRNA gene plotted against quantification cycle
(Cq) obtained from 10-fold serial dilutions of PCR products from pure culture of each
bacterial group. In order to prepare the standard curves, DNA was extracted from the pure culture
of each target bacteria (Lactobacillus, Bifidobacterium and E. coli) and conventional PCR was
used to amplify bacterial DNA. PCR products of the target bacteria were run in 1 g kg-1 agarose
gel and specific bands were purified using the MEGAquick-spin™ purification kit (iNtRON
Biotechnology, Korea). Purity and concentration of 16S rRNA gene in each sample was
measured using a Nanodrop ND-1000 spectrophotometer (Implen NanoPhotometer™, Germany).
The number of copies of the 16S rRNA gene per ml of elution buffer was calculated using the
following formula that is available online (http://web.uri.edu/gsc/dsdna-calculator/):
Number of copies
Amount of DNA
mg ml 1
Since the efficiency of amplification among primers and templates may be variable, the
amplification efficiency (E) of each primer-template combination was determined based on
the slope value of the linear regression of each standard curve calculated by the following
In this equation, E is 100% if a 10-fold dilution of DNA template results in a Cq difference
Real-time PCR was performed with a BioRad CFX96 Real-time PCR system (BioRad, USA)
using optical grade plates. Primers used in the quantification of different bacterial populations
are shown in Table 2. The real-time PCR reaction was performed on a total volume of 25 μl
using the Maxima SYBR Green qPCR Master Mix (Fermentas, USA). Each reaction consisted
of 12.5 μl of 2 × SYBR Green Master Mix, 1 μl of 10 μM forward primer, 1 μl of 10 μM reverse
Primer, 2 μl of DNA samples and 8.5 μl of nuclease-free water. Each sample was assayed with
triplicate reactions. No-template control was included in the real-time PCR amplification to
rule out any cross-contamination. Real-time PCR cycling conditions comprised an initial
denaturation at 94ÊC for 5 min, followed by 40 cycles of denaturation at 94ÊC for 20 s, primer
annealing at 58, 60 and 50ÊC for 30 s for Lactobacillus, Bifidobacterium and E. coli, respectively,
and extension at 72ÊC for 20 s. Upon completion of the amplification, the specificity of the
amplified product was confirmed by melting curve analysis. The real-time PCR products were
incubated by raising the temperature from 70 to 95ÊC in 0.5ÊC increments with a hold of 5 s at
each increment. The results were expressed as log10 copy number g-1 cecal content.
Serum lipid assay and relative weights of organs
Blood samples were allowed to settle at room temperature for 1 h, then centrifuged at 3000 × g
for 10 min. The serum was transferred into vials and stored at -20ÊC until use. Serum samples
were analyzed for total cholesterol, high density lipoprotein (HDL)-cholesterol, low density
lipoprotein (LDL)-cholesterol and triglycerides using an automatic clinical chemistry analyzer
The relative weight of organ was calculated as follows:
Weight of organ
Relative weight of organ
Live body weight
Villus height and crypt depth measurements
A 1-cm segment of the midpoint of the jejunum was cut, gently washed with PBS and fixed in
100 ml l-1 formalin. Samples were then dehydrated for 16 h in an automatic tissue processor
(Leica ASP 3000, Japan) and embedded in paraffin wax using a paraffin embedding system
GGG TGG TAA TGC CGG ATG
TAA GCC ATG GAC TTT CAC ACC
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(Leica EG 1160, Japan). Each sample was cut into 4 μm-thick sections using a rotary
microtome (Leica RM 2155, Japan). The sections were placed on glass slides, heated at 57ÊC until
dried, then stained with haematoxylin and eosin. The stained sections were examined using a
light microscope (Dialux, Leitz Wetzlar, Germany) fitted with a digital camera (Laica,
Germany). Villus height was measured from the tip of the villus to the villus-crypt junction, while
crypt depth was measured as the distance between the basement membrane and the mouth of
]. Fifteen measurements for villi and crypts were made for each sample.
β-Glucosidase and β-glucuronidase activity assays
One gram of cecal contents was suspended in 10 ml of PBS (pH 7.2) and centrifuged at 3000 ×
g for 5 min. The supernatant was used for analysis of harmful cecal bacterial enzyme
(β-glucosidase and β-glucuronidase) activities.
The assays for β-glucosidase and β-glucuronidase activities were according to that described
by Lee et al. [
] with modifications. Briefly, 0.8 ml of 2 mM
p-nitrophenyl-β-D-glucopyranoside (Sigma) (for β-glucosidase activity) or 2 mM p-nitrophenyl-β-D-glucuronide (Sigma) (for
β-glucuronidase activity) and 0.2 ml of sample were incubated at 37ÊC for 1 h. The reaction
was stopped by adding 1 ml of 0.5 mol l-1 NaOH, and the mixture was centrifuged at 4000 × g
for 10 min at room temperature. Enzyme activity of the supernatant was determined by
measuring absorbance at 405 nm using a spectrophotometer. Different concentrations (0, 0.1, 0.2,
0.5, 1 and 10 mmol l-1) of p-nitrophenol (Sigma) were used for preparation of a standard
curve. The enzyme activity was expressed as unit g-1 cecal contents. One unit is defined as the
activity required to release 1 μmol l-1 of p-nitrophenol in 1 h.
All the data were analyzed using one-way ANOVA procedure of SAS program (2008) version
] based on the completely randomized design, followed by comparison among means
using Duncan's new multiple range test. Differences were considered significant if P < 0.05.
Performance of broiler chickens
The effects of a mixture of L. salivarius CI1, CI2 and CI3 (LC) on body weight, body weight
gain, feed intake and FCR of broiler chickens are shown in Table 3. The body weights of broiler
chickens were not significantly different among the three dietary treatments at 1 and 21 d of
age. However, at 42 d of age, chickens fed 0.5 or 1 g kg-1 LC showed significantly (P < 0.01)
higher body weights (2164.3 and 2274.5 g, respectively) than control chickens (2017.3 g).
From 1 to 21 d of age, body weight gains of broiler chickens were not significantly different
among the dietary treatments, but from 22 to 42 and 1 to 42 d of age, broilers given 0.5 or 1 g
kg-1 LC had significantly (P < 0.01) higher body weight gains than control chickens. There
was no significant difference in feed intake of broilers in the three dietary treatments
throughout the experimental period. From 1 to 21 d of age, the FCRs of all broilers were not
significantly different. However, from 22 to 42 and 1 to 42 d of age, broiler chickens fed 0.5 or 1 g
kg-1 LC had significantly (P < 0.01) better FCR than control chickens. Mortality was observed
in the control and broilers supplemented with 0.5 g kg-1 LC (one chicken for each treatment
group during 42 days of experiment), but there was no mortality in broilers fed 1 g kg-1 LC.
During the experimental period, no significant difference (P > 0.05) was observed between the
two groups of broiler receiving LC (0.5 or 1 g kg-1 LC) in terms of body weight, weight gain,
feed intake or FCR.
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* Values are mean ± SD of 6 replicate cages, each with 15 chickens
a-b Means within a row with no common superscript are signi®cantly different (P < 0.01)
LC, mixture of L. salivarius CI1, CI2 and CI3 in the ratio of 1:1:1 (w:w:w); FCR, feed conversion ratio; control, basal diet; 0.5 g kg-1 LC, basal diet + 0.5 g kg-1
LC; 1 g kg-1 LC, basal diet + 1 g kg-1 LC
Enumeration of cecal bacteria
Conventional microbiological method. Fig 1 shows the results of bacterial enumeration
using the conventional spread plate method for lactobacilli, bifidobacteria, total aerobes and E.
coli. No Salmonella was detected in the cecal contents of broiler chickens in all three dietary
treatment groups throughout the experimental period. At 21 d of age, the population of
lactobacilli in cecal contents of broiler chickens fed 1 g kg-1 LC was significantly (P < 0.05) higher
than that of control chickens. Although the population of lactobacilli of chickens fed 0.5 g kg-1
LC was not significantly different (P > 0.05) from that of control chickens, it was numerically
higher. At 42 d of age, the populations of lactobacilli in broiler chickens given 0.5 or 1 g kg-1
LC were significantly (P < 0.05) higher than that of the control, and between the two
LCsupplemented groups, chickens given 1 g kg-1 LC showed significantly (P < 0.05) higher
lactobacilli population than those fed 0.5 g kg-1 LC. At 21 and 42 d of age, broiler chickens
supplemented with 0.5 or 1 g kg-1 LC had significantly (P < 0.05) higher populations of
bifidobacteria than the control. At both ages, the cecal bifidobacterial populations between broilers fed
0.5 or 1 g kg-1 LC were not significantly different. Birds fed dietary treatments supplemented
with 0.5 or 1 g kg-1 LC had significantly (P < 0.01) lower populations of total cecal aerobes
than the control at 21and 42 d of age.
Between the two supplemented groups, birds fed 0.5 g kg-1 LC had significantly (P < 0.01)
higher population of total cecal aerobes than those fed 1 g kg-1 LC at both ages. Broiler
chickens supplemented with 0.5 or 1 g kg-1 LC had significantly (P < 0.01) lower populations of E.
coli than control broilers at 21 and 42 d of age. At both ages, the E. coli populations of broilers
given 1 g kg-1 LC were significantly (P < 0.01) lower when compared to those fed 0.5 g kg-1
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Fig 1. Effects of dietary supplementations of a mixture of three L. salivarius strains (LC) on populations of cecal lactobacilli, bifidobacteria,
total aerobes and E. coli of broiler chickens at 21 and 42 d of age enumerated using the conventional spread plate method and expressed as
log10 CFU g-1. Columns represent means of six birds in each treatment group (one chicken per replicate cage) ± SD. Within each period, columns with
different letters differ significantly (P < 0.05). Control, basal diet; 0.05% LC, basal diet + 0.5 g kg-1 LC; 0.1% LC, basal diet + 1 g kg-1 LC.
Real-time PCR quantification. Since no Salmonella was detected in the cecal contents of
broiler chickens in all three dietary treatment groups at 21 and 42 d of age using the
conventional microbiological method, real-time PCR assay was not carried out for quantification of
Salmonella. Real-time PCR quantification was also not conducted for total aerobes due to no
existing designed primer for them. The standard curves for Lactobacillus, Bifidobacterium and
E. coli were constructed using the plot of copy numbers of 16S rRNA gene of each bacterial
group against its Cq values. The standard curves had high correlation coefficients of R2 =
0.988, 0.986 and 0.994 for Lactobacillus, Bifidobacterium and E. coli, respectively, indicating
that the Cq values were proportional to the copy numbers of 16S rRNA gene, for each target
bacterial group. From the slopes of the liner regressions of -3.407, -3.488 and -3.301,
amplification efficiencies were obtained 96.6, 93.5 and 100.9% for Lactobacillus, Bifidobacterium and E.
coli, respectively. The amplification curves for Lactobacillus, Bifidobacterium and E. coli were
8 / 20
constructed by plotting the cycle numbers against fluorescence signals (RFU, relative
fluorescence units). No fluorescence signals were detected from the no-template control. The melting
temperatures of 82.5, 86 and 79.5ÊC were detected at which the sets of primers were specific
for estimation of lactobacilli, bifidobacteria and E. coli, respectively.
The results from real-time PCR quantification of cecal lactobacilli, bifidobacteria and E. coli
populations of broiler chickens fed the three dietary treatments are shown in Fig 2. Cecal
lactobacilli populations were significantly (P < 0.05) higher in broilers fed diets containing 0.5 or 1
g kg-1 LC when compared to that in control broilers at 21 and 42 d of age, and there was no
significant difference between the cecal lactobacilli populations of broilers fed 0.5 or 1 g kg-1 LC.
At 21 and 42 d of age, broiler chickens fed 1 g kg-1 LC showed significantly (P < 0.05)
higher cecal bifidobacteria populations than the control. However, at both ages, the cecal
bifidobacteria populations of broilers supplemented with 0.5 g kg-1 LC were not significantly
different from that of control or broilers fed 1 g kg-1 LC. Broiler chickens supplemented with 0.5
or 1 g kg-1 LC had significantly (P < 0.05) lower populations of E. coli than those of control
broilers at 21 and 42 d of age, and the cecal E. coli populations between broilers fed 0.5 or 1 g
kg-1 LC were not significantly different at both ages.
Serum lipids and relative weights of organs
The results of serum lipid analysis of broilers fed the three dietary treatments at 21 and 42 d of
age are shown in Table 4. Serum total cholesterol, LDL-cholesterol and triglyceride
concentrations were significantly (P < 0.05) reduced in broiler chickens fed 0.5 or 1 g kg-1 LC when
compared to control broilers at 21 and 42 d of age, and there was no significant difference
between the two supplemented (0.5 or 1 g kg-1 LC) treatment groups at both ages.
HDL-cholesterol levels of broilers were not significantly different in all three dietary treatment groups at
21 and 42 d of age.
The relative weights of organs calculated as percentage of body weight of broilers are given
in Table 5. There were no significant differences in the relative weights of heart, liver, spleen,
bursa and pancreas of broiler chickens from the three dietary treatment groups at 21 and 42 d
Intestinal villus height and crypt depth
The effects of dietary treatments on intestinal villus height, crypt depth, and villus height:crypt
depth ratio are presented in Table 6. At 21 d of age, there was no significant difference
(P > 0.05) between the three dietary treatment groups in terms of villus heights and crypt
depths. In broilers fed 1 g kg-1 LC, the villus height:crypt depth ratio was significantly
(P < 0.05) higher than that of the control group (5.72 vs 5.15). However, birds fed 0.5 g kg-1
LC (5.54) did not showed any significant difference with the control birds for the villus height:
crypt depth ratio. At 42 d of age, broilers given 0.5 or 1 g kg-1 LC showed significantly
(P < 0.05) higher villus heights (1290.58 and 1312.50 μm, respectively) and villus height:crypt
depth ratios (6.52 and 6.76, respectively) than control broilers (1110.17 μm villus height and
5.41 villus height:crypt depth ratio), and there was no significant difference between those fed
0.5 or 1 g kg-1 LC. However, crypt depths were not significantly different in broilers from the
three dietary treatment groups. A representative photomicrograph of the intestinal villi and
crypts of broilers showing measurements of villus height and crypt depth is given in Fig 3.
Harmful cecal bacterial enzyme (β-glucosidase and β-glucuronidase) activities. The
cecal bacterial β-glucosidase and β-glucuronidase activities of broiler chickens fed the three
dietary treatments are shown in Table 7. Supplementation of 0.5 or 1 g kg-1 LC to broiler
chickens significantly (P < 0.01) decreased cecal β-glucosidase and β-glucuronidase activities
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Fig 2. Effects of dietary supplementations of a mixture of L. salivarius strains (LC) on populations of
cecal lactobacilli, bifidobacteria and E. coli of broiler chickens at 21 and 42 d of age quantified using
real-time PCR and expressed as log10 copy number g-1. Columns represent means of six birds in each
treatment group (one chicken per replicate cage) ± SD. Within each period, columns with different letters differ
significantly (P < 0.05). Control, basal diet; 0.05% LC, basal diet + 0.5 g kg-1 LC; 0.1% LC, basal diet + 1 g kg-1
10 / 20
at 21 and 42 d of age, and there was no significant difference in the bacterial enzyme activities
between broilers fed 0.5 or 1 g kg-1 LC.
Although improving effects of the mixture of three L. salivarius strains (CI1, CI2 and CI3) at
0.5 or 1 g kg-1 on performance of broilers from 1 to 21 d of age were not significant,
supplementation of the mixture significantly improved body weight of broilers at 42 d of age; it also
improved body weight gain and FCR of broilers from 22 to 42 and 1 to 42 d of age. A number
* Values are means ± SD of 6 replicate cages of 3 chickens each
LC, mixture of L. salivarius CI1, CI2 and CI3 in the ratio of 1:1:1 (w:w:w); control, basal diet; 0.5 g kg-1 LC, basal diet + 0.5 g kg-1 LC; 1 g kg-1 LC, basal diet
+ 1 g kg-1 LC
11 / 20
* Values are means ± SD of 6 replicate cages of 3 chickens each
aÐb Means within a row with no common superscript are signi®cantly (P < 0.05) different
LC, mixture of L. salivarius CI1, CI2 and CI3 in the ratio of 1:1:1 (w:w:w); control, basal diet; 0.5 g kg-1 LC, basal diet + 0.5 g kg-1 LC; 1 g kg-1 LC, basal diet
+ 1 g kg-1 LC
of studies had also shown improvements in body weight and FCR of broiler chickens fed diets
supplemented with a mixture of Lactobacillus strains [
] or with preparations
of lactobacilli and other bacteria [
]. However, there were also some studies which reported
no positive results in performance of broilers fed probiotic Lactobacillus supplemented feeds
]. The variations in the results from different studies could be due to differences in
the strains, sources, viability and concentrations of used bacteria, methods of administration,
and conditions of chickens.
Supplementation of the three L. salivarius strains had no effect on feed intake of broiler
chickens. Several other studies had also shown that feed intake of chickens was not affected by
supplementation of Lactobacillus or other bacteria [
]. At present, it is
not known why supplementation of Lactobacillus cultures to broiler chickens does not affect
their feed intake. In layers, it has been reported that supplementation of Lactobacillus cultures
stimulated their appetite [
]. However, this difference between broilers and layers may
be attributed to the fact that broilers have been genetically selected for having high feed intake
in comparison to layers, and as it has been reported by Ferket and Gernat [
], dietary factors
are less important than management and flock health issues for influencing feed intake in
broilers. Therefore, in unstressed broilers usually it is difficult to see the effects of dietary
supplements on feed intake.
In the present study, two methods, namely, the conventional microbiological method
(spread plate method) and the molecular technique (real-time PCR assay) were used to
estimate cecal microbial populations of broilers fed the three dietary treatments. One of the
weaknesses of the conventional microbiological method that has often been mentioned is that it
may underestimate microbial populations as some of the microbes may be clumped together
or lyzed during processing of samples. As real-time PCR assay measures microbial DNA, it
may be a better approach for estimation of microbial populations. However, the results showed
that population patterns of lactobacilli, bifidobacteria and E. coli obtained by the conventional
microbial method and the real-time PCR assay were comparable.
Both enumeration methods showed that the L. salivarius strains had beneficial modulatory
effects on the intestinal microflora of broilers fed 0.5 or 1 g kg-1 Lactobacillus strains, meaning
that the populations of cecal beneficial bacteria (lactobacilli and bifidobacteria) were
significantly increased, while populations of harmful bacteria (E.coli and total aerobes) were
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Fig 3. A representative photomicrograph showing intestinal villi and crypts of a broiler (fed diet
supplemented with 1 g kg-1 of a mixture of L. salivarius CI1, CI2 and CI3) at 42 d of age. Villus height was
measured from the top of the villus to the villus-crypt junction (long bar). Crypt depth was measured as the distance
between the basement membrane and the mouth of crypt (short bar).
13 / 20
* Values are mean ± SD of 6 replicate cages of 3 chickens each
a±b Means within a row with no common superscript are signi®cantly (P < 0.01) different
Unit, the activity required to release 1 μM of p-nitrophenol in 1 h; LC, mixture of L. salivarius CI1, CI2 and CI3 in the ratio of 1:1:1 (w:w:w); control, basal diet;
0.5 g kg-1 LC, basal diet + 0.5 g kg-1 LC; 1 g kg-1 LC, basal diet + 1 g kg-1 LC
decreased. Jin et al. [
] and Saminathan et al. [
] reported similar beneficial modulation of
intestinal microbial population in which there was an increase in intestinal lactobacilli and a
decrease in E. coli of broilers fed a mixture of 12 Lactobacillus cultures at 21 d of age. Ngoc Lan
et al. [
] also reported that two probiotic strains, L. agilis and L. salivarius, isolated from
chicken intestine, significantly increased the intestinal lactobacilli in the probiotic group in
comparison to the control group, after seven days of probiotic feeding. Gunal et al. [
reported that a probiotic mixture (Protexin) decreased the population of Gram-negative
bacteria in the ileal and cecal contents of broilers at 21 and 42 d of age. Mountzouris et al. [
used a probiotic mixture consisting of L. reuteri, Enterococcus faecium, Bifidobacterium
animalis, Pediococcus acidilactici and L. salivarius and found that the populations of
bifidobacteria, lactobacilli and gram-positive cocci were significantly higher in cecal contents of birds
received probiotic (1 g kg-1 of feed) compared with the control chickens (receiving no additive
in their feed) and chickens receiving antibiotic (avilamycin, 2.5 mg kg-1 of feed).
Although the precise mechanisms underlying the beneficial effects of probiotics are unclear,
one of the proposed modes of action of probiotics is their pathogen interference and
antagonistic activity, whereby probiotic strains inhibit the growth and colonization of other
microorganisms, such as pathogens [
]. This could be due to competitive exclusion by competing for
nutrients and attachment sites on the intestinal epithelial wall, or production of antimicrobial
substances by probiotic strains or a synergy of both actions [
]. As a result, probiotic strains
can help to maintain the gut health by providing a beneficial microbial balance in the GIT, and
a healthy, well functioning gut with reduced digestive disorders would ensure better utilization
and conversion of feeds, resulting in improved growth and vitality of the animal .
The results of the current study showed that both concentrations of the LC caused
significant reduction in the serum total cholesterol and triglyceride concentrations of broilers.
Similar hypocholesterolemic effects of probiotic bacterial strains on serum lipids of chickens had
been reported in other studies. Jin et al. [
] found significant reduction in the serum total
cholesterol level of broilers fed 1 g kg-1 of a multistrain probiotic comprising 12 Lactobacillus
strains. Kalavathy et al. [
], using the same multistrain probiotic mixture, also found
reductions in the serum total cholesterol and triglyceride levels of broilers fed 1 g kg-1 probiotic.
Mayahi et al. [
] fed 0.1% of two commercial probiotics, one consisting of E. faecium and
the other consisting of Bifidobacterium, to broilers and found significant reduction in their
serum total cholesterol and triglyceride concentrations. Mansoub [
] also reported significant
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decline in serum total cholesterol and triglycerides of broilers fed 0.5% L. casei, 1% L. casei,
0.5% L. acidophilus or 1% L. acidophilus.
Supplementation of the three L. salivarius strains to broilers significantly reduced their
serum LDL-cholesterol, but not their HDL-cholesterol levels at 21 and 42 d of age. Similar
results of reduction in LDL-cholesterol but not HDL-cholesterol were reported by Kalavathy
et al. [
] who fed 1 g kg-1 of a multistrain probiotic comprising 12 Lactobacillus strains to
broilers. Panda et al. [
] also found a decrease in serum LDL-cholesterol but not
HDLcholesterol in broilers supplemented with L. sporogenes at 100 or 200 mg kg-1 diet. In contrast,
Ashayerizadeh et al. [
] did not find significant differences in the serum HDL- and
LDL-cholesterol concentrations of chickens fed the commercial probiotic, PrimaLac, when compared
to control chickens.
Currently, the mechanism(s) responsible for the cholesterol-lowering effect of probiotic is
still unclear, but there are several mechanisms, based on reduction of cholesterol synthesis or
increase in degradation and excretion of cholesterol [
], that have been proposed. Some
probiotic strains with bile salt hydrolase (BSH) activity are able to reduce serum cholesterol
through deconjugation of bile salts [
]. Bile acids are secreted into the duodenum in their
conjugated forms, however, their deconjugated forms are less soluble and more likely to be
excreted from the body, and less likely to be absorbed into the intestine and enterohepatic
circulation. Since cholesterol is a precursor for hepatic synthesis of bile acids, the liver needs to
synthesise new bile acids from cholesterol in a homeostatic response, resulting in reducing
cholesterol. In addition, deconjugated bile acids are known to co-precipitate with cholesterol
resulting in more excretion of cholesterol from the body [
]. Gililand et al. [
] have also
proposed that some lactic acid bacteria are able to assimilate cholesterol into their cells resulting
in cholesterol reduction of surrounding environment. Another mechanism for
cholesterollowering effect of probiotics is their ability to produce intra- and extra-cellular cholesterol
dehydrogenase or isomerase for catalyzing the transformation of cholesterol into coprostanol
in the intestine [
]. The other enzymatic mechanism for cholesterol reduction activity of
probiotic strains is inhibition of HMG-CoA reductase enzyme, an important enzyme for
cholesterol synthesis, by probiotic strains [
]. The hypocholesterolemic effect of probiotic strains
could also be attributed to their ability to bind cholesterol to their cellular surface [
] and to
incorporate cholesterol into their cell membranes toward having a higher cellular resistance
against lysis [
Feeding 0.5 or 1 g kg-1 of a mixture of the three L. salivarius strains to broiler chickens in
the current study did not affect the relative weights of heart, liver, spleen, bursa and pancreas
at 21 and 42 d of age. This indicates that the three L. salivarius strains have no adverse effects
on the vital organs and the general health of the chickens. Other researchers [
using different probiotic strains also did not find any significant differences between the
relative organ weights of chickens in the control group and groups receiving probiotics.
In the present study, dietary supplementation of 0.5 or 1 g kg-1 of a mixture of the three L.
salivarius strains significantly increased intestinal villus heights of broiler chickens at 42 d of
age, and villus height:crypt depth ratios at 21 and 42 d of age. Similar result of increase in
intestinal villus heights of probiotic-fed broilers was reported by Awad et al. [
]. Peric et al. [
also found that broilers fed a probiotic blend containing Lactobacillus, Bifidobacterium,
Enterococcus and Pediococcus strains in water, and a probiotic consisting of E. faecium together with
prebiotics fructooligosacharides, cell wall fragments and phycophytic substances in feed,
showed significantly higher villi, deeper crypts and higher villus height:crypt depth ratio in
their jejunum than broilers of control group at 6 weeks of age, but not at 3 weeks of age.
The small intestine is an important digestive organ involved in nutrient absorption and its
development is essential to broiler performance. It has been suggested that probiotic cultures
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are able to reduce the damage of enterocytes and in turn the demand for enterocytes renewal
in the gut [
]. Due to the major role of intestinal microvilli in absorption of nutrients, an
increase in the villus height equates to an increase in surface area resulting in more effective
absorption of available nutrients [
]. Crypts are considered as origin area for production
of new epithelial and villus cells. Stem cells at the bottom of crypts divide to form daughter
cells, in which one of them is retained as a stem cell, and the other becomes an intestinal
epithelial cell. This newly formed cell has to pass the crypt walls and migrate up onto the villus,
where it will differentiate further to become a mature, absorptive epithelial cell. Therefore,
larger ratio of villus height:crypt depth will result in higher epithelial cell numbers  leading
to higher absorption of nutrients [
]. Then, higher amounts of absorbed nutrients lead to
higher performance and lower FCR [
]. On the contrary, decrease in villus height and
increase in crypt depth may lead to poor nutrient absorption, and lower performance [
Probiotics may also have effects on the poultry intestine by stimulating it to have more surface
for secretion of endogenous digestive enzymes, thus, improving feed digestion and growth
Production of harmful enzymes such as β-glucosidase and β-glucuronidase is a safety aspect
of probiotic bacteria that needs to be examined. These two enzymes are the major glycosidases
in the intestinal tract, which are produced by bacterial strains. These enzymes release toxic
metabolites from nontoxic glycosides and prolong the lifetime of toxicants in the body [
has been suggested that bacterial β-glucosidase is responsible for hydrolysis of amygdalin in
the gut to produce mandelonitrile, which in turn could be hydrolyzed to produce toxic cyanide
]. β-Glucuronidase could hydrolyze glucuronides in the gut that can potentially cause the
generation of toxic substances [
The results of the current study showed a significant reduction in both β-glucosidase and
βglucuronidase activities in the cecal contents of broilers receiving a mixture of L. salivarius
strains (0.5 or 1 g kg-1) as compared to control broilers. To date, there is very little information
on the effects of probiotics on the activities of β-glucuronidase in chickens. Cole et al. [
reported that young chickens fed yogurt-supplemented diet showed significantly reduced
βglucuronidase activity. Jin et al. [
] also reported that feeding Lactobacillus cultures to broilers
reduced significantly the intestinal and fecal β-glucuronidase, and fecal β-glucosidase activities,
but they had no effect on intestinal β-glucosidase activity. Gadelle et al. [
] tested 64
Lactobacillus strains and found none to be β-glucuronidase-producer. Drasar and Hill [
that almost all strains of E. coli were able to produce β-glucosidase, but less than 40% of
Lactobacillus strains had the ability to produce glucosidase. In a recent in vitro enzyme assay of the
three L. salivarius strains, we found that none of the strains produced β-glucosidase or
β-glucuronidase (unpublished data). Jin et al. [
] suggested that the reduction of harmful enzyme
activities by Lactobacillus strains in the intestine might be due to the partial replacement of the
intestinal microflora, especially E. coli which is a positive producer of the two enzymes, with
Lactobacillus strains. In the present study, the reduction of β-glucosidase and β-glucuronidase
activities in the cecal contents of broilers fed a mixture of three L. salivarius strains was
probably due to the same mode of action by the Lactobacillus strains as the results on the
enumeration of cecal microbes showed that there was a significant reduction of total aerobes and E. coli
populations and a significant increase of lactobacilli and bifidobacteria populations in the
The results of this study demonstrated that supplementation of a mixture of three L. salivarius
strains (CI1, CI2 and CI3) at a concentration of 0.5 or 1 g kg-1 diet to broiler chickens had
16 / 20
similar beneficial effects on them. It improved body weight, body weight gain and FCR,
reduced total cholesterol, LDL-cholesterol and triglycerides, increased populations of
beneficial bacteria such as lactobacilli and bifidobacteria, decreased harmful bacteria such as E. coli
and total aerobes, reduced harmful cecal bacterial enzymes such as β-glucosidase and
β-glucuronidase, and improved intestinal histomorphology of broiler chickens. Their remarkable
efficacy on broiler chickens in this preliminary experiment warrants the three L. salivarius strains
to be considered as good potential probiotic for chickens, and their benefits should be further
evaluated on a commercial scale.
Conceptualization: PS MFJ JBL KR CCS YWH.
Data curation: PS.
Formal analysis: PS MFJ.
Funding acquisition: JBL YWH.
Methodology: PS YWH.
Project administration: PS YWH.
Resources: JBL YWH.
Software: PS MFJ.
Validation: PS MFJ YWH.
Writing ± original draft: PS.
Writing ± review & editing: PS YWH.
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18 / 20
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