Effect of dietary probiotics and prebiotics on the performance

of broiler chickens

H. Al-Khalaifa,∗,1 A. Al-Nasser,∗ T. Al-Surayee,† S. Al-Kandari,‡ N. Al-Enzi,‡ T. Al-Sharrah,‡

G. Ragheb,∗ S. Al-Qalaf,∗ and A. Mohammed∗
∗
Desert Agriculture and Ecosystems Program (DAEP), KISR, Safat 13109, KUWAIT; † Biotechnology Program,
KISR, Safat 13109, KUWAIT; and ‡ Central Analytical Laboratory (CAL), KISR, Safat 13109, KUWAIT

ABSTRACT The prolonged use of antibiotics has led taste, and texture of the cooked meat were acceptable,
to the development of resistant bacteria and also led and that there were no significant differences between
to accumulation of antibiotic residue in the poultry the different groups. There was no significant effect of
feed, this ultimately led to the prohibition of antibi- the different diets on the biochemical parameters of the
otics as growth enhancers in animal production. Thus, blood among the experimental groups at 3- and 5-wk
there was a dire need for alternate sources to help of age. Phytohaemaglutinin test showed that dietary
in poultry production. Recently, probiotics and pre- FOS and MOS induced higher cellular response than
biotics claimed to be effective alternatives to antibi- the other treatments (P = 0.04) in the first cycle. In
otics in the poultry. The objective of this study is to the second cycle, the results revealed that dietary FOS
investigate the effect of different probiotics and pre- induced higher cellular response than the other treat-
biotics on the performance of broilers. The study in- ments (P = 0.019). The used experimental treatments
volved 2 broiler cycles, 1 during winter and 1 during have a positive effect on microbial count in 5-week-old
summer with a total of 425 1-day-old Cobb 500 broiler broilers. There was no Salmonella sp. recorded using
chicks for each cycle. They were allotted to 5 experi- the experimental treatments in the first cycle, and the
mental treatments. The probiotics were Bacillus coag- growth of E. coli was reduced significantly. In the sec-
ulans (1 g/kg dried culture) and Lactobacillus (1 g/kg ond cycle, all treatments in 3-week-old broilers did not
dried culture of 12 commercial strains). The prebiotics affect the count of both lactic acid bacteria (LAB) and
included fructo-oligosaccharides (FOS) (5 g/kg) and E. coli. At 5-week-old of the same cycle, the bacterial
mannan-oligosaccharide (MOS) derived from Saccha- count of E. coli increased even with control, whereas
romyces cerevisiae (5 g/kg). The results showed that Salmonella growth was inhibited. The pH value was
there was no effect of the different probiotics and pre- driven toward acidity in all of the treatments. Probi-
biotics on the production performance of broilers. There otics and prebiotics can be used in chicken feed safely
was increased weight of the thymus in the control group. and without any adverse effects on the productive pa-
In cycle 1, the panelists indicated that the smell, color, rameters and immune status of the flock.
Key words: antibiotic, prebiotic, probiotic, broiler, immune status
2019 Poultry Science 98:4465–4479
http://dx.doi.org/10.3382/ps/pez282

INTRODUCTION antibiotics as growth promoters was banned in 2006 by

the European Union (Castanon, 2007). In 2009, govern-
Antibiotics have been widely used since the 1940s to ment agencies in the United States of America, such as
build the immunocompetence of birds against infectious the Food and Drug Administration, concluded that the
diseases and as growth promoters. Using antibiotics for use of antibiotics for growth promotion should be elim-
long terms may lead to the development of bacteria re- inated (FDA, 2009). However, probiotics have not been
sistant to drugs, which can be transferred to humans approved by the FDA yet. Instead, they will remain
(Sweeney et al., 2018; Tania et al., 2018). For this rea- neither approved nor unapproved. As a result, effective
son, the World Health Organization (WHO, 1997) and dietary supplements, such as probiotics and/or prebi-
the Economic and Social Committee of the European otics, replaced antibiotics, which are supposed to be
Union (1998) concluded that the use of antimicrobials used at minimal levels. These replacement ingredients
in food animals is a public health concern. The use of are claimed to enhance growth and the immune sta-
tus against all infectious agents (Janvier et al., 2013;
Al-Khalaifah, 2018).

C 2019 Poultry Science Association Inc.
There are several definitions of probiotics in the liter-
Received December 16, 2018.
Accepted May 25, 2019. ature. Probiotics are “live microbial feed supplements,
1
Corresponding author: [email protected] which beneficially affect the host animal by improving
4465
4466 AL-KHALAIFA ET AL.

its intestinal microbial balance” (Fuller, 1989); “a live cost and improve production efficiency. The isolation
microbial feed that is beneficial to health” (Salminen rates of Campylobacter were found to be lesser in the
et al., 1998); or “live microorganisms which, when broilers raised in cages than in broilers raised on the
administered in adequate amounts, confer a health floor indicating the better hygienic conditions of birds
benefit on the host” (FAO/WHO, 2001). These live that are raised in cages than those raised on the floor.
microorganisms include strains of Lactobacillus, Bifi- No paw, leg issues, or high mortality rate were observed
dobacterium, and yeasts. Also, prebiotics have the abil- when chickens were reared in cages. Although chickens
ity to increase the levels of health-promoting bacte- are coprophagic, like many animals, this did not cause a
ria in the intestinal tract. When the prebiotic arrives problem during the course of the study as the bird drop-
in the colon, certain members of the indigenous mi- pings were collected into trays placed under the wires,
croflora ferment it selectively. The usual target for pre- this in turn helped produce birds that were healthy and
biotics is the 2 lactic acid bacterial (LAB) genera, Bifi- clean. The broiler chicks were fed a starter diet from
dobacterium and Lactobacillus. The enhancement of the hatch until 7 d of age (1 wk), a grower diet from 8 to
growth of these bacterial species also results in the pro- 21 d of age (2 to 3 wk), and a finisher diet from 22
duction of bacteriocins, which help inhibit the growth to 35 d of age (4 to 5 wk). The diet was corn and/or
of pathogenic bacteria (Alavi et al., 2012). One of the soy-based, which meets the rules and regulations of the
major causes for the deterioration of meat quality and National Research Council (NRC). The chickens were
reduction of the shelf life for meat and meat products is fed with the experimental diets from 1 d until slaugh-
the susceptibility of the lipid macronutrients to various ter at 35 d of age. The Environmental Life Sciences
medications. Prebiotics can alter lipid metabolism and Research Center (ELSRC) at the Kuwait Institute for
enhance the PUFAs ratio in chicken meat with benefits Scientific Research (KISR) approved all experimental
to human health but a greater risk for the shelf life of animal protocols prior to the onset of this study.
the meat. Oxidation of lipids and pigments of meat have The study involved 2 broiler cycles, 1 during winter
a negative effect on the consumers and will ultimately and 1 during summer. The building in which the chicks
lead to economic losses (Maiorano et al., 2017). were reared consisted of partial artificial and natural
The avian gastrointestinal tract consists of a di- environment. Cooling pads and fans were used for ven-
verse and dynamic population of microorganisms, liv- tilation. Due to the extreme Kuwait weather conditions
ing in symbiotic relationship with their host. This mu- during summers and winters, could have affected the
tualistic relationship is important for host nutrition, environment inside the poultry houses, even with the
metabolism, and immunity. The complex ecosystem presence of cooling pads and fan ventilation system.
works as a virtual organ system that aids in host home- During summer, the temperature in the poultry houses
ostasis. In adults, the intestinal microbial ecology is was between 30 and 35◦ C. During winter, the tempera-
highly stable; however, it may be influenced by either tures were found to be 20–25◦ C. The cooling pads and
feed or stress. fans were used to keep the temperature constant. Each
Recent research has focused on the importance of cycle involved a total of 425 broilers, which were dis-
probiotics and prebiotics as functional foods to influ- tributed in to 5 batteries, 85 birds per battery. Each
ence intestinal microflora, microbial profiles, and broiler battery consisted of 5 levels, and the space of each level
performance. Probiotics are found to beneficially affect was 0.85 m2 . There were 17 birds in each level, providing
the host animal by improving its intestinal microbial a space of 0.05 m2 for each bird. Five dietary treatments
balance and prebiotics benefit the host by selectively were randomly distributed among the 5 batteries, with
stimulating the growth and activity of one or a limited each level having different treatments.
number of bacteria in the colon. It is hypothesized that There were 5 experimental treatments (TRTs) in the
such supplements influence the intestinal microbiome study as follows: TRT 1, which was the control group,
and improve intestinal absorption, which altogether im- received a soybean basal diet (Table 1) without any
prove performance (Sohail et al., 2012). probiotic or prebiotic additives; TRT 2 group received
The aim of this research was investigating the effect the same basal diet as in TRT 1 plus 1 g/kg dried cul-
of different types of commercial dietary probiotic and ture of Bacillus coagulans (Cavazzoni et al., 1998; Zhou
prebiotic supplementations on the performance, meat et al., 2010); TRT 3 group received the same basal diet
quality, and immune status of broiler chickens raised. as in TRT 1, in addition, 1 g/kg dried culture of 12
commercial Lactobacillus strains was added to the diet
(Jin et al., 2000); TRT 4 group received the same basal
diet as in TRT 1, in addition, 5 g/kg of FOS extracted
MATERIALS AND METHODS from chicory root was added to the diet (Kannan et al.,
Animals, Housing, and Diets 2005); and TRT 5 group received the same basal diet
as in TRT 1, in addition, a Bio-MOS prebiotic level of
One-day-old Cobb 500 broiler chicks (Kuwait United 5 g/kg was added to the diet. Bio-MOS is a mannan-
Poultry Company, KW), vaccinated against Infectious oligosaccharide commercial prebiotic derived from the
Bronchitis, were used in this study. Water and feed were cell wall of the yeast Saccharomyces cerevisiae (Midilli
provided ad libitum. The broiler chickens were raised in et al., 2008; Janardhana et al., 2009). All prebiotics and
cages instead of floor pens to reduce the operational probiotics were obtained from Alltech, USA.
 DIETARY PROBIOTICS AND PREBIOTICS 4467
Table 1. Composition of the broiler feed ration (kg) for the basal house, especially during the summer time. Mortality
diet. was recorded daily. Broiler chicks were weighed regu-
Starter Grower Finisher
larly at hatch, after one week, and at the end of every
2 wk, thereafter until the end of the cycle after 35 d.
Ingredient 0–7 d 8–21 d 22–35 d

Corn 55.600 57.600 61.200

Soyabean meal 39.400 35.600 32.250 Sample Collection
Soya oil 1.350 3.200 3.350
Limestone 1.450 1.450 1.300 A representative sample of 5 chickens was used from
DiCalcium phosphate 1.400 1.400 1.200
Salt 0.210 0.210 0.200 each treatment group. Blood samples in triplicates of
L-Lysine 0.120 0.120 0.100 the broiler chickens were collected on weeks 3 and 5 of
DL-Methionine 0.270 0.270 0.260 the production cycle. Birds were sacrificed by stunning
Vitamin–Mineral Premix1 0.200 0.200 0.200
Total % 100 100 100 and bleeding. Blood was collected in heparinized tubes
Nutrient composition that had extra liquid heparin added to them to avoid
Chemical analysis clots.
Crude protein (CP) (%) 24.01 22.36 21.03
Metabolizable energy (kcal/kg) 2932.16 3054.44 3105.30 The thymic lobes were removed and transferred to a
Fat (g/kg) 3.860 5.748 6.002 tube filled with sterile ice cold phosphate buffered saline
Calculated analysis (PBS). The spleen was removed and placed in a tube
Calcium (g\kg) 0.989 0.98 0.87
Phosphorus (g\kg) 0.410 0.402 0.361 filled with sterile ice cold PBS. All fat and adherent tis-
Sodium (g\kg) 0.110 0.111 0.108 sues were removed from the thymus and spleen. Other
Lysine (g\kg CP) 1.45 1.34 1.23 organs, such as the liver, bursa of Fabricious, heart,
Methionine (g\kg CP) 0.665 0.642 0.615
Choline (mg\kg) 1420.73 1329.36 1260.18 and fat pads, were all removed and placed in PBS for
measuring weight.
1
Supplied per kg of premix: trans-retinol(A), 12,500,000 IU; chole-
calciferol(D3), 500,000 IU; α -tocopherol acetate(E), 75,000 mg; thi-
amine(B1), 4500 mg; riboflavin(B2), 8000 mg; pyridoxine(B6), 5000 mg;
vitamin B12, 22,000 mg; pantothenic acid, 20,000 mg; folic acid, 2000 mg; Hematological Measurements
biotin, 200,000 μ g; Fe, 100,000 mg; Co,250 mg; Mn, 100 mg; Cu,
10,000 mg; Zn, 80,000 mg; I, 1000 mg; Se, 300 mg; Mo, 0.5 mg; Ca, The haematocytometer, Cell-Dyn 3500 Hematol-
7.7%; P, 0.01%; Na, 0.18%; Ash, 97%. ogy System (Abbott Laboratories, Abbott Park, IL,
USA), was used to measure the total and differential
During the first 3 d of the brooding period, the 1-day- white blood cells (WBCs) and blood quality param-
old chicks were provided with 24 h of light to give them eters including red blood cells (RBC), haemoglobin
enough time to find out feed and water. After that, (HGB), haematocrit (HCT), mean corpuscular
a step down lighting program was followed. Artificial volume (MCV), mean corpuscular haemoglobin
bulbs were used as a source of light as natural light- (MCH), mean corpuscular haemoglobin concentration
ing was limited. Lighting is a crucial factor for rearing (MCHC), red cell distribution width (RDW), and
commercial broiler chickens as they play an important platelet count (PLT).
role on health, productivity, and welfare of the birds.
Poultry have been studied to possess highly specialized
vision and majority of their behavior is governed by Cellular Immune Response
this factor; they also have the most sophisticated reti- The phytohemagglutinin (PHA)-skin test involved
nal cones among vertebrates. This in turn influences subcutaneous injection of a mitogen and measurement
their physiological and behavioral process (Yang et al., of subsequent swelling as a surrogate of T-cell medi-
2016). There was a minimal vaccination of the broilers ated immunocompetence. The PHA was dissolved in
as per the guidelines of the company that supplied the cell-culture grade/pyrogen-free PBS, and the resulting
chicks. After mixing the feed rations with the differ- concomitant swelling was quantified at the site of in-
ent dietary treatments, and before feeding them to the jection over time. The resulting swelling, usually mea-
broiler chickens, all of the feed rations were analyzed for sured 24 h post-injection, was interpreted as an index
proximate analysis to check and maintain the quality of of cell-mediated immunocompetence (Goto et al., 1978;
feed. Corrier and DeLoach, 1990). At 5 wk of age, 5 chick-
ens were injected with PHA per treatment. The injec-
Production Performance Variables tion site (wattle) was marked prior to injection and the
in Broilers thickness of the injection site was measured by microm-
eter. Then, the birds were injected intradermally in the
Feed efficiency is the most important data required wattle with 0.5 mg of PHA-P in 0.1 mL of PBS. Post-
since it provides information on the efficiency of feed injection thickness was typically measured 24 h post-
utilization for the growth of chickens. Body weight and injection, yet 24 h does not reflect the peak of the re-
feed consumption were recorded. Temperature and rel- action, it could be measured (to the nearest 0.01 mm)
ative humidity were also recorded daily and adjusted at 0, 24, 48, and 72 h after PHA-P injection. Wattle
accordingly to avoid stressful conditions in the poultry swelling was calculated as the difference between the
4468 AL-KHALAIFA ET AL.

thickness of the wattle prior to and after the injection with a sterile scalpel and rinsed with 0.85% (w/v) NaCl
of PHA-P. sterile solution (1:9 v/v) to remove the content. Any
residual cecal content was removed gently by scraping
the cecal epithelium. The crude extract of the ceca was
Humoral Immune Response transferred into a sterile stomacher bag and homoge-
nized for 3 min. The collected crude extracts were used
This test was done at weeks three and five in each
directly for microbial analysis and pH measurements.
broiler cycle. Five chickens were used in each test.
Lactic acid bacteria, Escherichia coli, and Salmonella
This test involved the measurement of antibody titers
counts were determined using standard microbiological
against sheep RBC antigen. In this test, the chickens
methods as described by Lorch et al. (1995), and sam-
were challenged by injecting them with 1 mL of di-
ples were analysed by applying the spreading technol-
luted sheep RBC solution (7% vol/vol suspension in
ogy. Escherichia coli and Salmonella count experiments
0.9% NaCl). A week after the challenge, serum sam-
were conducted using Brilliance E. Coli selective and
ples were prepared from whole blood using the cen-
Xylose–Lysine–Desoxycholate agar media (Oxoid), re-
trifugation methods, and the total and differential anti-
spectively, while LAB experiments were conducted us-
body titers (immunoglobulins IgA, IgY, and IgM) were
ing de Man, Rogosa, Sharpe (MRS) media (Oxoid).
measured using commercial enzyme-linked immunosor-
For the analysis, serial dilution was made from the
bent assay (ELISA) kits (Bioassay Technology Lab-
crude samples. About 0.1 mL of the prepared sample
oratory, Shanghai, China). In the 96-well tray, 50 μL
was spread onto the surface of the media with a ster-
of the respective standards were added into each well.
ile spreader. The plates were incubated aerobically for
Then, 40 μL of each sample was added to sample wells,
24 h at 37◦ C for both E. coli and Salmonella, whereas
followed by the addition of 10 μL of biotin-conjugate
for LAB, the plates were incubated anaerobically for
anti-chicken antibody into sample wells. Next, 50 μL of
48 h at 37◦ C. The colonies were counted at the end
streptavidin-HRP was added to sample and standard
of the incubation periods and were transformed to log
samples carefully avoiding the blank control wells. The
values.
reagents were then mixed well. The plate was covered
with a sealer and then incubated for 60 min at 37◦ C.
Post incubation, sealer was removed and the plate was Hindgut Acidosis
washed 5 times with wash buffer; the wells were over-
filled and soaked for at least 30 s to 1 min. The plate This generally refers to the lowering of pH (an in-
was blotted onto paper towels following each washing. crease in acidity) in the cecum and/or colon. Hindgut
Then, 50 μL of substrate solution A was added to each acidosis was measured and compared between the con-
well, followed by the addition of 50 μL of substrate so- trol and the experimental groups to investigate the ef-
lution B (care must be taken not to expose substrate fect of prebiotics and probiotics on the acidity of the
solution B to light as it is light sensitive). The plate was hindgut, which is considered an indicator of chicken’s
sealed with a new sealer and incubated for 10 min in health, well-being, and performance, and may even be
the dark at 37◦ C. Upon the addition of 50 μL of stop the precursor to more serious health risks. The pH was
solution to each well, the blue color immediately turned measured by a pH probe. This test was done at weeks
to yellow. Lastly, the optical density (OD) value was 3 and 5 in each broiler cycle. Five chickens from each
determined at 40 nm within 30 min after adding the treatment were used. The GI tissues from the duode-
stop solution, using a microplate reader. num to the ileum and ceca were separated from the
birds. The hindgut was defined as the portion of the
intestine from the yolk sac diverticulum to the ileocecal
Microbial Counts in the Chickens’ Gut junction. Hindgut digesta were emptied into tubes. A
pH probe was placed directly into the digesta, and pH
The chickens were dipped and washed in 1:2 diluted
was recorded.
disinfectants. The abdomen area was de-feathered. The
de-feathered area was sprayed with 70% ethanol to en-
sure the sterility of the area before dissecting. The Volatile Fatty Acid Profile in the Hindgut
chicken skin was cut using a sterile scissor and re-
moved away from the abdomen area with sterile forceps. This test was done at weeks 3 and 5 in each broiler
The covering membrane was cut carefully using sterile cycle. Five chickens from each treatment were used. Ap-
scissors or scalpel to reach the chicken’s digestive sys- proximately, 1.50 g of thawed digesta was diluted with
tem. Then, the lower intestine was surgically exposed, distilled water (1:1 wt/vol) in a screw-capped tube. Af-
the ceca were removed using sterile scissors, and their ter homogenization and centrifugation, 1 mL of clear
weights were recorded. In order to do the microbial supernatant was used to analyze the volatile fatty acid
analysis (E. coli, LAB, and Salmonella), cecum con- profile using gas chromatography (GC) technique. The
tents were extracted as described by Schoeni and Doyle main principle underlying the separation of fatty acids
(1992). The content of each cecum was squeezed in a using GC is that they differ in the temperature at which
sterile Petri dish, and then the ceca was split lengthwise they become volatile. This depends on the carbon chain
 DIETARY PROBIOTICS AND PREBIOTICS 4469
length, number, and the position of double bonds in the Fatty Acid Analysis by Gas
molecules. The injection temperature was set at 140˚C Chromatography
and helium was the carrier gas with a pre-column split
ratio of 50:1 and a head pressure of 37.34 psi. These set- Fatty acid methyl esters were analyzed using a
tings were shown to be optimum for the needed fatty Hewlett Packard (HP) 6890 series GC system (HP,
acids in chicken intestinal fluid samples. In addition, Basingstoke, UK). For transmethylation, 400 μL
the following temperature program was found to be the toluene (+BHT 50 mg/L) and 800 μL of 1.5% sulfu-
best for the optimal separation of methyl esters: ric acid in methanol were added to the dried samples.
The tubes were vortexed and heated in a water bath
1. The oven was initially heated to 100˚C and held for at 70◦ C for 1 h. After cooling, 2 mL neutralizing agent
1 min, and the temperature was then increased to (0.1 M K2 CO3 , 0.1 M KHCO3 ) and 2 mL hexane were
240◦ C at 4◦ C per min. added and the suspension was mixed and centrifuged at
2. The FAME was diluted in 100 μL of hexane, 1 μL of 1162 × g for 10 min. The upper layer was transferred to
which was injected onto the column using an Agilent a glass tube and evaporated to dryness under nitrogen.
7683 series autosampler. The extract was suspended in 100 μL hexane, vortexed,
3. The FAME was identified by comparing the re- and transferred to a GC vial. Samples were run against
tention time against a known standard (Supelco a known standard.
Volatile Free Acid Mix, Sigma-Aldrich, Wyoming,
USA). FAMEs were detected using a flame ioniza- Statistical Analysis
tion detector.
4. The chromatograms were plotted and analyzed us- The overall differences between the dietary treat-
ing the HP Chemstation software package. ments were analyzed using one-way analysis of vari-
ance (ANOVA), and the general linear model procedure
The machine (Agilent Technologies GC system, of Minitab was applied. Differences between the treat-
Wilmington, USA) was standardized using the short ment groups were considered statistically different at
chain volatile fatty acid standard mixes. All of the re- P ≤ 0.05. When significant differences occurred (P ≤
quired fatty acid peaks were obtained and standardized. 0.05), treatment means’ differences were identified by
The machine was ready to analyze the samples. pairwise comparison using the Bonferroni test.

Organoleptic Study RESULTS AND DISCUSSION

This included taste/acceptability measures of the
meat samples to determine the effect of dietary treat-
Production Performance Parameters
ments on the sensory quality of the produced meat. The The broilers were raised successfully, following op-
meat was simply cooked with minimal spices to ensure timal managerial practices including strict biosecurity
that the chicken meat was edible and acceptable by pan- measures and nutritional practices. Tables 1 and 2 show
elists. Sensory evaluation parameters included accept- the routine proximate analysis of the feed ration fed to
ability and palatability tests of the poultry meat. This broilers in cycles 1 and 2. This analysis involved crude
was done by taste paneling and visual observations. protein percentage, crude fat percentage, ash, and mois-
There were 16 panelists in the panel. The panelists who ture tests. Results provided in these tables show that
participated in the sensory evaluation had been trained the feed rations fed to the broilers contained normal
on the method before they began the procedure. Each ranges of protein and fat. This ensures the healthy feed-
group of meat samples were labeled randomly with a ing of broilers with optimal ranges of vital levels of in-
two-digit code number. Each batch of samples was pre- gredients. Hence, no outstanding mortality, outbreaks,
sented in a randomized order, and the panelists were or abnormal growth were observed.
asked to assign scores for color, smell, texture, taste, Broiler Cycle 1 The first broiler cycle was conducted
and general acceptability on a structured 9-point he- in the summer. Table 3 shows the effect of different
donic scale (1 to 9, where 1 indicates the worst qual- probiotics and prebiotics on the body weight of broil-
ity and 9 indicates the best quality). In this method, ers. There was no significant effect of these probiotics
the stimuli (meat samples) are presented singly and are and prebiotics, at different weeks, on the body weight.
rated on a scale where the 9 categories range from “dis- On the contrary, Sarangi et al. (2016) fed 360 one-day-
like extremely” to “like extremely”. Major advantages old VenCobb broilers with diets consisting of control
of the method are as follows: participant can respond and basal diets supplemented with prebiotics and probi-
meaningfully without prior experience, it is suitable for otics. The results showed lower body weight in broilers
use with a wide range of populations, the data can be fed diets supplemented with probiotics and prebiotics
handled by the statistics of variables, and results are than those fed control diets.
meaningful for indicating general levels of preference The effect of different probiotics and prebiotics on the
(Peryam and Pilgrim 1957). feed consumption of broilers, as seen in Table 4, shows
4470 AL-KHALAIFA ET AL.
Table 2. Proximate analysis of the feed ration fed to broilers in cycle 1.

Parameter
Crude protein % Crude fat% Ash Moisture

Control (Starter) 25.01 ± 0.02 4.85 ± 0.07 4.56 ± 0.57 60.09 ± 0.05
Control (Grower) 23.10 ± 0.03 5.31 ± 0.42 5.01 ± 0.05 62.10 ± 0.15
Control (Finisher) 21.00 ± 0.06 5.88 ± 0.03 5.21 ± 0.03 63.98 ± 0.12
B. coagulans (Starter) 24.03 ± 0.05 4.78 ± 0.53 5.49 ± 0.09 62.98 ± 0.77
B. coagulans (Grower) 22.09 ± 0.06 5.13 ± 0.15 4.29 ± 0.07 63.45 ± 0.19
B. coagulans (Finisher) 20.94 ± 0.05 5.38 ± 0.21 5.04 ± 0.64 64.08 ± 0.08
Lactobacillus (Starter) 24.00 ± 0.03 4.01 ± 0.98 4.89 ± 0.09 61.98 ± 0.09
Lactobacillus (Grower) 21.57 ± 0.08 4.87 ± 0.09 4.98 ± 0.24 62.01 ± 0.04
Lactobacillus (Finisher) 21.02 ± 0.07 5.01 ± 0.29 4.87 ± 0.09 62.89 ± 0.09
FOS (Starter) 24.09 ± 0.05 4.99 ± 0.08 5.01 ± 0.13 62.09 ± 0.15
FOS (Grower) 20.87 ± 0.09 4.89 ± 0.05 5.18 ± 0.23 62.01 ± 0.04
FOS (Finisher) 20.92 ± 0.10 5.19 ± 0.18 4.98 ± 0.07 63.02 ± 0.05
MOS (Starter) 23.98 ± 0.04 4.00 ± 0.97 5.01 ± 0.07 62.97 ± 0.04
MOS (Grower) 22.09 ± 0.10 4.97 ± 0.51 5.19 ± 0.21 62.00 ± 0.03
MOS (Finisher) 20.55 ± 0.07 5.53 ± 0.13 5.03 ± 0.10 62.53 ± 0.05

% On Dry matter Basis ± S.D∗

Mean ± ∗ S.D. = Standard Deviation

Table 3. Effect of different probiotics and prebiotics on the body weight (g) of broilers (cycle 1).

Age Control B. coagulans Lactobacillus FOS MOS SEM P-value

1 d 45.04 45.4 44.24 44.4 44.96 0.66 0.717

1 wk 125.8 128.64 122.92 128.8 126.52 2.55 0.489
2 wk 304.00 296.00 290.0 307.20 307.00 5.13 0.132
3 wk 644.0 626.40 611.2 619.2 612.80 11.71 0.306
4 wk 1091.2 1084.0 1085.6 1078.4 1084.80 23.30 0.997
5 wk 1672 1688.8 1646.40 1664.8 1584.80 40.15 0.427

B = Bacillus coagulans (1 g/kg dried culture); L = Lactobacillus (1 g/kg dried culture of 12 commercial
strains); FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from
Saccharomyces cerevisiae (5 g/kg); All treatment groups received a soybean basal diet; Differences between
the treatment groups are statistically different at P ≤ 0.05, n = 5, 17 birds in each replicate.

Table 4. Effect of different probiotics and prebiotics on the feed consumption (g) of b Broilers
(cycle 1).

Age (Weeks) Control B. coagulans Lactobacillus FOS MOS SEM P-value

1 113.6 117.6 114.6 117.20 117.8 3.27 0.844

2 192.3 191.3 192.1 198.7 203.0 4.30 0.259
3 484.0 487.2 483.6 488.8 482.6 4.18 0.809
4 840.8 644.0 641.60 641.20 645.2 11.24 0.998
5 907.00 930.0 949.6 918.8 925.2 27.77 0.861

B = Bacillus coagulans (1 g/kg dried culture); L = Lactobacillus (1 g/kg dried culture of 12 commercial
strains); FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from
Saccharomyces cerevisiae (5 g/kg); All treatment groups received a soybean basal diet; Differences between
the treatment groups are statistically different at P ≤ 0.05, n = 5, 17 birds in each replicate.

no significant effect through the different weeks of age. showed that these supplementations significantly in-
A similar study was conducted on 350 one-day-old male creased (P ≤ 0.05) feed efficiency at different ages.
Ross 308 broiler chickens that were randomly assigned Table 6 shows the effect of different probiotics and
different dietary treatments consisting of a control diet, prebiotics on the weekly body weight gain of broilers.
basal diet supplemented with black cumin seed, a pre- There is no significance observed for body weight gain
biotic, a probiotic, and a SYN. The results showed that in the broilers. However, in a study by Ipek et al. (2016),
the feed consumption of the broilers was not affected at 720 one-day-old Cobb 500 broiler chicks were assigned
all by any of the treatments (Ghasemi et al., 2014). into 4 treatment groups that were administered diets
The effect of different probiotics and prebiotics on consisting of control, and 3 groups of different measures
feed efficiency in broilers is shown in Table 5. There of probiotics and prebiotics (PPS), which included live
is no significance observed at different weeks in the Saccharomyces cerevisiae strain. It was observed that in
broilers. A study by Mookiah et al. (2014) showed con- all of the 3 groups supplemented with PPS, body weight
trasting results wherein broiler chickens were fed diets gain was found to be significantly higher in the broilers.
supplemented with varying levels of prebiotics, probi- Table 7 shows the effect of different probiotics and
otics, and synbiotics (SYN). The results in Table 5 prebiotics on body weight gain, total feed consump-
 DIETARY PROBIOTICS AND PREBIOTICS 4471
Table 5. Effect of different probiotics and prebiotics on the feed efficiency of broilers (cycle 1).

Age (Weeks) Control B. coagulans Lactobacillus FOS MOS SEM P-value

1 1.42 1.42 1.46 1.40 1.45 0.05 0.942

2 1.08 1.14 1.15 1.12 1.13 0.02 0.487
3 1.43 1.48 1.52 1.57 1.59 0.05 0.308
4 1.45 1.42 1.37 1.42 1.40 0.08 0.974
5 1.58 1.56 1.75 1.58 1.93 0.15 0.392

B = Bacillus coagulans (1 g/kg dried culture); L = Lactobacillus (1 g/kg dried culture of 12 commercial
strains); FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from
Saccharomyces cerevisiae (5 g/kg); All treatment groups received a soybean basal diet; Differences between
the treatment groups are statistically different at P ≤ 0.05, n = 5, 17 birds in each replicate.

Table 6. Effects of different probiotics and prebiotics on the weekly body weight gain (g) of
broilers (cycle 1).

Age (Weeks) Control B. coagulans Lactobacillus FOS MOS SEM P-value

1 80.76 83.24 78.68 84.4 81.56 2.60 0.583

2 178.2 167.36 167.92 178.4 180.45 5.18 0.246
3 340.0 330.4 320.36 312.0 305.8 11.62 0.268
4 447.2 457.6 474.4 459.2 472.0 24.45 0.930
5 581.6 604.8 560.8 586.4 500.0 36.06 0.319

B = Bacillus coagulans (1 g/kg dried culture); L = Lactobacillus (1 g/kg dried culture of 12 commercial
strains); FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from
Saccharomyces cerevisiae (5 g/kg); All treatment groups received a soybean basal diet; Differences between
the treatment groups are statistically different at P ≤ 0.05, n = 5, 17 birds in each replicate.

Table 7. Effects of different probiotics and prebiotics on total body weight gain, total feed con-
sumption, and overall feed efficiency of broilers (cycle 1).

Parameters Control B. coagulans Lactobacillus FOS MOS SEM P-value

Body weight gain 1627.80 1643.4 1602.2 1620.4 1539.8 40.28 0.432
Total feed consumption 2337.70 2370.10 2381.50 2345.20 2387.00 40.63 0.951
Overall feed efficiency 1.44 1.45 1.42 1.46 1.40 0.05 0.671

B = Bacillus coagulans (1 g/kg dried culture); L = Lactobacillus (1 g/kg dried culture of 12 commercial
strains); FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from
Saccharomyces cerevisiae (5 g/kg); All treatment groups received a soybean basal diet; Differences between
the treatment groups are statistically different at P ≤ 0.05, n = 5, 17 birds in each replicate.

tion, and overall feed efficiency of broilers. There was received diets with all the other dietary treatments.
no significance seen for any of the parameters in the However, feed consumption was significantly higher in
broilers. In a study, 200 one-day-old Hubbard broiler birds fed diets with MOS than in those fed diets with
chicks were randomly divided into 4 groups, which were B. coagulans.
fed diets consisting of a control and diets with varying The effects of different probiotics and prebiotics on
percentages of the prebiotic β -galacto-oligosaccharides the feed efficiency and weekly body weight gain are
(β -GOS). It was observed that body weight gain and shown in Tables 11 and 12, respectively. The results of
feed efficiency were significantly higher (P < 0.05) Table 11 show no effect of the different probiotics and
for broilers fed β -GOS supplemented diet; however, prebiotics on the feed efficiency of broilers. Similarly,
there was no significance observed for feed consump- there was no significant effect of the dietary treatments
tion (Yousaf et al., 2016). on the weekly body weight gain of broilers. Table 13
Broiler Cycle 2 Broiler cycle no. 2 was conducted in shows the effects of different probiotics and prebiotics
the winter time. Table 8 shows the proximate analysis on the body weight gain, total feed consumption, and
of the feed ration fed to broilers in cycle 2. The effect of overall feed efficiency of broilers. The results of Table 13
different probiotics and prebiotics on the body weight show that the different probiotics and prebiotics did
of broilers is shown in Table 9. There was no significant not significantly affect the body weight gain, total feed
effect of the different probiotics and prebiotics on the consumption, and overall feed efficiency of broilers.
body weight of broilers.
Table 10 shows the effect of different probiotics and
prebiotics on the feed consumption of broilers. At weeks Hematological Measurements
1, 4, and 5, there was no effect of the different probi-
otics and prebiotics on the feed consumption of broil- Broiler Cycle 1 Table 14 shows the haematological
ers. At week 2, feed consumption of the broilers was and biochemical parameters of 5-week-old broiler fed
similar for the groups of the control, and the birds different probiotics and prebiotics. All broilers appeared
4472 AL-KHALAIFA ET AL.
Table 8. Proximate analysis of the feed ration fed to broilers in cycle 2.

Parameter
Crude protein % Crude fat% Ash Moisture

Control (Starter) 24.52 ± 0.05 5.03 ± 0.03 5.01 ± 0.12 62.01 ± 0.08
Control (Grower) 23.11 ± 0.06 5.12 ± 0.08 5.00 ± 0.09 61.13 ± 0.09
Control (Finisher) 20.90 ± 0.03 4.50 ± 0.08 5.91 ± 0.07 62.08 ± 0.13
B. coagulans (Starter) 23.93 ± 0.02 5.00 ± 0.03 5.50 ± 0.11 61.98 ± 0.09
B. coagulans (Grower) 21.87 ± 0.05 4.98 ± 0.07 4.98 ± 0.09 62.95 ± 0.10
B. coagulans (Finisher) 21.03 ± 0.10 4.89 ± 0.08 5.00 ± 0.32 63.99 ± 0.09
Lactobacillus (Starter) 24.61 ± 0.02 5.01 ± 0.09 5.63 ± 0.08 62.08 ± 0.10
Lactobacillus (Grower) 22.01 ± 0.06 4.99 ± 0.04 4.99 ± 0.10 63.01 ± 0.09
Lactobacillus (Finisher) 21.64 ± 0.05 5.15 ± 0.03 5.00 ± 0.06 63.01 ± 0.10
FOS (Starter) 24.13 ± 0.05 5.01 ± 0.12 5.11 ± 0.10 62.87 ± 0.13
FOS (Grower) 21.08 ± 0.09 5.11 ± 0.09 5.09 ± 0.09 63.02 ± 0.12
FOS (Finisher) 21.13 ± 0.08 4.89 ± 0.07 4.99 ± 0.09 62.99 ± 0.09
MOS (Starter) 23.99 ± 0.02 5.06 ± 0.29 4.91 ± 0.08 61.87 ± 0.14
MOS (Grower) 22.64 ± 0.12 4.99 ± 0.12 5.10 ± 0.10 63.00 ± 0.07
MOS (Finisher) 21.05 ± 0.09 5.03 ± 0.08 5.08 ± 0.09 62.98 ± 0.12

% on dry matter Basis ± S.D∗

Mean ± ∗ S.D. = Standard Deviation

Table 9. Effects of different probiotics and prebiotics on the body weight (g) of broiler (broiler
cycle 2).

Age Control B. coagulans Lactobacillus FOS MOS SEM P-value

1 d 44.84 44.2 43.88 43.32 42.32 0.65 0.119

1 wk 130.68 120.68 122.6 124.04 124.04 3.44 0.342
2 wk 304.80 292.0 292.80 293.6 302.80 7.02 0.569
3 wk 553.6 544.80 530.40 548.0 554.4 15.73 0.820
4 wk 939.2 938.4 906.4 946.4 904.8 29.20 0.763
5 wk 1425.6 1407.2 1425.6 1432.8 1403.2 34.38 0.965

B = Bacillus coagulans (1 g/kg dried culture); L = Lactobacillus (1 g/kg dried culture of 12 commercial
strains); FOS = Fructo-oligosaccharides (5 g/kg), MOS (Bio-MOS) = mannan-oligosaccharide derived from
Saccharomyces cerevisiae (5 g/kg); All treatment groups received a soybean basal diet; Differences between
the treatment groups are statistically different at P ≤ 0.05, n = 5, 17 birds in each replicate.

Table 10. Effects of different probiotics and prebiotics on the feed consumption (g) of broilers
(broiler cycle 2).

Age (Weeks) Control B. coagulans Lactobacillus FOS MOS SEM P-value

1 110.4 115.0 108.6 105.20 109.2 5.00 0.734

2 228.60a,b 215.0b 225.40a,b 227.80a,b 231.6a 3.27 0.019
3 478.6 480.4 478.6 476.4 473.80 3.26 0.380
4 671.00 668.2 670.00 665.20 671.00 3.72 0.783
5 926.4 915.20 915.2 911.60 928.4 21.87 0.974

Bacillus coagulans (1 g/kg dried culture); Lactobacillus (1 g/kg dried culture of 12 commercial strains)’
FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from Saccha-
romyces cerevisiae (5 g/kg); All treatment groups received a soybean basal diet; Differences between the
treatment groups are statistically different at P ≤ 0.05, n = 5, 17 birds in each replicate.

healthy, and no significant mortality occurred through- otics on all of the biochemical parameters of the blood
out the experimental period. Interestingly, there was no among the experimental groups of broilers at 5 wk
significant effect of the different probiotics and prebi- of age, as shown in Table 15. These parameters were
otics on all of the biochemical parameters of the blood WBC, neutrophils (%), lymphocytes (%), monocytes
among the experimental groups of broilers at 5 wk (%), eosinophils (%), basophils (%), RBC, HGB, HCT
of age, as shown in Table 13. These parameters were %, MCV, MCH, MCHC, and RDW (%).
WBC, neutrophils (%), lymphocytes (%), monocytes
(%), eosinophils (%), basophils (%), RBC, HGB, HCT
%, MCV, MCH, MCHC, and RDW (%). Cellular Immune Response
Broiler Cycle 2 Table 15 shows the hematological
and biochemical parameters of 5-week-old broilers fed Broiler Cycle 1 Wattle swelling changes as affected
different probiotics and prebiotics. All broilers appeared by the different dietary treatments in cycle 1 are shown
healthy and no significant mortality occurred through- in Table 16. The results revealed that dietary FOS and
out the experimental period. Interestingly, there was no MOS induced higher cellular response than the other
significant effect of the different probiotics and prebi- treatments (P = 0.04).
 DIETARY PROBIOTICS AND PREBIOTICS 4473
Table 11. Effects of different probiotics and prebiotics on the feed efficiency (kg feed/kg body
gain) of broilers (broiler cycle 2).

Age (Weeks) Control B. coagulans Lactobacillus FOS MOS SEM P-value

1 1.31 1.51 1.38 1.32 1.34 0.08 0.449

2 1.32 1.26 1.33 1.38 1.29 0.06 0.747
3 1.95 1.92 2.02 1.87 1.91 0.10 0.860
4 1.76 1.71 1.86 1.71 1.93 0.12 0.630
5 1.94 2.05 1.78 1.92 1.89 0.17 0.851

Bacillus coagulans (1 g/kg dried culture); Lactobacillus (1 g/kg dried culture of 12 commercial strains);
FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from Saccha-
romyces cerevisiae (5 g/kg); All treatment groups received a soybean basal diet; Differences between the
treatment groups are statistically different at P ≤ 0.05, n = 5, 17 birds in each replicate.

Table 12. Effects of different probiotics and prebiotics on the weekly body weight gain (g) of
broilers (broiler cycle 2).

Age (Weeks) Control B. coagulans Lactobacillus FOS MOS SEM P-value

1 85.84 76.48 78.72 80.72 81.72 3.45 0.414

2 174.12 171.32 170.2 169.56 178.76 6.93 0.878
3 248.80 252.80 237.60 254.40 251.60 13.41 0.906
4 385.6 393.60 376.00 398.40 350.40 26.70 0.732
5 486.4 468.80 519.20 486.40 498.40 34.60 0.882

Bacillus coagulans (1 g/kg dried culture); Lactobacillus (1 g/kg dried culture of 12 commercial strains);
FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from Saccha-
romyces cerevisiae (5 g/kg); All treatment groups received a soybean basal diet; Differences between the
treatment groups are statistically different at P ≤ 0.05, n = 5, 17 birds in each replicate.

Table 13. Effects of different probiotics and prebiotics on the body weight gain, total feed con-
sumption, and overall feed efficiency of broilers (broiler cycle 2).

Parameters Control B. coagulans Lactobacillus FOS MOS SEM P-value

Body weight gain (g) 1380.8 1363.0 1375.4 1378.8 1379.4 34.24 0.968
Total feed consumption (g) 2415.0 2395.8 2402.4 2708.2 2399.0 25.64 0.830
Overall feed efficiency (Kg/Kg gain) 1.66 1.69 1.67 1.70 1.68 0.04 0.949

Bacillus coagulans (1 g/kg dried culture); Lactobacillus (1 g/kg dried culture of 12 commercial strains),
FOS = Fructo-oligosaccharides (5 g/kg), MOS (Bio-MOS) = mannan-oligosaccharide derived from Saccha-
romyces cerevisiae (5 g/kg); All treatment groups received a soybean basal diet; Differences between the
treatment groups are statistically different at P ≤ 0.05, n = 5, 17 birds in each replicate.

Broiler Cycle 2 Wattle swelling changes as affected etary treatments that included a basal diet (BD), BD
by the different dietary treatments in cycle 2 are shown + prebiotic, BD + probiotic, BD + symbiotic, and
in Table 17. The results revealed that dietary FOS BD + antibiotic. The chicks were also administered
induced higher cellular response than the other treat- vaccines against Marek’s disease, coccidiosis, and New-
ments (P = 0.019). castle’s disease. Serum titers of antibodies against New-
castle were collected at 14 d and then later subjected
Humoral Immune Response to ELISA. It was observed that broilers fed diets with
prebiotics presented better feed gain ratio, along with
Broiler Cycle 1 Tables 18 and 19 show the effect of an increase in antibody production against Newcastle
different dietary treatments on the total chicken anti- disease virus (NDV). No significance was observed for
bodies in 3- and 5-wk-old broiler chickens (cycle 1), re- the rest of the diets. On the contrary, a study was con-
spectively. Results of Table 18 show significant effect of ducted to determine the effects of dietary probiotics on
the dietary treatments on the total titers of antibodies. natural IgM levels binding keyhole limpet hemocyanin
Feeding the broilers on diets supplemented with FOS (KLH) in indigenous chicken (IC) in Kenya. One hun-
significantly increased the titers of IgY, compared to dred and fifty 2-month-old chickens were randomly di-
the other dietary treatments. IgA titers in broilers fed vided into 5 treatments of 25 birds each, and 5 mL
on diets supplemented with B. coagulans were higher of the probiotic Molaplus was dissolved in 250, 500,
than those observed in birds fed on the control and the 1000, 1500, and 2000 mL of drinking water. The lev-
Lactobacillus diets, but significantly similar to those fed els of IgM were determined using the indirect ELISA
the FOS- and MOS-supplemented diets. However, there technique. The results showed that dietary probiotic
was no significant effect of the different dietary treat- supplementations did not significantly affect the levels
ments on IgM titers. of IgM binding the KLH (Khobondo et al., 2015).
In a study by Murarolli et al. (2014), 1400 one-day- An experiment was conducted to determine the ef-
old male Cobb 500 chicks were given five different di- fects of probiotics and yeast-derived carbohydrates
4474 AL-KHALAIFA ET AL.
Table 14. Haematological and biochemical parameters of 5-week-old broiler fed different probiotics
and prebiotics (cycle 1).

Variables Control B L FOS MOS SEM P-value

WBC 51.83 64.10 38.83 47.83 41.97 10.72 0.526

Neutrophils 9.63 20.60 28.48 14.84 11.88 5.90 0.236
Lymphocytes 43.63 38.15 47.07 36.73 37.73 8.95 0.903
Monocytes 29.50 23.19 16.06 25.47 32.70 8.05 0.655
Eosinophils 0.08 0.14 0.08 0.06 0.03 0.03 0.159
Basophils 17.10 17.90 8.32 22.88 17.68 8.93 0.840
RBC 2.44 2.40 2.48 1.66 2.40 0.38 0.542
HGB 13.13 13.45 13.50 13.70 13.10 0.47 0.870
HCT 35.23 34.95 35.47 24.77 33.83 5.67 0.641
MCV 144.33 146.00 143.00 99.33 141.00 22.34 0.557
MCH 54.03 56.15 54.40 37.83 54.67 8.52 0.550
MCHC 37.47 38.40 38.07 25.33 38.80 5.69 0.439
RDW 13.23 13.00 13.27 8.60 14.40 2.02 0.354
PLT 26.65 4.73 1.99 2.22 0.81 7.84 0.180
B = Bacillus coagulans; L = Lactobacillus; FOS = Fructo-oligosacharride; MOS = mannan-oligosaccharide;
WBC = White Blood Cell, RBC = Red Blood Cell; HGB = Hemoglobin; HCT = Hematocrit; MCV =
Mean Corpuscular Volume; MCH = Mean Corpuscular Hemoglobin; MCHC = Mean Corpuscular Hemoglobin
Concentration; RDW = Red blood cell distribution width; PLT = Platelet count.

Table 15. Haematological and biochemical parameters of 5-week-old broiler fed different probiotics
and prebiotics (cycle 2).

Control B L FOS MOS SEM P-value

WBC 65.19 55.70 63.60 18.90 79.67 23.74 0.181

Neutrophils 6.65 7.01 7.57 7.67 11.60 3.19 0.218
Lymphocytes 21.43 23.67 20.27 23.20 30.47 10.83 0.468
Monocytes 20.43 23.67 20.27 23.20 30.47 9.51 0.282
Eosinophils 0.03 0.02 0.02 0.04 0.04 0.02 0.346
Basophils 16.65 17.00 17.87 25.77 30.50 11.16 0.414
RBC 2.79 2.78 2.78 2.79 2.53 0.13 0.585
HGB 15.77 15.17 15.20 15.70 14.07 0.71 0.483
HCT 36.80 36.98 37.50 37.30 34.40 1.86 0.768
MCV 131.67 132.67 135.33 134.00 136.33 2.57 0.707
MCH 56.43 54.57 54.70 56.47 55.67 0.61 0.142
MCHC 42.83 41.13 40.53 42.20 40.83 0.75 0.227
RDW 11.63 11.97 12.00 11.33 11.63 0.34 0.624
PLT 5.50 2.40 0.89 3.58 1.87 1.39 0.244
B = Bacillus coagulans; L = Lactobacillus; FOS = Fructo-oligosacharride; MOS = mannan-oligosaccharide;
WBC = White Blood Cell; RBC = Red Blood Cell; HGB = Hemoglobin; HCT = Hematocrit; MCV =
Mean Corpuscular Volume; MCH = Mean Corpuscular Hemoglobin; MCHC = Mean Corpuscular Hemoglobin
Concentration; RDW = Red blood cell distribution width; PLT = Platelet count.

Table 16. Wattle swelling changes as affected by the different dietary treatments (cycle 1).

Treatment
Control B. coagulans Lactobacillus FOS MOS SEM P-value
b b b a a
Wattle swelling (mm) 1.45 1.37 1.87 2.20 2.19 0.12 0.04
a-b
Means within rows with no common superscripts are significantly different (P ≤ 0.05); Values are ex-
pressed as means (n = 5 for each dietary treatment); pooled standard error of means (SEM); MOS = mannan-
oligosaccharide; FOS = fructo-oligosacharride.

(YDC), a SNB, on serum IgG concentrations, tious bronchitis virus (IBV); and in experiment three,
maternal-derived antibody (MDA) decay, and spe- birds were immunized intramuscularly with sheep red
cific antibody-mediated immune response in chick pul- blood cells and bovine serum albumin. The results sug-
lets post immunization with T-cell dependent antigens. gested that diets containing SNB increased serum IgG,
A total of 300 chicks were fed a basal diet (control) but the decay rates of MDA against NDV and infectious
and diets containing YDC and SNB (Lactobacillus aci- bursal disease virus were not affected. High specific an-
dophilus, L.casei, Streptococcus faecium, and Bacillus tibody response against IBV was observed for birds fed
subtilis and YDC). In the first experiment, serum was YDC in week 4, but it decreased in week 6. There was
analyzed by ELISA for total IgY and MDA against no effect of specific antibody response against sheep
NDV and infectious bursal disease virus. The second ex- red blood cells and bovine serum albumin. In conclu-
periment analyzed the specific antibody against infec- sion, SNB enhanced humoral immunity by increasing
 DIETARY PROBIOTICS AND PREBIOTICS 4475
Table 17. Wattle swelling change as affected by the different dietary treatments (cycle 2).

Treatments
Control B. coagulans Lactobacillus FOS MOS SEM P-value

Thickness (mm) 2.10b 2.35b 2.20b 3.17a 2.12b 0.459 0.019

B = Bacillus coagulans (1 g/kg dried culture); L = Lactobacillus (1 g/kg dried culture of 12 commercial
strains); FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from
Saccharomyces cerevisiae (5 g/kg); All treatment groups received a soybean basal diet; Differences between
the treatment groups are statistically different at P ≤ 0.05, n = 5, 17 birds in each replicate.

Table 18. Effect of different dietary treatments on chicken an- Table 20. Effect of different dietary treatments on chicken an-
tibodies in 3-week-old broiler chickens (cycle 1). tibodies in 3-week-old broiler chickens (cycle 2).

Antibody concentration Antibody concentration

Treatments IgY IgA IgM Treatments IgY IgA IgM

Control 0.56 0.03 0.002 Control 0.10 0.02 0.002

B. coagulans 0.27 0.03 0.003 B. coagulans 0.23 0.03 0.002
Lactobacillus 0.47 0.03 0.002 Lactobacillus 0.31 0.03 0.002
FOS 0.38 0.02 0.002 FOS 0.19 0.03 0.003
MOS 0.24 0.03 0.003 MOS 0.17 0.02 0.002
SEM 0.095 0.008 0.0007 SEM 0.053 0.008 0.0003
P-value 0.246 0.918 0.521 P-value 0.113 0.344 0.500

Bacillus coagulans (1 g/kg dried culture), Lactobacillus (1 g/kg Bacillus coagulans (1 g/kg dried culture), Lactobacillus (1 g/kg
dried culture of 12 commercial strains); FOS = Fructo-oligosaccharides dried culture of 12 commercial strains); FOS = Fructo-oligosaccharides
(5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from Sac- (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from Sac-
charomyces cerevisiae (5 g/kg); IgY = Immunoglobulin Y; IgA = Im- charomyces cerevisiae (5 g/kg); IgY = Immunoglobulin Y; IgA = Im-
munoglobulin A; IgM = Immunoglobulin M; Differences between the munoglobulin A; IgM = Immunoglobulin M; Differences between the
treatment groups are statistically different at P ≤ 0.05, n = 5. treatment groups are statistically different at P ≤ 0.05, n = 5.

Table 19. Effect of different dietary treatments on chicken an- Table 21. Effect of different dietary treatments on chicken an-
tibodies in 5-week-old broiler chickens (cycle 1). tibodies in 5-week-old broiler chickens (cycle 2).

Antibody concentration Antibody concentration

Treatments IgY IgA IgM Treatments IgY IgA IgM

Control 0.54 0.05 0.000

Control 0.38b 0.03a,b 0.004
B. coagulans 0.33 0.03 0.003
B. coagulans 0.75b 0.05a 0.003
Lactobacillus 0.44 0.03 0.000
Lactobacillus 0.66b 0.03b 0.002 FOS 0.25 0.05 0.004
FOS 0.78a 0.04a,b 0.000 MOS 0.36 0.03 0.003
MOS 0.71b 0.04a,b 0.004 SEM 0.113 0.009 0.001
SEM 0.078 0.003 0.001 P-value 0.490 0.239 0.248
P-value 0.033 0.025 0.385
Bacillus coagulans (1 g/kg dried culture), Lactobacillus (1 g/kg
Bacillus coagulans (1 g/kg dried culture), Lactobacillus (1 g/kg dried culture of 12 commercial strains); FOS = Fructo-oligosaccharides
dried culture of 12 commercial strains); FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from Sac-
(5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from Sac- charomyces cerevisiae (5 g/kg); IgY = Immunoglobulin Y; IgA = Im-
charomyces cerevisiae (5 g/kg); IgY = Immunoglobulin Y; IgA = Im- munoglobulin A; IgM = Immunoglobulin M; Differences between the
munoglobulin A; IgM = Immunoglobulin M; Differences between the treatment groups are statistically different at P ≤ 0.05, n = 5.
treatment groups are statistically different at P ≤ 0.05, n = 5.

results show that the addition of lactobacillus reduced

IgG concentration in serum and modulated adap- the E. coli count, whereas the E. coli growth was inhib-
tive antibody-mediated immune response against IBV ited with the addition of FOS. All dietary treatments
(Alizadeh et al., 2016). did not affect the count of both LAB and E. coli in
Broiler Cycle 2 The effect of different dietary treat- 5-week-old broilers. All selected samples did not have
ments on chicken antibodies in 3- and 5-wk-old broiler Salmonella in their bacterial count. The 5 treatments,
chickens (cycle 2) is shown in Tables 20 and 21, respec- including control, reduced the pH value slightly to the
tively. As shown in the tables, there was no significant acidic.
effect of the different dietary treatments on total anti- There was no Salmonella sp. recorded using the 4
body titers in the broilers. dietary treatments in the first cycle, and the growth of
E. coli was reduced significantly. However, LAB number
Microbial Counts and Hindgut Acidosis was not affected with the same treatments.
Results of the gut’s acidity show that the pH value
Broiler Cycle 1 Microbial count in cycle 1 with was driven toward acidity in all of the treatments, but
five different treatments, including control, is shown in it failed to reach significance, as it is only a numerical
Table 22. (The data is presented in log10 scale). The difference. The acidic pH results from the production of
4476 AL-KHALAIFA ET AL.
Table 22. Effect of different dietary treatments on microbial count in 5-week-old broiler chickens
(cycle 1).

Treatments
Analysis Control B. coagulans Lactobacillus FOS MOS SEM P-value

Log LAB 7.233 7.78 7.523 7.160 7.460 0.264 0.507

Log E.coli 3.592 3.433 1.767 0.000 3.801 1.608 0.443
Log Salmonella 0.000 0.000 0.000 0.000 0.000 0.022 0.564
pH 7.18 6.92 6.84 6.98 6.89 0.281 0.918

B = Bacillus coagulans (1 g/kg dried culture); L = Lactobacillus (1 g/kg dried culture of 12 commercial
strains); FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from
Saccharomyces cerevisiae (5 g/kg); Differences between the treatment groups are statistically different at P ≤
0.05, n = 5; values presented in log10 scale.

Table 23. Effect of different dietary treatments on microbial count in 3-week-old broiler chickens
(cycle 2).

Treatments
Analysis Control B. coagulans Lactobacillus FOS MOS SEM P-value

Log LAB 7.174 6.683 6.555 6.991 6.708 0.487 0.886

Log E.coli 2.500 2.500 0.000 3.443 5.400 2.208 0.584
Log Salmonella 2.050a 0.000b 0.000b 0.000b 0.000b 0.022 < .001
pH 7.230 6.955 5.975 6.295 6.085 0.299 0.104

B = Bacillus coagulans (1 g/Kg dried culture); L = Lactobacillus (1 g/Kg dried culture of 12 commercial
strains); FOS = Fructo-oligosaccharides (5 g/Kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from
Saccharomyces cerevisiae (5 g/kg); Differences between the treatment groups are statistically different at P ≤
0.05, n = 5; values presented in log10 scale.

undissociated volatile fatty acids in the caeca (acetic, physiology of the bacterial cell (Kuruti et al., 2017).
butyric, propionic, and lactic acids) and antimicrobial Based on the aforementioned data, it can be concluded
substances that effectively exclude or kill many differ- that probiotic supplementation affects the balance of
ent pathogens (Sarra et al., 1992). cecal volatile fatty acids, which in turn exert antimi-
In newly hatched chicks in commercial hatcheries, the crobial effect in poultry.
volatile fatty acid concentration and pH are not suffi- Broiler Cycle 2 Microbial count in cycle 2 for the
cient to chemically suppress pathogens (Barnes et al., 5 different treatments, including control, after 3 wk
1979, 1980), and therefore, supplementation of probi- of the experiment reflects the broilers’ diet as shown
otic microorganisms will be beneficial. It is critical to in Table 23. The additions of lactobacilli inhibited the
apply probiotic products as early as possible to achieve growth of both E. coli and Salmonella. The number of
the best results in the poultry industry (Casas et al., Salmonella varied significantly (P < 0.001) and gave
1993, 1998). As soon as a chicken hatches into an en- zero count, related to control, whereas the pH values
vironment that is heavily contaminated by bacteria, were clearly reduced from alkaline to acidic in all treat-
viruses, and protozoans, it must begin to develop pro- ments.
tective gut microflora. Under normal conditions, a 3 to Microbial count in cycle 2 after 5 wk reflects the diet
5 wk period is required for the development of a stable as shown in Table 24. The addition of lactobacillus in-
population of gut-associated bacteria, and it is in the hibited the growth of Salmonella, whereas E. coli count
ceca where the greatest numbers reside. In the ceca, an was not affected. All dietary treatments administered
anaerobic environment develops and favors the growth in 3-week-old chickens fed diets containing probiotics
of organisms such as Bifidobacterium spp. and Bacte- did not affect the count of both LAB and E. coli, and
riodes spp. Along with these bacteria, other LAB cre- their counts were significantly similar. The number of
ate a micro-ecology, which can be characterized by an Salmonella varied significantly (P < 0.001) and gave
acid pH resulting from the production of undissociated zero count, related to control. The pH values slightly
volatile fatty acids in the caeca (acetic, butyric, pro- reduced from alkaline to acidic in all treatments.
pionic, and lactic acids) and antimicrobial substances In conclusion, all dietary treatments in 3-week-old
that effectively exclude or kill many different pathogens broilers did not affect the count of both LAB and E.
(Sarra et al., 1992). The effect of undissociated short coli, whereas Salmonella growth was significantly in-
chain volatile fatty acids is well documented in many hibited. At 5-week-old of the same cycle, the bacterial
studies. They are present in high concentrations with count of E. coli increased even with control, whereas
acidic pH and exert antimicrobial effect. These acids Salmonella growth was inhibited. All treatments inhib-
are lipophilic penetrating the bacterial cell wall and ited the growth of Salmonella and reduced or inhibited
produce H+ ions, which in turn destroy the internal E. coli, depending on time or diet treatment. Results
 DIETARY PROBIOTICS AND PREBIOTICS 4477
Table 24. Effect of different dietary treatments on microbial count in 5-week-old broiler chickens
(cycle 2).

Treatments
Analysis Control B.coagulans Lactobacillus FOS MOS SEM P-value

Log LAB 5.983 6.994 7.206 7.028 6.690 0.507 0.525

Log E.coli 7.148 7.361 6.981 7.303 7.489 0.426 0.921
Log Salmonella 3.145a 0.000b 0.000b 0.000b 0.000b 0.002 < .001
pH 6.870 6.850 6.940 6.865 6.945 0.080 0.862

C = Control group; B = Bacillus coagulans (1 g/kg dried culture); L = Lactobacillus (1 g/kg dried culture of
12 commercial strains); FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide
derived from Saccharomyces cerevisiae (5 g/kg); Differences between the treatment groups are statistically
different at P ≤ 0.05, n = 5; values presented in log10 scale.

Table 25. Short fatty acid composition of 5-week-old broilers fed different probiotics and prebiotics
(broiler cycle 1).

Treatments
Fatty Acids Control B. coagulans Lactobacillus FOS MOS SEM P-value

Acetic acid 46.237 37 .77 53.92 45.62 51.01 5.938 0.457

Butyric acid 2.064 1.709 1.960 1.63 1.32 1.758 0.998
Total 48.30 39.47 55.88 47.26 52.33 6.67 0.548

B = Bacillus coagulans (1 g/Kg dried culture), L = Lactobacillus (1 g/Kg dried culture of 12 commercial
strains), FOS = Fructo-oligosaccharides (5 g/Kg), MOS (Bio-MOS) = mannan-oligosaccharide derived from
Saccaromyces cerevisiae (5 g/Kg). All treatment groups received a soybean basal diet. Differences between the
treatment groups are statistically different at P ≤ 0.05, n = 5.

Table 26. Short Fatty acid composition of 3-week-old broilers fed different probiotics and prebi-
otics (broiler cycle 2).

Treatments
Fatty Acids Control B. coagulans Lactobacillus FOS MOS SEM P-value

Acetic acid 58.95 116.24 70.32 40.79 47.54 22.57 0.275

Propionic acid 1.08 0.00 0.00 0.00 0.00 0.48 0.486
Butyric acid 0.00 4.50 2.74 1.26 0.00 1.06 0.110
Isovaleric acid 1.66 1.70 1.64 0.50 1.97 0.97 0.839
Valeric acid 0.46 0.72 1.80 0.44 0.65 0.53 0.448

B = Bacillus coagulans (1 g/Kg dried culture), L = Lactobacillus (1 g/Kg dried culture of 12 commercial
strains), FOS = Fructo-oligosaccharides (5 g/Kg), MOS (Bio-MOS) = mannan-oligosaccharide derived from
Saccharomyces cerevisiae (5 g/Kg). All treatment groups received a soybean basal diet. Differences between
the treatment groups are statistically different at P ≤ 0.05, n = 5.

of the gut’s acidity show that the pH value was driven 3-week-old broilers in cycle 2 are shown in Table 26. Re-
toward acidity in all of the treatments, but it failed to sults in Table 26 show no significant effect of these pro-
reach significance, as it is only a numerical difference. biotics and prebiotics on volatile fatty acid composition.
However, in a contrasting study, broilers were fed pro-
biotics, prebiotics, and synbiotics. These dietary sup-
Volatile Fatty Acid Profile in the Hindgut plementations resulted in improved weight gain, feed
Broiler Cycle 1 The results of the effect of different conversion ratio, and also significantly increased caecal
probiotics and probiotics on the short fatty acid com- VFA at 21 and 42 d of age (Mookiah et al., 2014).
position of broilers are shown in Table 25. There was
no significance observed for the effect of these probi- Immune Tissue Weight
otics and prebiotics on the volatile fatty acid composi-
tion in the broilers. In another similar study, 294 one-d- Broiler Cycle 1 The results of the effect of feeding
old Cobb broiler chickens were fed 6 dietary treatments broilers different probiotic and prebiotic diets on the
consisting of basal diets and 4 strains of the probiotic tissue weight of 5-week-old broiler chickens in cycle 1
Lactobacillus. The results showed that no significance (Table 27) showed a significant effect of the different
was observed for growth performance as well as no ef- dietary treatments on thymus (P = 0.030). The thymus
fect was seen on pH and concentrations of short chain weights in the groups fed B. coagulans and FOS were
fatty acids (Olnood et al., 2015). similar to each other and to the other treatment groups.
Broiler Cycle 2 The results of the effect of different The broiler groups fed Lactobacillus and MOS gained
prebiotics and probiotics on the volatile fatty acids of similar thymus weights, which are significantly lower
4478 AL-KHALAIFA ET AL.
Table 27. Effect of different dietary treatments on tissue weight in 5-week-old broiler chickens
(broiler cycle 1).

Dietary treatment Abdominal fat pad Heart Spleen Liver Bursa Thymus Breast Leg+Thigh
g/kg % of Body Weight

Control 0.86 0.59 0.12 2.92 0.20 0.30a 11.82 8.09

B. coagulans 1.49 0.63 0.13 3.35 0.17 0.40a,b 11.22 7.56
FOS 1.53 0.65 0.11 3.33 0.19 0.40a,b 8.60 7.31
Lactobacillus 1.55 0.53 0.10 3.03 0.22 0.50b 9.97 7.57
MOS 0.74 0.58 0.09 3.17 0.25 0.50b 10.63 7.07
SE Mean 0.35 0.05 0.01 0.17 0.05 0.03 0.79 0.52
P-value 0.389 0.543 0.453 0.413 0.826 0.030 0.179 0.725

B = Bacillus coagulans (1 g/kg dried culture); L = Lactobacillus (1 g/kg dried culture of 12 commercial
strains); FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from
Saccharomyces cerevisiae (5 g/kg); All treatment groups received a soybean basal diet; Differences between
the treatment groups are statistically different at P ≤ 0.05, n = 5, 17 birds in each replicate.

Table 28. Effect of different dietary treatments on tissue weight in 5-week-old broilers (broiler
cycle 2).

Diet Abdominal fat pad Heart Spleen Liver Bursa Thymus Breast Leg+ Thigh
(g/kg) % of Body Weight

Control 1.10 0.52 0.10 1.97 0.21 0.52b 6.59 1.10

B. coagulans 1.22 0.53 0.12 1.92 0.16 0.55b 6.16 1.22
Lactobacillus 1.15 0.54 0.10 2.10 0.20 0.66a 7.59 1.15
FOS 1.01 0.50 0.09 2.25 0.17 0.50b 7.35 1.01
MOS 0.93 0.55 0.13 2.32 0.20 0.49b 7.29 0.93
SE Mean 0.27 0.04 0.01 0.17 0.02 0.05 0.48 0.27
P-Value 0.944 0.959 0.239 0.481 0.503 0.027 0.272 0.944

Bacillus coagulans (1 g/kg dried culture); Lactobacillus (1 g/kg dried culture of 12 commercial strains);
FOS = Fructo-oligosaccharides (5 g/kg); MOS (Bio-MOS) = mannan-oligosaccharide derived from Saccha-
romyces cerevisiae (5 g/kg); All treatment groups received a soybean basal diet; Differences between the
treatment groups are statistically different at P ≤ 0.05, n = 5, 17 birds in each replicate.

CONCLUSION
than those of the control group (Table 27). This increase
in the thymus weight of the control group may indicate The use of the prebiotic FOS produced significant re-
an increase in the T-immune cells in the thymus. These sults for all of the production in cycle 2 and in cycle 1
T- cells are responsible for the specific cellular response. for the parameter of feed consumption whereas the pro-
On the other hand, the results of the effect of feeding biotic B. coagulans was better than FOS for the other
broilers different probiotic and prebiotic diets on the parameters. The use of FOS induced a higher immune
tissue weight of 5-week-old broiler chickens (Table 27) response in cycle 1 and MOS in cycle 2. The use of
showed that the thymus weight of the group fed Lac- probiotics and prebiotics in chicken feed can improve
tobacillus was significantly higher than that of all the the immune status of the flock by reducing harmful mi-
other groups. This may indicate an increase of the T- crobes in their intestine and thus reducing the use of
immune cells in the thymus, which are responsible for antibiotics.
the specific cellular response.
Broiler Cycle 2 The effect of different dietary treat-
ACKNOWLEDGMENTS
ments on the tissue weight of 5-week-old broilers The authors would like to extend their sincere thanks to
(broiler cycle 2) is shown in Table 28. The results in the Kuwait Institute for Scientific Research (KISR) and
Table 28 show a significant effect on thymus (P = Kuwait Foundation for the Advancement of Sciences
0.027). The thymus weight in the group fed Lactobacil- (KFAS) for their technical and financial support of the
lus was significantly higher than that in the group fed current study. .
FOS. This may indicate an increase in the T-immune
cells in the thymus, which are responsible for the spe- REFERENCES
cific cellular response. The thymus weight in both the Al-Khalaifah, H. S. 2018. Benefits of probiotics and/or prebiotics for
Lactobacillus and the FOS groups was statistically sim- antibiotic-reduced poultry. Poult. Sci. 97:588–593.
Alavi, S. A. N., A. Zakeri, B. Kamrani, and Y. Pourakbari. 2012. Ef-
ilar to that of the other experimental groups. There fect of prebiotics, probiotics, acidfire, growth promoter antibiotics
was no significant effect of the experimental diets ob- and synbiotic on humural immunity of broiler chickens. Global
served on tissue weight for abdominal fat, heart, spleen, Eterinaria. 8:612–617.
liver, bursa, breast, and leg + thigh (Table 28). This in- Alizadeh, M., P. Munyaka, A. Yitbarek, H. Echeverry, and
J. Rodriguez-Lecompte. 2016. Maternal antibody decay and
crease in the thymus weight may indicate an increase antibody-mediated immune responses in chicken pullets fed pre-
in the T-immune cells in the thymus. These T-cells are biotics and synbiotics. Poult. Sci. 96:58–64.
responsible for the specific cellular response. Barnes, E. M., C. Impey, and B. Stevens. 1979. Factors affecting
the incidence and anti-salmonella activity of the anaerobic caecal
 DIETARY PROBIOTICS AND PREBIOTICS 4479
efficacy of commercial prebiotics on broiler performance and ox-
flora of the young chick. J. Hyg. 82:263–283. idative stability of meat. Poult. Sci. 96:511–518.
Barnes, E. M., C. S. Impey, and D. M. Cooper. 1980. Manipulation Midilli, M., M. Alp, N. Kocabagli, O. H. Muglal, N. Turan, H. Yli-
of the crop and intestinal flora of the newly hatched chick. Am. maz, and S. Cakir. 2008. Effects of dietary probiotic and prebiotic
J. Clin. Nutr. 33:2426–2433. supplementation on growth performance and serum IgG concen-
Casas, I. A., F. W. Edens, W. J. Dobrogosz, and C. R. Parkhurst tration of broilers. S. Afr. J. Anim Sci. 38:21–27.
1993. Performance of GAIAfeed and GAIAspray: A Lactobacillus Mookiah, S., C. C. Sieo, K. Ramasamy, N. Abdullah, and Y. W. Ho
reuteri-based probiotic for poultry. Prevention and Control of 2014. Effects of dietary prebiotics, probiotic and synbiotics on
Potentially Pathogenic Microorganisms in Poultry and Poultry performance, caecal bacterial populations and caecal fermenta-
Meat Products Proceedings. tion concentrations of broiler chickens. J. Sci. Food Agric. 94:341–
Casas, I. A., F. W. Edens, and W. J. Dobrogosz 1998. Lactobacillus 348.
reuteri: an effective probiotic for poultry and other animals. J. Murarolli, V., M. Burbarelli, G. Polycarpo, P. Ribeiro, M. Moro,
Food. Sci. Technol. 475–518. and R. Albuquerque. 2014. Prebiotic, probiotic and symbiotic as
Castanon, J. 2007. History of the use of antibiotic as growth pro- alternative to Antibiotics on the Performance and Immune Re-
moters in European poultry feeds. Poult. Sci. 86:2466–2471. sponse of Broiler Chickens. Revista Brasileira de Ciência Avı́cola.
Cavazzoni, V., A. Adami, and C. Castrovilli. 1998. Performance of 16:279–284.
broiler chickens supplemented with Bacillus coagulans as probi- Olnood, C. G., S. S. Beski, M. Choct, and P. A. Iji. 2015. Novel pro-
otic. Br. Poult. Sci. 39:526–529. biotics: Their effects on growth performance, gut development,
Corrier, D., and J. DeLoach. 1990. Evaluation of cell-mediated, cu- microbial community and activity of broiler chickens. Anim. Nu-
taneous basophil hypersensitivity in young chickens by an inter- trit. 1:184–191.
digital skin test. Poult. Sci. 69:403–408. Peryam, D. R., and F. J. Pilgrim. 1957. Hedonic scale method of
Economic and Social Committee of the European Union. 1998. measuring food preferences. Food Technol. 11(Suppl.):9–14.
Opinion on resistance to antibiotics as a threat to public Salminen, S., A. C. Ouwehand, and E. Isolauri. 1998. Clinical appli-
health. No. ESC-98-016-EN. http://eescopinions.eesc.europa.eu/ cations of probiotic bacteria. Int. Dairy J. 8:563–572.
EESCopinionDocument.aspx?identifier=ces\anciennes sections\ Sarangi, N. R., L. K. Babu, A. Kumar, C. R. Pradhan, P. K. Pati,
envi\envi471\ces1118-1998 ac.doc&language=EN.Research.Gate, and J. P. Mishra. 2016. Effect of dietary supplementation of
Accessed on June 12, 2007. prebiotic, probiotic, and synbiotic on growth performance and
FAO/WHO. 2001. Health and nutritional probiotics in food includ- carcass characteristics of broiler chickens. Vet. World. 9:313–
ing powder milk with live lactic acid bacteria. Joint FAO/WHO 319.
expert consultation. Cordoba, Argentina, 1 - 4 October 2001. Sarra, P., L. Morelli, and V. Bottazzi. 1992. The lactic microflora
FDA. 2009. Summary Report on Antimicrobials Sold or Distributed of fowl The Lactic Acid Bacteria Volume 1. (pp. 3–19), Springer,
for Use in Food Producing Animals. Food and Drug Administra- Boston, MA, US.
tion, Available at http://www.fda.gov/downloads/ForIndustry/ Schoeni, J. L., and M. P. Doyle. 1992. Reduction of Campylobacter
UserFees/AnimalDrugUserFeeActADUFA/UCM231851.pdf, Ac- jejuni colonization of chicks by cecum-colonizing bacteria pro-
cessed on August 31, 2011. ducing anti-C. jejuni metabolites. Appl. Environm. Microbiol.
Fuller, R. 1989. Probiotics in man and animals. J. Appl. Bacteriol. 58:664–670.
66:365–378. Sohail, M., M. Hume, J. Byrd, D. Nisbet, A. Ijaz, A. Sohail, M.
Ghasemi, H. A., N. Kasani, and K. Taherpour. 2014. Effects of black Shabbir, and H. Rehman. 2012. Effect of supplementation of pre-
cumin seed (Nigella sativa L.), a probiotic, a prebiotic and a syn- biotic mannan-oligosaccharides and probiotic mixture on growth
biotic on growth performance, immune response and blood char- performance of broilers subjected to chronic heat stress. Poult.
acteristics of male broilers. Livest. Sci. 164:128–134. Sci. 91:2235–2240.
Goto, N., H. Kodama, K. Okada, and Y. Fujimoto. 1978. Suppression Sweeney, M. T., B. V. Lubbers, S. Schwarz, and J. L. Watts. 2018.
of phytohemagglutinin skin response in thymectomized chickens. Applying definitions for multidrug resistance, extensive drug
Poult. Sci. 57:246–250. resistance and pandrug resistance to clinically significant live-
Ipek, A., A. Sozcu, and V. Akay. 2016. Effects of dietary inclusion of stock and companion animal bacterial pathogens. J. Antimicrob.
probiotics and prebiotics (SynerAll) on growth performance and Chemother. 73:1460–1463.
serum biochemical parameters in broilers. J. Anim. Sci. 94:484– Tania, C., L. Ines, F. Ricardo, N. Jasmin, and A. Adelaide. 2018.
485. Frequency and antibiotic resistance of bacteria implicated in com-
Janardhana, V., M. M. Broadway, M. P. Bruce, J. W. Lowenthal, munity Urinary Tract Infections in North Aveiro between 2011
M. S. Geier, R. J. Hughes, and A. G. D. Bean. 2009. Prebiotics and 2014. Microb. Drug Resist. 24:493–504.
modulate immune responses in the gut-associated lymphoid tissue WHO. 1997. The medical impact of the use of antimicrobials
of chickens. J. Nutr. 139:1404–1409. in food animals. World Health Organization, Report of a
Janvier, A., J. Lantos, and K. Barrington. 2013. The politics of probi- WHO meeting, Berlin, Germany, Available at http://whqlibdoc.
otics: probiotics, necrotizing enterocolitis and the ethics of neona- who.int/hq/1997/WHO EMC ZOO 97.4.pdf, Accessed on June
tal research. Acta Paediatr. 102:116–118. 23, 2007.
Jin, L. Z., Y. W. Ho, N. Abdullah, and S. Jalaludin 2000. Digestive Yang, Y., Y. Yu, J. Pan, Y. Ying, and H. Zhou. 2016. A new method
and bacterial enzyme activities in broilers fed diets supplemented to manipulate broiler chicken growth and metabolism: Response
with Lactobacillus cultures. Poult. Sci. 79:886–891. to mixed LED light system. Scientific Reports. 6:25972.
Kannan, M., R. Karunakaran, V. Balakrishnan, and T. G. Prab- Yousaf, M., A. Ijaz, K. Ashraf, M. Rasheed, A. Hafeez, H. Zaneb,
hakar. 2005. Influence of prebiotics supplementation on lipid pro- E. Dar, R. Naseer, I. Rabbani, and J. Zentek. 2016. Compar-
file of broilers. Int. J. Poult. Sci. 4:994–997. ative effects of different dietary concentrations of β -galacto-
Khobondo, J., P. Ogore, J. Atela, P. Onjoro, J. Ondiek, and A. oligosaccharides on growth performance, feed conversion effi-
Kahi 2015. The effects of dietary probiotics on natural IgM anti- ciency and organs development in broilers. J. Anim. Plant Sci.
body titres of Kenyan indigenous chicken. Livest. Res. Rural Dev. 26:1603–1608.
27:230. Zhou, X., Y. Wang, Q. Gu, and W. Li. 2010. Effect of dietary probi-
Kuruti, K., S. Nakkasunchi, S. Begum, S. Juntupally, V. Arelli, otic, Bacillus coagulans, on growth performance, chemical com-
and G. R. Anupoju. 2017. Rapid generation of volatile fatty position, and meat quality of Guangxi Yellow chicken. Poult. Sci.
acids (VFA) through anaerobic acidification of livestock organic 89:588–593.
waste at low hydraulic residence time (HRT). Bioresour. Technol.
238:188–193.
Lorch, H., G. Benckieser, and J. C. G. Ottow 1995. Basic methods
for counting microorganisms in soil and water. Methods Appl.
Soil Microbiol. Biochem. 146–161.
Maiorano, G., K. Stadnicka, S. Tavaniello, C. Abiuso, J. Bogucka,
and M. Bednarczyk. 2017. In ovo validation model to assess the