animals

Article
The Efficiency of Probiotics Administrated via Different Routes
and Doses in Enhancing Production Performance, Meat Quality,
Gut Morphology, and Microbial Profile of Broiler Chickens
Elham A. Soumeh 1, * , Astrid Del Rocio Coba Cedeno 1,2 , Shahram Niknafs 2 , Jacoba Bromfield 1,3
and Louwrens C. Hoffman 2,4

1 School of Agriculture and Food Sciences, Gatton Campus, University of Queensland,

Gatton, QLD 4343, Australia; [email protected] (A.D.R.C.C.); [email protected] (J.B.)
2 Food Innovation, University of Queensland, Brisbane, QLD 4343, Australia; [email protected] (S.N.);
[email protected] (L.C.H.)
3 Bioproton Pty Ltd., Acacia Ridge, Brisbane, QLD 4110, Australia
4 Department of Animal Sciences, Stellenbosch University, Stellenbosch 7906, South Africa
* Correspondence: [email protected]; Tel.: +61-7-5460-1308

Simple Summary: Antimicrobial growth promoters (AGPs) have been used in the animal production
industry around the world for decades, with the consequence of a high potential of antibiotic-resistant
bacteria transfer to humans. Efficiently raising broiler chickens in an antibiotic-free production system





 is a challenge, and identifying an effective nutritional alternative to support growth performance, gut
Citation: Soumeh, E.A.; Cedeno, health, and functionality without administrating AGPs is of essence. Several antimicrobial alternative
A.D.R.C.; Niknafs, S.; Bromfield, J.; options that are commercially available include herbal essential oils, exogenous enzymes, organic
Hoffman, L.C. The Efficiency of acids, plant secondary metabolites, probiotics, and prebiotics. Probiotics in animal feed is projected
Probiotics Administrated via to attain a massive global growth, reaching USD 6.24 billion by 2026. This study tested the efficiency
Different Routes and Doses in of probiotics when supplemented via different administration routes (feed or water) and doses, or in
Enhancing Production Performance, combination with prebiotics, on growth performance, meat quality, gut morphology, and microbial
Meat Quality, Gut Morphology, and profile of broiler chickens. The outcomes revealed that probiotics enhance production performance,
Microbial Profile of Broiler Chickens.
and compared to AGPs, do not reduce the beta-diversity of the gut microbial community. Water-
Animals 2021, 11, 3607. https://
soluble probiotics seemed to be more effective in improving growth performance.
doi.org/10.3390/ani11123607

Abstract: To study the efficiency of Bacillus spp. probiotics administered via different routes and
Academic Editor: Velmurugu
doses, a 6-week grow-out trial was conducted using a total of 378 day-old mixed-sex ROSS308 broiler
Ravindran
chickens in a completely randomized block design. Six experimental diets included probiotics added
Received: 2 November 2021 at two different inclusion rates into the feed (250 g/ton; PRO250, or 500 g/ton; PRO500), or in the
Accepted: 18 December 2021 drinking water (25 g/L; PRO-WS), or as a feed synbiotic (250 g probiotic + 250 g/ton prebiotic; SYN),
Published: 20 December 2021 compared to a negative (NC; without additives) and positive control (PC; with antibiotics) diets. The
PRO-WS enhanced feed intake (p < 0.05) and tended to improve average daily gain and final body
Publisher’s Note: MDPI stays neutral weight (p = 0.14). Broiler gut morphology in the duodenum including the villus height (p = 0.04),
with regard to jurisdictional claims in villus width (p = 0.05) and crypt depth (p = 0.02) were improved by PRO500. Firmicutes was the most
published maps and institutional affil- abundant phylum, followed by Bacteroidetes. Streptococcaceae, Lachnoospiraceae, Peptostreptococcaceae,
iations. Ruminococcaceae, and Erysipe-lotrichaceae were the top five most abundant families. Antibiotic inclusion
in PC reduced microbial beta-diversity and increased similarity compared to probiotic inclusion
(p = 0.05). Probiotic inclusion reduced the relative abundance of Bacteroides fragilis, which is a
commonly isolated pathogen and is considered as a marker for antimicrobial resistance. Overall,
Copyright: © 2021 by the authors. probiotic supplementation via feed or water may potentially improve the production performance of
Licensee MDPI, Basel, Switzerland. the broiler chickens, and water-soluble probiotics are potentially more effective. Probiotics, especially
This article is an open access article
when added to water, suggest a promising feed additive to support gut microbial maturation and
distributed under the terms and
diversity, and may reduce resistant bacteria in broiler chickens. However, it is suggested that the best
conditions of the Creative Commons
route for the administration of probiotics be further examined under commercial conditions to find
Attribution (CC BY) license (https://
the most effective and practical application method that yields the most consistent results.
creativecommons.org/licenses/by/
4.0/).

Animals 2021, 11, 3607. https://doi.org/10.3390/ani11123607 https://www.mdpi.com/journal/animals

Animals 2021, 11, 3607 2 of 20

Keywords: antibiotics; broiler chicken; growth performance; gut morphology; nutrient digestibility;
probiotics in feed or water

1. Introduction
According to OECD-FAO Agricultural Outlook 2020–2029, global chicken meat con-
sumption is projected to increase to 145 million tons by 2029, thereby accounting for 50% of
the increased global meat consumption [1]. The intensification of meat chicken farming has
led to an increase in the use of antibiotics to improve growth performance and control the
spread of diseases. The prolonged use of antibiotics at subtherapeutic levels has resulted
in bacterial resistance and accumulation of residues in meat products having detrimental
effects on animal and human health [2]. Therefore, multiple countries have banned subther-
apeutic use of antibiotics as a growth promoter in animal production resulting in the search
for alternative practices to support healthy and efficient chicken production in an antibiotic-
free system. Probiotics are mono- or mixed-cultures of live beneficial bacteria which have
been used in poultry production to establish a healthy and diverse gut microbial ecosystem
which competitively excludes pathogens [3]. The use of commensal bacteria induces an
immune response in poultry through fortification of the mucosal barrier and stimulating
innate immune responses, thereby enhancing growth performance and nutrient digestibil-
ity [4–6]. Prebiotics are the non-digestible fermentable fraction of feed ingredients, mostly
natural oligosaccharides, and/or small sugar molecules, and/or soluble fibre fractions
which selectively stimulate the growth and activity of the beneficial gut bacteria [7,8]. Syn-
biotics, a combination of probiotics and prebiotics, provide the beneficial bacteria together
with nourishing material thereby improving the survival and establishment of the directly
fed microbes in the gastrointestinal tract [9,10]. Different routes of administrating probiotic
preparations to the broiler chickens are used, including via feed, water, gavage including
droplet or inoculation, spray, or litter [11]. Although feed supplementation is the most used
route of probiotic administration in poultry production, the survival rates of the bacteria
in pelleted diets are low, although spore-forming bacteria strains have higher stability to
heat, acidic pH, and the harsh environments typically encountered during the pelleting
procedures [12,13]. The water-soluble probiotics are the next generation of probiotics with
a new formula, ensuring a quick and homogeneous distribution and release of the probiotic
spores in water and have been reported to be more efficient in improving broiler chickens’
growth performance [4,14]. The overall number of bacteria in the gastrointestinal tract
(GIT) exceeds that of the host body’s eukaryotic cells. There are three types of bacteria in
the host: dominant bacteria (more than 106 CFU/g sample), subdominant bacteria (103 to
106 CFU/g sample), and residual bacteria (less than 103 CFU/g sample) [15]. From the
crop to the lower ileum, the poultry GIT is dominated by Gram-positive, mostly facultative
anaerobes, whilst the ceca are dominated by Lactobacillus, Enterococcus, coliforms, and
yeasts. Low pH induces bacterial population reduction in the proventriculus and gizzard,
where in the duodenum, enzymes, high oxygen pressure, and bile salts reduce the microbial
concentration. The conditions of the lower small intestine and large intestine are suitable
for the formation of diverse microbiota. Establishing a healthy diverse gut microbiota plays
a protective role as the first line of defence against pathogenic bacteria, enhances the gut
structure integrity, and assists in specific metabolic activities [16]. It is reported that the
bacterial communities in the hindgut are dynamic and change or diversify with diet and
age [17]. The current study aimed to compare the efficiency of probiotics administration
added at two different inclusion rates into the feed, or as a water-soluble probiotic in the
drinking water, or as a feed synbiotic in combination with prebiotics, compared to negative
(without additives) and positive control (with antibiotics) diets.
Animals 2021, 11, 3607 3 of 20

2. Materials and Methods

All experimental procedures, including animal handing and husbandries, were ap-
proved by the Animal Ethics Committee of Research Ethics Unit of The University of
Queensland, AEC Approval Number SAFS/579/18.

2.1. Birds, Diets and Experimental Design

A total of 378 1-day old mixed gender broiler chickens (ROSS 308) were purchased
from a commercial hatchery (Woodlands Hatchery, QLD) and transferred to the Queens-
land Animal Science Precinct (QASP) facility at the University of Queensland (Gatton
Campus, Qld 4343, Australia). Birds were vaccinated for Marek’s disease and infectious
bronchitis before transportation. On arrival at the facilities, all birds were weighed indi-
vidually and distributed randomly into 54 floor pens. Pens were randomly assigned to
one of six experimental groups in a completely randomized block design (CRBD), with
nine replicate pens per experimental group and seven birds in each (n = 63 chickens per
experimental group).
A basal wheat–corn–soybean meal diet was purchased from a commercial feed miller,
Allora Grains & Milling (Down Holdings Pty Ltd, Ellinthrope QLD 4362, Australia) and
transported to the facility. The feed-supplemented probiotic and synbiotic products were
added to the basal diet in the facility prior to the start of the experiment. The experimen-
tal treatments (Table 1) included a standard mash wheat–corn–soybean diet (Negative
Control; NC); NC + 200 g/t Virginiamycin (Positive Control; PC); NC + 250 g/t Probiotic
(PRO250); NC + 500 g/t Probiotic (PRO500); NC + 0.25 g/L Probiotic in Water (PRO-WS);
and NC + 250 g/t probiotic + 250 g/t prebiotic (SYN). The probiotic added to all exper-
imental diets was Natupro®and the water-soluble probiotic was Natupro W®(supplied
by Bioproton Pty Ltd (Acacia Ridge, Queensland 4110, Australia). Natupro®contains a
collection of multiple Bacillus strains (Table 2) and the prebiotic, a commercial yeast cell
wall (YCW) product containing 20% β-glucan and 20% mannan-oligosaccharides (MOS),
the latter with the commercial name of X-MOS supplied by Algebra Bio Pty Ltd (Balmain,
NSW 2041, Australia).

Table 1. Experimental treatments’ specification fed to broiler chickens for 42 days.

Diet Code Diet Short Description Diet Specification

NC NC Negative Control: no Probiotic, no Antibiotic
PC PC (NC + 200 g/t Virginiamycin) Negative Control + Antibiotic as Growth Promoter
PRO250 NC + 250 g/t Probiotic Negative Control + Probiotics 250 g/t
PRO500 NC + 500 g/t Probiotic Negative Control + Probiotics 500 g/t
PRO-WS NC + 0.25 g/L Probiotic in Water Supplied (WS) Negative Control + Probiotics in water 0.25 g/L
SYN NC + 250 g pre + 250 g pro Negative Control + Probiotics 250 g/t + Prebiotics 250 g/t

Table 2. Different Bacillus strains and specifications in Natupro® probiotic.

Probiotic Strains Specification

Bacillus subtilis 1.5 × 108 CFU/g
Bacillus licheniformis 1.5 × 108 CFU/g
Bacillus amyloliquefaciens 3 × 108 CFU/g

The PRO-WS was made twice per week to be served to the birds as fresh as possible.
Two tanks of 40 L were used to make the solution and the pens on the PRO-WS treatment,
had bell drinkers (5 L) filled from the tank containing the PRO-WS. Each replicate group
of broiler chickens was reared in floor pens (120 × 120 × 80 cm) with carboard beddings
for a total of 42 days and had ad libitum access to feed and water for the entire trial
period. The grow-out period was divided into three phases (starter: day 1–14; grower: day
14–28; finisher: day 28–42), and nutrient levels were adjusted accordingly as per ROSS
308 guidelines (Table 3). Calculated and analysed nutrient composition of experimental
Animals 2021, 11, 3607 4 of 20

diets (as-is) is presented in Table 3. Throughout the trial, the ROSS 308 management
guidelines were followed to meet the broiler nutrient recommendations and the appropriate
environmental conditions including temperature, relative humidity, and lighting [18]. The
lighting program provided 23 h of light at a 30–40 lux intensity and 1 h of dark (less than
0.4 lux) for the first 7 days and a minimum of 4 h darkness and a light period of 10 lux
intensity after 7 days. Temperature was set at 32 ◦ C and 40% relative humidity for the
first 7 days and a 2 ◦ C reduction per week after 7 days until the temperature reached
24 ◦ C at 27 days and 40% relative humidity. This temperature and relative humidity were
maintained until the end of the trial. Each pen (except for the PRO-WS replicates) had two
nipple drinkers adjusted weekly to the birds’ height and one cone feeder.

Table 3. Ingredient composition, calculated and analysed nutrients composition of the basal diet 1 .

Grow-Out Phases
Diet Composition
Starter (1–14 Days) Grower (15–28 Days) Finisher (29–42 Days)
Ingredient Composition (%)
Wheat 40.00 40.00 45.00
Corn 20.46 20.00 21.02
Soybean meal 29.82 29.37 23.80
Soybean oil 4.27 5.89 5.84
L-Lysine HCL 0.50 0.35 0.34
DL-Methionine 0.41 0.34 0.31
L-Threonine 0.26 0.17 0.16
Limestone 1.52 1.38 1.26
Mono-Calcium Phosphate 1.74 1.51 1.32
Sodium bicarbonate 0.32 0.24 0.25
Salt 0.15 0.20 0.20
Vitamin—trace Mineral Premix 0.50 0.50 0.50
Coccidiostat 0.05 0.05 0.00
Calculated Nutrients Composition
ME 2 (MJ/kg) 11.92 12.34 12.55
Crude protein (%) 21.00 20.50 18.50
SID 3 Lysine (%) 1.28 1.15 1.02
SID Met + Cys (%) 0.95 0.87 0.80
SID Threonine (%) 0.86 0.77 0.68
Calcium (%) 0.96 0.87 0.78
Avail. Phosphorous (%) 0.48 0.44 0.39
Sodium (%) 0.16 0.16 0.16
Potassium (%) 0.90 0.89 0.79
Chloride (%) 0.23 0.23 0.23
Analysed Nutrients Composition
Dry matter (%) 89.66 90.80 90.93
Crude protein (%) 20.52 21.84 19.30
Ash (%) 8.14 7.25 7.25
1 Calculated nutrients composition is on “as is” basis and analysed nutrient composition is on “DM” basis. 2 ME: metabolisable energy.
3 SID: standardised ileal digestible.

2.2. Chemical Analyses

The experimental diets were analysed for dry matter (DM), total nitrogen (N), and
ash contents following the Association of Official Agricultural Chemists (AOAC) (2005).
Dry matter content was determined by drying the sub-samples in an oven at 105 ◦ C for
24 h. Total N content of feed samples was analysed using a LECO CNS928 carbon/nitrogen
combustion analyser 1.0 (Leco, St. Joseph, MI, USA) following the instructions of the
manufacturer, and the crude protein (CP) was calculated (6.25 × N).
Animals 2021, 11, 3607 5 of 20

2.3. Growth Performance

Body weight (BW) of individual birds and feed intake (FI) of each replicate pen were
recorded weekly and average daily gain (ADG), average daily feed intake (ADFI) and feed
conversion ratio (FCR: feed intake/live weights of birds per replicate) were calculated.
Chicken mortality was recorded daily during morning and afternoon inspection
and used for chicken-day calculations. The feed intake and FCR were corrected for
mortality accordingly.

2.4. Slaughtering, Carcass Composition, and Sample Collection

On the last day of the trial (42 days), 1 bird per replicate was arbitrarily chosen,
weighed and euthanised by electrical stunning (240 V for 10 s) and exsanguination to
collect blood samples. The carcass was then soaked in a scalding tank for 2 min (water tem-
perature 60 ◦ C) followed by defeathering and evisceration. Dressed carcass weights were
recorded before dissection followed by recording the removed internal organs’ weights
(heart, liver, proventriculus, gizzard, spleen, bursa of Fabricius, pancreas, and abdomi-
nal fat pad). The relative weights of individual organs were calculated in ratio to final
body weight. Additionally, the small intestine of each bird was sampled to study gut
pH and histomorphology. After the evisceration process, the birds’ carcasses were indi-
vidually weighed before and after chilling at 4 ◦ C for 24 h before commencing the meat
quality analyses.

2.5. Meat Quality Analysis

The right breast meat portion from all slaughtered birds was removed from the
carcass by cutting around the furcula or wishbone alongside the keel bone, and then
weighed. Where applicable, sub-samples/steaks of the breast muscle were cut for various
measurements.

2.5.1. Breast Meat pH

The pH of the right breast meat was measured by positioning the portable meat pH
meter (Hanna, HI 98163, Keysborough, VIC, Australia) 1 cm deep into the centre of the
breast meat tissue and the readings recorded for all samples. The pH meter was calibrated
with pH 4.01 and 7.01 standards according to the manufacturer’s instructions and cleaned
using distilled water between measurements.

2.5.2. Colorimetry
The calorimetric characteristics, L* (lightness), a* (red index) and b* (yellow index) of
the right breast meat were measured [19] using a Konica Minolta Chromameter CR-400/410
(Thermo Fisher Scientific Pty Ltd., Waltham, MA, USA) set at d:0◦ (diffuse illumination/0◦
viewing angle; specular component included), with a standard observer angle of CIE: 2◦ .
The colorimeter light source was a pulsed xenon lamp. Three readings, each at a different
position, were taken, and the mean of the lab ordinates was automatically calculated and
used in further statistical analyses. To calculate the hue and chroma to determine the
precise meat colour, the following equations were used:
q
Chroma (C∗) = (a∗)2 + (b∗)2 (1)

b∗
   
Hue angle hab = tan −1 (2)
a∗

2.5.3. Water Holding Capacity

The water holding capacity (WHC) of the breast meat samples was measured using
the filter paper press method (FPPM) [20]. A small sample of 1 g was cut from each of the
breast meat samples and placed between two filter papers. The sample and filter paper
altogether were placed between two Perspex plates and then pressed using a high-pressure
Animals 2021, 11, 3607 6 of 20

(588 Newton (N) for 1 min. A digital photograph was taken of the filter paper to show the
amount of water expelled from the sample. Finally, ImageJ for Mac OS X, a java-based
image processing program, was used to determine the ratio of water (outer area) and
meat (inner area) of the sample to calculate the WHC. In addition, the difference between
the outer and inner areas was used to predict the drip loss of the samples following the
equation below [20]:

Water Holding Capacity = total water − loose water %

Loose Water = (b − a) × 0.0084
1 × 100%

(3)
b : area enclosed by the outer front cm2

a : area enclosed by the inner front cm2

2.5.4. Cooking Loss

Cooking loss was calculated by subtracting the individual breast meat sample post-
cooking weight from the pre-cooking weight of the breast meat sample after cooking it
in a zip-lock bag in an 80 ◦ C water-bath for 45 min. The cooked breast meat sub-samples
were removed and allowed to cool down to room temperature for 10 to 15 min in a cold-
water bath. The cooled cooked sub-samples were blotted dry with absorbent paper towels
and weighed.

% Cooking Loss = ((weight before − weight after)/(weight before)) × 100 (4)

2.5.5. Warner–Bratzler Shear Force

The tenderness of the cooked breast meat samples was analysed using the Warner–Bratzler
Shear Force (WBSF) test [21]. From the centre of the cooked breast sub-sample, two adjacent
1 × 1 cm, minimum of 2 cm long, meat strips were cut parallel to the muscle fibre’s
orientation using pre-set scalpel blades (1 cm apart). A universal texture analyser (Instron
5543 model, 15 Stud Road Baywater, Melbourne, Victoria, Australia) equipped with a
Warner–Bratzler (WB) blade was used to measure the force (N) needed to shear a strip of
cooked breast meat perpendicular to the muscle fibre’s orientation. The WB blade was
a 1 mm thick isosceles triangle with sides 45 mm long and a 60◦ cutting angle. A 2 kN
load cell was used, and the crosshead speed was set at 200 mm/minute. The maximum
shear force value of all cooked breast meat samples was measured in N, where a higher
value indicated a tougher breast meat. A minimum of three measurements per sample was
recorded, and the average was used as the WBSF reading.

2.5.6. Meat Chemical Composition

The breast meat sub-samples were stored at −20 ◦ C. Breast sub-samples were later
defrosted at 4 ◦ C for 6 h, the meat samples ground and a subsample taken for moisture
determination in an oven at 105 ◦ C for 48 h. The remaining meat samples were freeze-
dried for 3 days in a freeze dryer. The dry meat sub-samples were ground again in a
blender. The fat content of the meat was determined in a Soxhlet apparatus using ether as
solvent [22]. The ash content of the sub-samples was determined after combustion at 550 ◦ C
for eight hours. The N content of the sub-samples was determined using a LECO CN928
Carbon/Nitrogen combustion analyser and multiplied by 6.25 to calculate crude protein
content. The combustion temperature was 1100 ◦ C, and about 0.3 g of the sub-sample was
used for the analysis.

2.6. Gut Morphology

During the dissection, the small intestine samples were divided into the following
segments: duodenum, jejunum, and ileum. Each segment was flushed with distilled water,
and a small section (~1 cm length) was cut from the mid-region of each segment. Samples
were immersed in 10% neutral buffered formalin solution to preserve the tissue samples for
morphology analysis. The pH values from the contents of the duodenum, jejunum, ileum,
and caecum segments were recorded using the same procedure described and equipment
Animals 2021, 11, 3607 7 of 20

used for the meat pH readings. Fixed tissues were loaded into appropriate size cassettes for
further gut histo-morphological analysis. Each fixed intestinal tissue sample was dipped
in wax and a 5 mm section was cut and embedded in paraffin. Embedded intestinal
segments were cut at a thickness of 6µm (Leica semi-automated RM2245 rotary microtome,
Leica Microsystems, VIC, Australia) and mounted onto slides. The slides were stained by
Hematoxylin and Eosin (HE), dried in an oven overnight at 37 ◦ C, and cleared by xylene for
2 min to be scanned by light microscopy. The slides were scanned by an Aperio ScanScope
XT (Leica Microsystems, VIC, Melbourne, Australia) and the villus height, crypt depth,
villus width, and the number of goblet cells determined. The villus surface area and the
villus height to crypt depth ratio were calculated. Villus height was measured from the tip
of the villus to the crypt between two individual villi. Crypt depth was measured from the
valley between the bases of the villi to the submucosa. Villus width was calculated from
the mean value of the villus’ width at one-third and villus’ width at two thirds of the height
of the villus. The area between the four villi was used from three cuts per sample to count
the number of goblet cells. The average of the three measurements was then reported as
the number of goblet cells per surface area.

2.7. Microbial Profile

2.7.1. DNA Extraction and 16S rRNA Gene Amplicon Sequencing
Caecal content samples were collected from the sacrificed birds at 42 days using
sterile scissors. Samples were immediately flash frozen in liquid nitrogen and stored in
−20 freezer until DNA extraction. Caecal samples were thawed on ice, and 50 mg of
each sample was transferred into sterile screw-cap tube containing sterile 0.1 and 1.0 mm
zirconia beads (total weight 0.4 g; ratio 1:1). For complete genomic DNA (gDNA) extraction,
microbial cells were lysed using Lysate buffer (Promega, AS1010, Madison, WI, USA) with
a Qiagen TissueLyser II (Qiagen, Hilden, Germany; 30 Hz, 60 sec, 1 repetition settings).
Following lysis, tubes were left for 2 min to allow the lysate to phase separate, and each
sample supernatant (600 µL) was processed for gDNA extraction and purification using
the Maxwell 16 blood DNA purification kit (Promega, AS1010, Madison, WI, USA) and the
automated Maxwell 16 MDx instrument (Promega, Alexandria, NSW, Australia), according
to the manufacturer’s instructions. Quality and quantity of extracted gDNA were assessed
using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Brisbane, Australia). DNA
samples were sent to the Australian Genome Research Facility (AGRF Ltd., Melbourne,
VIC, Australia) for amplicon sequencing.
The V3-V4 region of the 16SrRNA gene was amplified using specific primers (F: 50 -
CCTAYGGGRBGCASCAG-30 , R: 50 -GGACTACNNGGGTATCTAAT-30 ). The PCR condition
were one cycle of 2 min at 95 ◦ C, 30 cycles of 20 s each at 95 ◦ C, 55 ◦ C for 15 s, and 72 ◦ C
for 5 min, followed by one cycle of 10 min at 72 ◦ C. PCR products (300 bp) were sequenced
on Illumina MiSeq platform (AGRF, Melbourne, VIC, Melbourne, Australia).

2.7.2. Bioinformatics Analysis

Raw data files were provided by AGRF as fasta files. Pair-end sequence data un-
derwent standard demultiplexing, which reads/determines quality control, operational
taxonomic units (OTU) clustering (≥97% similarity), and taxonomic classification. Micro-
bial profiling was performed with QIIME 2 2019.7 [23]. The demultiplexed raw reads were
primer trimmed and quality filtered using the CUTADAPT plugin followed by denoising
with DADA2 [24]. Taxonomy was assigned to amplicon sequences variant (ASV) using the
q2-featureclassifier [25] and classify-sklearn naive Bayes taxonomy classifier against SILVA
v128 database [26].
Alpha-diversity analysis was performed using EstimateS v9.1.0 [27]. The OTUs with
average relative abundance less than 0.005% were discarded for alpha-diversity analysis.
Diversity indices of Chao1, Shannon, and Simpson were calculated, and the averages were
compared between treatments. Beta-diversity analysis was performed in PAST v4.03 [28].
Bray–Curtis metric was calculated and PERMANOVA with 1000 permutations were run
Animals 2021, 11, 3607 8 of 20

to compare the beta-diversity between samples and treatment groups. Principle coordi-
nate analysis (PCoA) was performed to visualize the between group diversity. Relative
abundance of microorganisms at different taxonomical levels (reported as percentage) was
statistically compared between treatments using ANOVA via General Linear Model (GLM)
procedure of SAS 9.4. p < 0.05 was used as significant threshold in Tukey’s test.

2.8. Statistical Analyses

The growth performance parameters, organs’ development, meat quality, and gut
morphology data were analysed using Mixed Models of SAS [29], in a randomized block
design with six experimental diets as class effects and blocks as random effects. Each
pen was used as the experimental unit for the analysis of performance data, while the
individual bird was used as the experimental unit for the analysis of other parameters. All
p ≤ 0.05 values were deemed statistically significant. The reported least square means were
separated using Fisher’s least significant differences as the post hoc test.

3. Results
The basal feed was mixed and supplied externally by a local feed producer based on
the provided formulation. However, the feed chemical composition analyses showed some
discrepancies between the calculated and analysed values for the crude protein content
(Table 3), which may have affected the outcomes of the study. In addition, in the second
week of the trial, there was an Escherichia coli outbreak in the shed which caused a higher
mortality rate than expected, especially for treatments PRO-WS (11.90%) and SYN (11.90%)
groups compared to NC (8.33%), PRO250 (7.14%), PC (5.95%), and PRO500 (5.95%). During
the challenge, the broiler chickens were regularly checked by the veterinary services of
the University of Queensland and as the infection was not severe, no medication was
prescribed. Birds recovered within the next week (week 3) and the original experiment
followed the original design; however, this incident has introduced a challenge to the trial
which may also have affected the outcomes of the trial; this has been discussed in more
detail where relevant.

3.1. Growth Performance, Carcass Composition, and Organ Weights

The growth performance traits (Table 4) indicated that the PRO-WS improved ADFI
(p = 0.02) compared to PC and PRO500, but was not different when compared to NC,
PRO250, and SYN. Final body weight (p = 0.14), and ADG (p = 0.14) tended to be
improved by PRO-WS followed by PRO250 and SYN. The FCR was not affected by
experimental treatments.

Table 4. Effects of probiotic, prebiotic and symbiotic in feed or water on growth performance of broiler chickens.

Experimental Diets 2
Parameters 1 SEM 3 p Value
NC PC PRO250 PRO500 PRO-WS SYN
FBW (g) 2365 2326 2389 2265 2405 2377 39 0.14
ADG (g) 55.32 54.40 55.92 52.96 56.32 55.63 1.0 0.14
ADFI (g) 93.07 ab 90.65 bc 92.63 ab 88.27c 96.0 a 92.44 ab 1.54 0.02
FCR-c (g) 1.57 1.56 1.55 1.57 1.59 1.56 0.02 0.85
Mortality (%) 8.33 5.95 7.14 5.95 11.90 11.90 3.54 0.65
1FBW: body weight; ADG: average daily gain; ADFI: average daily feed intake; FCR-c: mortality-corrected feed conversion ratio; 2 NC:
Negative Control; PC: Positive Control: NC + 200 g/t Virginiamycin as Antibiotic Growth Promoter (AGP); PRO250: NC + 250 g/t Probiotic;
PRO500: NC + 500 g/t Probiotic; WS: NC + 0.25g /L Probiotic in Water Supplied; and SYN: NC + 500 g/t Synbiotic (composed of a mix e of
250 g Probiotic + 250 g Prebiotic/t of feed). 3 SEM: standard error of means. a–c Means with different superscripts differ (p ≤ 0.05).

The effects of the experimental treatments on the broiler chickens’ organs relative
weight are presented in Table 5. The addition of antibiotics and or probiotics in different
doses into the feed, or water, and in combination with the prebiotic, did not affect the carcass
weight or composition in terms of breast and abdominal fat pad weights in ratio to final
Animals 2021, 11, 3607 9 of 20

BW. The relative weight of internal organs including heart, liver, bursa of Fabricius, spleen,
gizzard, pancreas, and proventriculus were not affected by the experimental treatments.

Table 5. Effects of probiotic, prebiotic and symbiotic in feed or water on broiler chickens’ organs relative weight to final
body weight at day 42.

Experimental Diets 1
Parameters SEM p Value
NC PC PRO250 PRO500 PRO-WS SYN
Relative Organ weight 2
Carcass Weight 724.7 728.5 726.6 713.7 730.1 718.4 9.92 0.84
Breast Muscle Weight, (g) 245.0 229.4 234.9 233.6 242.0 236.0 5.57 0.35
Heart, (g) 3.86 4.09 4.18 3.82 4.33 4.02 0.16 0.19
Liver, (g) 16.69 16.39 17.93 16.38 16.27 16.0 0.73 0.47
Bursa of Fabricius, (g) 1.73 1.88 1.73 1.89 1.92 1.64 0.17 0.81
Spleen, (g) 0.85 1.03 0.94 0.95 1.09 0.97 0.06 0.09
Gizzard, (g) 21.25 24.22 22.14 22.22 22.27 22.41 1.12 0.58
Pancreas, (g) 1.79 2.06 2.01 1.83 1.88 1.85 0.09 0.24
Abdominal Fat, (g) 4.85 6.12 4.69 3.88 5.50 3.95 0.77 0.19
Proventriculus, (g) 4.99 5.58 4.83 5.00 5.22 5.19 0.35 0.73
1NC: Negative Control; PC: Positive Control: NC + 200 g/t Virginiamycin as Antibiotic Growth Promoter (AGP); PRO250: NC + 250 g/t
Probiotic; PRO500: NC + 500 g/t Probiotic; WS: NC + 0.25 g /L Probiotic in Water Supplied; and SYN: NC + 500 g/t Synbiotic (composed
of a mix e of 250 g Probiotic + 250 g Prebiotic/t of feed). SEM: Standard error of means. 2 Relative weight = absolute weight (g)/body
weight (kg).

3.2. Meat Quality

The effects of probiotic, prebiotic and synbiotic in feed or water on meat quality
parameters of broiler chickens at day 42 are reported in Table 6. All meat quality traits
were similar for the birds on the different treatments except for the breast colour where
the breast lightness (L*) differed (p = 0.02) among experimental groups; birds on PRO250
had lighter breasts than birds on NC, PRO500 or PRO-WS and SYN, but were similar
to PC. As pertaining to the proximate chemical composition of the breast, the moisture
content was higher (p = 0.03) in SYN fed chickens compared to those receiving NC, PC,
and PRO-WS, but similar to PRO250 and PRO500 fed broilers. The Ash content tended
to be higher (p = 0.09) in NC followed by PRO-WS and PRO500. The breast weight and
pH, water holding capacity percentage, Shear force, cooking water loss, breast’s a* and b*,
Hue, moisture percentage, crude protein, and crude fat contents did not differ among all
experimental treatments.

Table 6. Effects of probiotic, prebiotic and symbiotic in feed or water on meat quality parameters of broiler chickens
at day 42.

Experimental Diets 1
Parameters SEM 2 p Value
NC PC PRO250 PRO500 PRO-WS SYN
Breast Meat Weight (% of BW) 25.4 22.9 23.5 23.4 24.2 23.6 0.56 0.35
Breast Meat pH 5.74 5.76 5.73 5.69 5.80 5.73 0.04 0.41
Water Holding Capacity % 68.00 68.97 69.53 69.64 68.71 68.92 0.61 0.42
Shear Force (N) 22.34 20.45 21.25 21.42 20.37 22.58 2.87 0.86
Cooking Water Loss (%) 21.93 19.73 19.83 21.08 21.35 21.33 1.98 0.51
Breast Meat L* 53.71 ab 52.43 bc 50.77 c 54.94 a 53.12 ab 53.03 ab 0.95 0.02
Breast Meat a* 2.51 2.64 2.56 2.49 2.73 2.38 0.35 0.98
Breast Meat b* 5.75 5.85 5.26 6.24 5.59 5.57 0.38 0.59
Hue (◦) 66.22 65.75 62.94 69.39 64.56 67.22 2.89 0.60
Moisture % 73.34 b 74.35 b 74.71 ab 74.62 ab 74.47 b 75.01 a 0.16 0.03
Crude Protein % (as is) 22.50 22.78 22.69 22.50 22.69 22.36 0.22 0.77
Crude Fat % (as is) 1.76 1.60 1.51 1.59 1.57 1.46 0.09 0.29
Ash % (as is) 1.59 1.54 1.53 1.55 1.56 1.48 0.04 0.09
1 NC: Negative Control; PC: Positive Control: NC + 200 g/t Virginiamycin as Antibiotic Growth Promoter (AGP); PRO250: NC + 250 g/t
Probiotic; PRO500: NC + 500 g/t Probiotic; WS: NC + 0.25g /L Probiotic in Water Supplied; and SYN: NC + 500 g/t Synbiotic (composed
of a mix e of 250 g Probiotic + 250 g Prebiotic/t of feed). 2 SEM: Standard error of means. a–c Means with different superscripts differ
(p ≤ 0.05).
Animals 2021, 11, 3607 10 of 20

3.3. Gut Morphology and Excreta pH

The effects of the different experimental treatments on gut morphology traits of
broiler chickens are presented in Table 7. Villus height in the duodenum was higher in
PRO500 (p = 0.04) compared to all the other experimental groups. Crypt of the duodenum
was deeper (p = 0.02) in PRO500 compared to NC, PRO250, PRO-WS, and SYN, but not
different to PC. Duodenal villus width was wider (p = 0.05) in PRO500 compared to PRO250
and PRO-WS, but did no differ from NC, PC, and SYN. Villus length, VH:CD ratio and
the number of goblet cells in the duodenum of the chickens were not affected by the
experimental treatments. In the jejunum segment, the villus width of the chickens in
PRO-WS group showed a higher (p = 0.03) surface area, when compared with NC and PC
treatments, but was similar to PRO250, PRO500, and SYN. However, all the other studied
parameters in the jejunum, including villus height, crypt depth, villus length, VH:CD
ratio and number of goblet cells were similar between the different treatments. In the
ileum segment of the small intestine, villus height, crypt depth, villus width, villus length
and number of goblet cells were not affected by the dietary treatments, however, VH:CD
ratio was significantly greater in the PRO-WS group (p = 0.02), compared to all the other
experimental treatments.

Table 7. Gut morphological parameters of broilers fed with different experimental diets at day 42.

Experimental Diets 1
Parameter SEM 2 p Value
NC PC PRO250 PRO500 PRO-WS SYN
Duodenum
Villus height (µm) 1222 b 1208 b 1101 b 1474 a 1170 b 1113 b 95.18 0.04
Crypt depth (µm) 51.00 b 57.71 ab 48.66 b 68.12 a 48.58 b 55.87 b 5.21 0.02
Villus width (µm) 160.0 a 152.7 ab 149.1 b 184.4 a 126.8 c 164.1 a 13.17 0.05
Villus length (µm) 62.92 64.22 60.38 59.58 62.55 62.30 1.78 0.48
VH:CD 3 26.96 21.98 23.75 22.54 26.82 21.10 3.02 0.49
No. of goblet cells 29.0 23.25 24.92 26.67 24.33 23.50 2.34 0.50
Duodenum pH 6.0 6.09 6.01 6.07 6.05 6.06 0.05 0.72
Jejunum
Villus height (µm) 598 644 642 691 630 647 47.45 0.85
Crypt depth (µm) 43.37 44.21 44.06 52.87 42.47 38.14 3.76 0.16
107.28 132.88
Villus width (µm) 99.55 c bc 127.52 ab 130.04 ab 146.8 a ab 10.73 0.03
Villus length (cm) 53.33 45.40 48.87 44.50 47.43 45.75 2.91 0.30
VH:CD 2 14.26 13.36 16.53 14.26 16.12 17.47 1.93 0.62
No. of goblet cells 15.33 17.05 13.67 15.92 14.50 15.75 1.31 0.43
Jejunum pH 5.83 6.03 5.95 5.92 5.91 5.94 0.07 0.43
Ileum
Villus height (µm) 566 490 539 533 574 512 39.29 0.52
Crypt depth (µm) 44.27 44.92 46.45 51.87 38.68 45.84 3.40 0.19
Villus width (µm) 123.94 104.44 139.77 117.56 121.12 117.66 9.64 0.14
Villus length (cm) 78.75 81.17 70.75 74.50 77.54 78.92 3.01 0.15
VH:CD 2 13.35 b 11.53 b 12.24 b 11.40 b 16.85 a 11.27 b 1.47 0.04
No. of goblet cells 15.03 12.67 15.83 15.58 17.25 14.50 1.17 0.09
Ileum pH 6.23 6.09 6.09 6.30 6.30 6.43 0.17 0.41
Caecum
Caecum pH 5.96 5.93 5.99 5.87 6.01 5.88 0.09 0.85
1NC: Negative Control; PC: Positive Control: NC + 200 g/t Virginiamycin as Antibiotic Growth Promoter (AGP); PRO250: NC + 250 g/t
Probiotic; PRO500: NC + 500 g/t Probiotic; WS: NC + 0.25 g /L Probiotic in Water Supplied; and SYN: NC + 500 g/t Synbiotic (composed
of a mix e of 250 g Probiotic + 250 g Prebiotic/t of feed). 2 SEM: Standard error of means. 3 VH:CD: villus height to crypt depth ratio. a–c
Means with different superscripts differ (p ≤ 0.05).

The pH of excreta at different gut segments, including the duodenum, jejunum, ileum,
and caecum of the broiler chickens at day 42, was not affected by probiotic inclusion into
the feed at different doses, nor in the water, or in combination with prebiotics.
Animals 2021, 11, 3607 11 of 20

3.4. Microbial Profile

On average, a total of 243,294 read sequences (0.146 GB) were yielded from each caecal
sample. Following sequencing quality control, a total of 203,946 sequence reads remained,
of those, 199,375 and 139,650 reads were denoised and non-chimeric, respectively.
Alpha diversity metrics (Chao1, Shannon, and Simpson), which are indications of
richness and evenness of the samples, are shown in Figure 1A. There were no differences
(p > 0.05) in Chao1, Shannon, and Simpson indices between treatment groups. Beta-
diversity results showed that PC (positive control with antibiotics) had less diversity
(higher similarity index) compared to PRO250 (p = 0.050). In addition, PRO500 compared
to PRO250 showed a significantly higher similarity index (p < 0.05), but PC and NC groups
did not differ (p > 0.05).
The relative abundance of different bacteria at both phylum and family levels is pre-
sented in Figure 2. Firmicutes was the most abundant phylum, followed by Bacteroidetes
(Figure 2A). Streptococcaceae, Lachnoospiraceae, Peptostreptococcaceae, Ruminococcaceae, and
Erysipe-lotrichaceae were the top five most abundant families (Figure 2B).
Table 8 shows the relative abundance of bacteria at both family and genus level. A
Total of 88 families were identified, out of which one family differed (p < 0.05) and one
showed a tendency (p = 0.565) to differ in the relative abundance between treatment groups.
These two families were Peptococcaceae and Tannerellaceae, respectively. Relative abundance
of Peptococcaceae in NC and PRO-WS groups compared to PC was higher (p < 0.05). There
was no difference in the abundance of Peptococcaceae between PC and other groups (PRO250,
PRO500, SYN; p > 0.05). As for Tannerellaceae, the NC group tended to have higher relative
abundance compared to PRO500 (p = 0.057). There was no difference between the negative
and positive controls.

Figure 1. Alpha-diversity (A) and Beta-diversity (B) and comparison between different treatments fed to broiler chickens.
NC: Negative control; PC: Positive control (NC plus antibiotics 200 g/t); PRO250: NC plus probiotics 250 g/t; PRO500:
NC plus probiotics 500 g/t; PRO-WS: NC plus probiotics in water 0.25 g/L; SYN: NC plus probiotics 250 g/t plus
prebiotics 250 g/t.
Animals 2021, 11, 3607 12 of 20

Figure 2. Relative abundance of OTUs at phylum (A) and family (B) levels between experimental treatments. NC: Negative
control; PC: Positive control (NC plus antibiotics 200 g/t); PRO250: NC plus probiotics 250 g/t; PRO500: NC plus probiotics
500 g/t; PRO-WS: NC plus probiotics in water 0.25 g/L; SYN: NC plus probiotics 250 g/t plus prebiotics 250 g/t.

Table 8. Relative abundance (%) of different bacteria at Family and Genus levels in caecum content of the chickens fed
experimental treatments.

Experimental Diets 1
Level Name
NC PC PRO250 PRO500 PRO-WS SYN SEM p Value
Family
Bacteria- Firmicutes- Clostridia-
0.093 a 0.034 b 0.074 ab 0.065 ab 0.102 a 0.061 ab 0.013 0.015
Clostridiales_Peptococcaceae
Bacteria-Bacteroidetes-Bacteroidia-Bacteroidales-
0.279 0.170 0.096 0.075 0.137 0.110 0.030 0.057
Tannerellaceae
Genus
Bacteria_Firmicutes_Clostridia_Clostridia-
0.091 a 0.033 b 0.073 ab 0.065 ab 0.101 a 0.061 ab 0.013 0.017
les_Peptococcaceae_ uncultured
Bacteria_Firmicutes_Clostridia_Clostridia-
0.096 ab 0.067 b 0.106 ab 0.116 ab 0.162 a 0.124 ab 0.017 0.019
les_Lachnospiraceae_Lachnoclostridium
Bacteria_Firmicutes_Clostridia_Clostridia-
les_Clostridiales 0.0030 a 0.0007 ab 0.0008 ab 0.000 b 0.0007 ab 0.0005 ab 0.0006 0.072
vadinBB60 group_gut metagenome
1NC: Negative control; PC: Positive control (NC plus antibiotics 200 g/t); PRO250: NC plus probiotics 250 g/t; PRO500: NC plus probiotics
500 g/t; PRO-WS: NC plus probiotics in water 0.25 g/L; SYN: NC plus probiotics 250 g/t plus prebiotics 250 g/t. a,b Means with different
superscripts differ (p ≤ 0.05).

At genus level, relative abundance of three genera was significantly affected by the
treatment groups (Table 8). The first genus belongs to the family of Peptococcaceae and was
not identified and is thus classified as “uncultured”. The abundance of Lachnoclostridium in
Animals 2021, 11, 3607 13 of 20

the PRO-WS was higher than the PC (p < 0.05) treatment. As for the Clostridiales vadinBB60
group, the relative abundance in PRO500 was lower than the NC (p < 0.05).
Table 9 shows the comparison between treatments at species level. More than
400 species were identified, among which seven were affected by the treatment groups
(p < 0.05; Table 9). Species of Erysipelatoclostridium were more abundant in the PC com-
pared to the PRO500 group (p < 0.05). Rusminococcaceae species, GCA-900066225_uncultured
bacterium (p < 0.05) and GCA-900066225 (p < 0.03), were higher in the NC group compared
to SYN and PRO-WS, respectively. Bacteroides fragilis was more abundant (p < 0.05) in
NC compared to the probiotic treatments (PRO250, PRO500, PRO-WS, and SYN). As for
Eubacterium sp. Marseille-P3202, the relative abundance was higher (p < 0.05) in PRO-WS
compared to PC.

Table 9. Relative abundance (%) of different bacteria at Species levels in caecum content of the chickens fed
experimental treatments.

Experimental Diets 1
Name
NC PC PRO250 PRO500 PRO-WS SYN SEM p Value
Species
Bacteria_Firmicutes_Erysipelotri-
chia_Erysipelotrichales_ 0.058 ab 0.12 a 0.061 ab 0.046 b 0.053 ab 0.077 ab 0.018 0.050
Erysipelotrichaceae_Erysipelatoclostridium
Bacteria_Firmicutes_Clostridia_Clos-
tridiales_Ruminococcaceae_GCA- 0.011 a 0.001 ab 0.004 ab 0.002 ab 0.000 b 0.003 ab 0.002 0.030
900066225
Bacteria_Firmicutes_Clostridia_Clos-
tridiales_Ruminococcaceae_GCA-
0.032 a 0.009 ab 0.013 ab 0.009 ab 0.015 ab 0.005 b 0.005 0.049
900066225_uncultured
bacterium
Bacteria_Firmicutes_Clostridia_Clos-
tridiales_Peptococcaceae_uncultured_un- 0.086 ab 0.031 b 0.071 ab 0.062 ab 0.098 a 0.056 ab 0.013 0.018
cultured bacterium
Bacteria_Firmicutes_Clostridia_Clos-
tridiales_Lachnospiraceae_
0.037 ab 0.028 ab 0.031 ab 0.064 a 0.035 ab 0.006 b 0.013 0.047
Lachnospiraceae NC2004
group_uncultured bacterium
Bacteria_Bacteroidetes_Bacteroidia_Bac-
teroidales_Bacteroidaceae_Bac- 0.280 a 0.131 ab 0.093 b 0.036 b 0.071 b 0.088 b 0.035 0.0008
teroides_Bacteroides fragilis
Bacteria_Firmicutes_Clostridia_Clostri
iales_Lachnospiraceae_ Lachnoclostri 0.092 ab 0.067 b 0.106 ab 0.116 ab 0.162 a 0.124 ab 0.017 0.018
ium_Eubacterium sp. Marseille-P3202
1NC: Negative control; PC: Positive control (NC plus antibiotics 200 g/t); PRO250: NC plus probiotics 250 g/t; PRO500: NC plus probiotics
500 g/t; PRO-WS: NC plus probiotics in water 0.25 g/L; SYN: NC plus probiotics 250 g/t plus prebiotics 250 g/t. a,b Means with different
superscripts differ (p ≤ 0.05).

4. Discussion
Probiotics are commonly added to poultry feed as a natural and green feed addi-
tive, with reported beneficial effects on growth performance, nutrient digestion, immune
response, and gut morphology and microbiota [5,6,30,31]. The aim of the current study
was to compare the effectiveness of probiotics added into the feed in two different doses
(PRO250 & PRO500) or in water (PRO-WS), or as a combination with prebiotics (SYN) on
the production efficiency and gut morphology of commercially reared chicken broilers,
whilst a diet with no addition (NC) and a second diet with antibiotics (PC) would serve as
control diets. The results of the current study revealed no differences between NC and PC
in growth performance traits indicating the birds performed as efficient without needing
antibiotics as growth promoters. It is argued that the high hygienic standards of the ex-
perimental facility and the low stocking density of the birds in the research layout do not
Animals 2021, 11, 3607 14 of 20

introduce a health challenge that requires antibiotics. This argument is borne out further in
that neither dose of probiotics in the feed, nor water soluble probiotics or synbiotics had
any effects on the feed efficiency of the broiler chickens in the current study. However,
the unexpected E. Coli outbreak created a challenge across the shed for all treatments and
caused higher mortality rates than normal. Although the mortality rate was not statistically
different among the experimental groups, birds on PC and PRO500 treatments had the
lowest mortality rate (5.95%) compared to the other groups (NC: 8.33%, PRO250: 7.14%,
PRO-WS: 11.90%, and SYN: 11.90%), indicating a potentially better immune response.
Birds recovered after a week without any medication, and the trial followed its original
plan; however, this challenge may have impacted the growth performance of birds.
Considering the overall 42-day growth performance, the PRO-WS significantly im-
proved ADFI (p = 0.02) when compared to PRO500 and PC; however, PRO-WS did not
differ from the NC, PRO250, and SYN treatments for these performance traits. The FCR in
the current study are similar to the outcomes of a recent study [30] where no differences in
production performance of birds that received probiotics in the feed or water compared
to the negative and positive control groups were noted. Similarly, the addition of two
different probiotic preparations, including Bacillus coagulans (1 g Bacillus/kg dried cul-
ture) and Lactobacillus (1 g/kg dried culture of 12 commercial Lactobacillus strains) and
fructo-oligosaccharides or mannan-oligosaccharide prebiotics (each added at 5 g/kg), had
no effects on the growth performance of broiler chickens [32]. In line with our findings,
no significant improvement in body weight, feed intake, and FCR of broiler chickens was
reported for a 5-week experimental period regardless of the route of probiotics adminis-
tration [11]. However, several studies have reported improved growth performance and
reduced mortality rates of broiler chickens when probiotics were supplemented into the
feed [14,33]. Another study [29] reported an enhanced performance of broilers fed probiotic
containing three Bacillus spp. and noted that among the three Bacillus spp., B. coagulans
remarkably improved the growth performance. Some reports indicate that the addition of
different probiotics in water improves feed intake in broilers [3,34], which is in agreement
with ADFI data for PRO-WS group in the current trial. In another study, ADG and feed
efficiency were enhanced for the first 3 weeks, but not for the second 3 weeks, when broilers
were fed with L. fermentum and S. cerevisiae probiotics [35]. Few reports stated either no
impact or a smaller benefit on the growth performance of broilers by water-based probiotics
compared to those added into the feed, leaving the research question with no conclusive an-
swer [11,33,36]. There are numerous factors contributing to the effectiveness of probiotics’
administration in broiler chickens’ diet which makes it challenging to compare different
studies and assess overall outcomes. These factors include bacterial strains, stability, and
viability of the species in the feed or water, administration route, basal diet ingredient
composition, bird flock specification such as age, breed, and overall health status, farm or
facility hygiene standards, and environmental stressors [14,30,37].
It was hypothesized that water-soluble probiotics might be more effective, as probiotic
spores may remain unchanged while passing the bird’s upper GIT mainly due to the
quicker transit time. In addition, the water may restrict the destruction impacts of gastric
acid secretion on the microorganisms [38].
In modern intensive poultry farming systems, eggs are hatched in the hatchery facility
and transferred to the grow-out sheds, in a way that the newly hatched chicken has no
contact with its mother hen. Therefore, it is believed that the intestine of a newly hatched
chicken is nearly sterile and gut microbiota originates from the faecal and/or environmental
contaminants around the egg [39]. Recent studies, however, have reported that the egg
white and embryo show a similar microbial profile and that the egg white is the source of
the intestinal microbiota of the chicken embryo prior to hatching [40]. Some researchers
have suggested that the colonization of the reproductive tract of mother hens by pathogenic
bacteria such as Salmonella species is vertically transmitted to their chickens [41,42]. As a
result, using probiotics in broiler breeder hen nutrition, hatchery facilities, transportation
Animals 2021, 11, 3607 15 of 20

vehicles, and broiler chicken diets may be more successful in establishing the diverse and
healthy gut ecosystem and improving broiler production efficiency.
Our findings showed that the probiotics in feed or water and synbiotics in feed did
not affect the relative organ weights of the broiler chickens after feeding the birds for
42 days in a typical production cycle. These findings are in line with multiple studies that
have used different strains of probiotics for varying durations via different routes, with
no effects on relative organ weights [11,14,33,34,40,43]. Although an increase in pancreas
weight after 21 days of probiotics administration has been reported [11], the reason for this
increase was unknown. Other studies [44,45] reported a greater carcass and breast muscle
yield when probiotics were added to the feed, which is not supported by the current study.
These inconsistent results may also depend on administration level or route, basal diet
composition, strains and probiotic concentrations as discussed previously.
Meat quality traits of broiler chickens were not affected by probiotics administration
in feed or water for 42 days. Similar to the current study, no effects of probiotics addition
to the feed or water on meat tenderness traits including water holding capacity, cooking
loss, and shear force were reported [46]. It has been reported that probiotics enhance
meat quality traits, including colour, oxidation stability, water holding capacity, flavour
and juiciness [47–50]; however, Bacillus subtillis-based probiotics had negligible effects on
the texture of the cooked meat. Meat colour, tenderness, and water holding capacity are
important quality traits. The change in pH is one of the most significant changes that can
affect meat quality characteristics which attribute to consumer acceptance [51].
The chicken breast meat lightness (L*) values can be classified as follows: lighter than
normal (light, L* > 53), normal (48 < L* < 53), and darker than normal (dark, L* < 46) [51].
In the current study, the breast meat lightness was classified as lighter than normal for
all experimental groups except for PRO250 and antibiotic groups. Consumers may reject
chicken meat in which the quality varies from the expected normal appearance. In agree-
ment with our results a lighter breast colour in broiler chicken fed diets supplemented with
probiotics, AGPs, or the combination of probiotics and AGPs compared to the control diet
has also been reported previously [51]. However, another study [46] reported a decrease in
breast meat lightness of broiler chickens when fed with probiotics. There were minimal
differences in the proximate chemical composition of the breast meat of the chickens fed
the different dietary treatments. In this experiment, only the moisture content of the breast
meat of the chickens fed PRO250, PRO500, SYN was higher than that of the NC, PC, and
PRO-WS treatments. No differences in moisture, crude protein, and ash content were
observed; however, a reduced fat content of the breast meat of birds fed with probiotics,
AGPs, and the combination compared to a control diet have been observed [51].
In the current study, the breast pH was not affected by the dietary treatments. Overall,
these results suggest that probiotic supplementation had no negative effects on meat
quality traits.
The addition of probiotics has been reported to alter gut histomorphology; however,
the degree of change varies depending on the strain. In the present study, intestinal
morphology was improved when probiotics were added into the feed or water. Previous
studies on dietary supplementation of Bacillus spp. probiotics including B. subtilis and
B. licheniformis have reported increased VH:CD and improved function of intestinal barrier
leading to a greater nutrient absorption [31,52]. Different strains of probiotics have been
studied for their influence on gut histomorphology and have been found to affect the gut
morphological measurements differently. A previous study on the effects of a probiotic
composed of different Bacillus species on intestinal morphology of broiler chickens found
that B. coagulans improved the intestinal morphometric parameters the most [31]. It
has been reported that probiotics containing Lactobacillus spp. including L. casei and
L. acidophilus, Bifidobacterium thermophilum, and Enterococcus faecium improved the villus
height and decreased the crypt depth in the jejunum [53]. However, the administration
of L. johnsonii through feed, water or litter did not change the ileal morphology of birds
on day 7 and 21 [11]. Our results showed a positive influence on the morphological
Animals 2021, 11, 3607 16 of 20

measurements of the small intestinal mucosa such as increased villus height, villus width,
and VH:CD, suggesting that the addition of probiotics can enhance the intestinal mucosal
architecture.
The data from the current study indicate no changes in the pH of the excreta in the
different intestine segments of the duodenum, jejunum, ileum, and caecum of broilers
when receiving probiotics with or without prebiotics in feed or water. These findings
are in accordance with a previous study [11], which reported an unchanged pH load in
chickens fed different probiotics and/or prebiotics. It is believed that probiotics alter
the gastrointestinal pH and have a great impact on composition and function of the gut
microbiome to favour an increased activity of intestinal enzymes leading to increased
digestibility of nutrients. However, the effectiveness of the probiotics to reduce gut pH is
variable and depends on the bacterial strain, inclusion dose, and the survival rate of the
beneficial bacteria or their spore in the acidic upper GIT of the chicken. Multiple other
factors can also affect gut pH such as environmental conditions of the poultry shed, feed
stuff sanitation and feed processing hygiene at the feed mill [54,55].
The GIT of broilers is a favourable environment for the growth of diverse microbiota.
The microbial profile of the gut is, however, dynamic and factors such as age, especially
the early stages of life, genotype, farming conditions/environment, and diet/feed addi-
tives all influence the makeup of the chicken’s gut microbiota [56]. Various stressors can
alter the gut microbiota composition, leading to dysbiosis, and can consequently impact
the functionality of the intestine, e.g., increased permeability, increased risk of bacterial
infection, sepsis, inflammation, and slower digestion [57]. A study of the diversity of gut
flora in the ileum and caeca of broiler chickens when fed with a corn–soybean meal diet
with no additives revealed that ileum is mostly colonized by Lactobacillus (70%), with the
rest of the bacteria belonging to Clostridiaceae (11%), Streptococcus (6.5%), and Enterococcus
(3.5%) families (6.5%). Conversely, in the caecum, Clostridiaceae (65%) were the most abun-
dant group followed by Fusobacterium (14%), Lactobacillus (8%), and Bacteroides (6%) [58].
Probiotics can enhance the balance of microbiota by competitive exclusion of pathogens
through occupying binding sites and receptors on the intestinal mucosa and suppression
of the growth of other microbes by producing antimicrobial agents [6,59].
Although many papers have been published on probiotics in broiler chicken nutrition,
literature regarding probiotics administration in feed or water and its effects on broiler
intestinal microbiota has only been studied sparsely using 16S rRNA gene amplicon
sequencing and molecular methods. The findings of the current study revealed no changes
in microbial alpha-diversity across all treatments, with no significant prevalence of any
bacterial species being linked to any experimental treatment. These findings were similar
to that of Zhu and co-workers [60], who reported no significant effects of antibiotic or heat-
inactivated compound probiotics treatments on alpha-diversity, including observed species,
Chao 1, Shannon index, Simpson index, Goods coverage, ACE, and PD whole tree (p > 0.05).
In the present investigation, the beta-diversity of the microbial profile of the chicken’s
caecum content was slightly reduced in the PC group compared to other treatments,
indicating a potentially reduced microbial diversity when birds received antibiotics in
feed at subtherapeutic levels; however, this needs to be verified in a repeated experiment,
possibly with a more pathogenic challenging environment. Comparable results were also
reported by Zhu and co-workers [60] where the beta-diversity index of cecal microbiota was
significantly higher than that of PC group at the same sampling timepoints. Accordingly,
Gao et al. [61] reported opposing effects of probiotic and antibiotic administrations on the
age-dependent maturation of intestinal microbiota. The probiotic treatment showed an
early-maturing trend, reaching a plateau at day 15 and indicating an accelerated maturation
of the gut microbiota, whereas antibiotics showed a delayed microbiota development, and
the beta-diversity of intestinal microbiota changed more heavily in the antibiotic group
from day 1 to 42. This indicates that antibiotics destroy a portion of the gut microflora,
causing diminished levels of microbial diversity.
Animals 2021, 11, 3607 17 of 20

The most abundant phylum was Firmicutes, followed by Bacteroidetes. There was no
significant difference between the treatments regarding relative abundance of the phyla.
At the family level, Peptococcaceae were significantly reduced by the antibiotic and not
by the probiotic treatments when administered via water and not through feed. This
could indicate an interaction between the microbial community and the administration
method. At the genus level, three genera were identified that were significantly affected
by the treatment groups. Genus Lachnoclostridium was significantly increased by PRO-WS
compared to the antibiotic positive control. This genus includes bacteria from several
clostridial clusters which are known to have anti-inflammatory effects and play important
role in homeostasis [61]. Gao et al. [62] found eight genera that were significantly changed
on day 28; however, no genera were found significantly changed on day 42. In the present
study, the relative abundance at species level showed seven species that were significantly
affected by the treatment groups. However, only Bacteroides fragilis could be detected to the
species level. The remaining were unknown. Bacteroides fragilis was significantly higher
in NC compared to PRO250, PRO500, PRO-WS, and SYN, but not different from the PC.
This indicates that the tested probiotic product, reduced the abundance of this organism in
the chicken’s cecum. Bacteroides fragilis which accounts for 0.5% of the colonic flora in the
human, is the most isolated pathogen [63]. In chickens, B. fragilis species has a different
pattern than in the human and showed high antimicrobial resistance [64]. This might be
the reason why the antibiotic group did not reduce this species compared to the negative
control. However, these results indicate that the probiotic used in this study has the
potential to reduce the risk of Bacteroides fragilis. This could also have health implications
for humans since Bacteroides fragilis operate as an amphibiotic organism in the colon, and as
a result, they are key markers and sources of antimicrobial resistance genes during endogen
infections [64]. Meat chickens are an important source of protein for humans, but they are
also a source of antimicrobial resistance genetic determinants that can be passed to other
bacterial species in the human digestive tract following ingestion [65].

5. Conclusions
In the present study, the positive and negative control groups performed similarly,
which indicates an ideal environment with insufficient challenges where birds did not
need any antibiotics. It is recommended to test the probiotics in a more challenging
environment to evaluate the efficacy of the positive control and other treatments. Although
an unexpected E. coli outbreak introduced a mild health challenge to the study on the
second week, however, birds recovered without any medication; it is assumed that this
environmental challenge was not severe enough to test the efficacy of the treatments.
Nonetheless, based on the outcomes of this study, probiotic supplementation via feed
or water could partially enhance the production performance and gut morphometry
parameters in broiler chickens; however, the lack of consistent improvement in the different
studied parameters from a specific treatment makes it difficult to conclude on a dose or
administration route. The results obtained from the current experiment are evidence that
supplementation of specific probiotics mixtures can modulate the microbiota that colonizes
the gut and increase the diversity of the bacterial community where antibiotics reduced
it. In addition, the relative abundance of Bacteroides fragilis, which is a marker of bacterial
resistance, was reduced in probiotic-containing treatments. Further research is required
to study probiotics supplementation and its effects on the bird’s immune response in
challenge with non-resistant pathogens.

Author Contributions: E.A.S.: conceptualization, methodology, investigation, writing—original

draft, supervision, formal analysis, funding acquisition. A.D.R.C.C.: data curation, writing, review.
J.B.: project administration, supervision, data curation, review. S.N.: microbial profile bioinformatics,
review. L.C.H.: methodology, supervision, data curation, review and editing, formal analyses. All
authors have read and agreed to the published version of the manuscript.
Animals 2021, 11, 3607 18 of 20

Funding: This project was funded by Bioproton Pty Ltd. (grant number 2020000073) and the
University of Queensland.
Institutional Review Board Statement: All experimental procedures, including animal handing
and husbandries, were approved by the Animal Ethics Committee of Research Ethics Unit of The
University of Queensland, AEC Approval Number SAFS/579/18.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors greatly acknowledge and appreciate the technical support from
the staff of QASP facility, professional staff, and the postgraduate students at the University of
Queensland.
Conflicts of Interest: Jacoba Bromfield declares employment at Bioproton during the trial period
and manuscript development. Elham Assadi Soumeh declares financial support from Bioproton Pty
Ltd. to conduct the farm trial and laboratory analyses.

References
1. OECD/FAO. OECD-FAO Agricultural Outlook 2020–2029; FAO, Rome/OECD Publishing: Paris, France, 2020. [CrossRef]
2. Aarestrup, F.M.; Wegener, H.C.; Collignon, P. Resistance in bacteria of the food chain: Epidemiology and control strategies. Expert
Rev. Anti-Infective Ther. 2008, 6, 733–750. [CrossRef] [PubMed]
3. Kalavathy, R.; Abdullah, N.; Jalaludin, S.; Ho, Y.W. Effects of Lactobacillus cultures on growth performance, abdominal fat
deposition, serum lipids and weight of organs of broiler chickens. Br. Poult. Sci. 2003, 44, 139–144. [CrossRef] [PubMed]
4. Immerman, H.M.; Veldman, A.; van den Elsen, E.; Rombouts, F.M.; Beynen, A.C. Mortality and Growth Performance of Broilers
Given Drinking Water Supplemented with Chicken-Specific Probiotics. Poult. Sci. 2006, 85, 1383–1388. [CrossRef] [PubMed]
5. Kabir, S.M.L. The Role of Probiotics in the Poultry Industry. Int. J. Mol. Sci. 2009, 10, 3531–3546. [CrossRef]
6. El-Hack, M.E.A.; El-Saadony, M.T.; Shafi, M.E.; Qattan, S.Y.A.; Batiha, G.E.; Khafaga, A.F.; Abdel-Moneim, A.-M.E.; Alagawany, M.
Probiotics in poultry feed: A comprehensive review. J. Anim. Physiol. Anim. Nutr. 2020, 104, 1835–1850. [CrossRef]
7. Spring, P.; Wenk, C.; Dawson, K.A.; Newman, K.E. The effects of dietary mannaoligosaccharides on cecal parameters and the
concentrations of enteric bacteria in the ceca of salmonella-challenged broiler chicks. Poult. Sci. 2000, 79, 205–211. [CrossRef]
8. Flickinger, E.A.; Van Loo, J.; Fahey, G.C. Nutritional Responses to the Presence of Inulin and Oligofructose in the Diets of
Domesticated Animals: A Review. Crit. Rev. Food Sci. Nutr. 2003, 43, 19–60. [CrossRef] [PubMed]
9. Dunislawska, A.; Slawinska, A.; Stadnicka, K.; Bednarczyk, M.; Gulewicz, P.; Józefiak, D.; Siwek, M. Synbiotics for broiler
chickens—In Vitro design and evaluation of the influence on host and selected microbiota populations following In Ovo delivery.
PLoS ONE 2017, 12, e0168587. [CrossRef]
10. Jiang, S.; Mohammed, A.; Jacobs, J.; Cramer, T.; Cheng, H. Effect of synbiotics on thyroid hormones, intestinal histomorphology,
and heat shock protein 70 expression in broiler chickens reared under cyclic heat stress. Poult. Sci. 2020, 99, 142–150. [CrossRef]
11. Olnood, C.G.; Beski, S.S.; Iji, P.A.; Choct, M. Delivery routes for probiotics: Effects on broiler performance, intestinal morphology
and gut microflora. Anim. Nutr. 2015, 1, 192–202. [CrossRef]
12. Nathanon, T.; Panomkorn, W.; Moongngarm, A.; Suttajit, M. Stability of Freeze-Dried Lactobacillus acidophilus in Banana, Soybean
and Pearl Barley Powders. J. Biol. Sci. 2008, 8, 119–124.
13. Cutting, S.M. Bacillus probiotics. Food Microbiol. 2011, 28, 214–220. [CrossRef]
14. Khan, S.H.; Yousaf, B.; Mian, A.A.; Rehman, A.; Farooq, M.S. Assessing the effect of administering different probiotics in drinking
water supplement on broiler performance, blood biochemistry and immune response. J. Appl. Anim. Res. 2011, 39, 418–428.
[CrossRef]
15. Aland, A.; Madec, F. Sustainable Animal Production; Wageningen Academic Publishers: Wageningen, The Netherlands, 2009.
16. Oviedo-Rondón, E.O.; Hume, M.E.; Hernández, C.; Clemente-Hernández, S. Intestinal microbial ecology of broilers vaccinated
and challenged with mixed Eimeria species, and supplemented with essential oil blends. Poult. Sci. 2006, 85, 854–860. [CrossRef]
17. Knarreborg, A.; Simon, M.A.; Engberg, R.M.; Jensen, B.B.; Tannock, G.W. Effects of dietary fat source and subtherapeutic levels
of antibiotic on the bacterial community in the ileum of broiler chickens at various ages. Appl. Environ. Microbiol. 2002, 68,
5918–5924. [CrossRef] [PubMed]
18. Aviagen. Aviagen Management Handbook. 2019. Available online: http://es.aviagen.com/assets/Tech_Center/Ross_Broiler/
RossBroilerNutritionSpecs2019-EN.pdf (accessed on 21 January 2021).
19. American Meat Science Association. Meat Color Measurement Guidelines; American Meat Science Association: Champaign, IL,
USA, 2012.
20. Trout, G.R. Techniques for measuring water-binding capacity in muscle foods—A review of methodology. Meat Sci. 1988, 23,
235–252. [CrossRef]
Animals 2021, 11, 3607 19 of 20

21. Honikel, K.O. Reference methods for the assessment of physical characteristics of meat. Meat Sci. 1998, 49, 447–457. [CrossRef]
[PubMed]
22. Sawicka-Kapusta, K. Fat extraction in the Soxhlet apparatus. In Methods for Ecological Bioenergetics; Grodzinski, W., Klekowski, R.Z.,
Duncan, A., Eds.; Blackwell: London, UK, 1975; pp. 288–292.
23. Bolyen, E.; Rideout, J.R.; Dillon, M.R.; Bokulich, N.A.; Abnet, C.C.; Al-Ghalith, G.A.; Alexander, H.; Alm, E.J.; Arumugam, M.;
Asnicar, F.; et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat. Biotechnol. 2019,
37, 852–857. [CrossRef]
24. Callahan, B.J.; Mcmurdie, P.J.; Rosen, M.J.; Han, A.W.; Johnson, A.J.A.; Holmes, S.P. DADA2: High-resolution sample inference
from Illumina amplicon data. Nat. Methods 2016, 13, 581–583. [CrossRef]
25. Bokulich, N.A.; Dillon, M.R.; Zhang, Y.; Rideout, J.R.; Bolyen, E.; Li, H.; Albert, P.S.; Caporaso, J.G. q2-longitudinal: Longitudinal
and Paired-Sample Analyses of Microbiome Data. mSystems 2018, 3, e00219-18. [CrossRef] [PubMed]
26. Quast, C.; Pruesse, E.; Yilmaz, P.; Gerken, J.; Schweer, T.; Yarza, P.; Peplies, J.; Glöckner, F.O. The SILVA ribosomal RNA gene
database project: Improved data processing and web-based tools. Nucleic Acids Res. 2013, 41, D590–D596. [CrossRef] [PubMed]
27. Colwell, R.K. EstimateS: Statistical Estimation of Species Richness and Shared Species from Samples. Version 9.1.0. 2019. Available
online: http://viceroy.colorado.edu/estimates/index.html (accessed on 11 January 2021).
28. Hammer-Muntz, O.; Harper, D.; Ryan, P.D. PAST: Paleontological statistics software package for education and data analysis.
Palaeontol. Electron. 2001, 4, 4.
29. SAS Institute Inc. SAS User’s Guide; Statistics Version 9.1; SAS Institute Inc.: Cary, NC, USA, 2003.
30. Atela, J.A.; Mlambo, V.; Mnisi, C.M. A multi-strain probiotic administered via drinking water enhances feed conversion efficiency
and meat quality traits in indigenous chickens. Anim. Nutr. 2019, 5, 179–184. [CrossRef]
31. Li, C.-L.; Wang, J.; Zhang, H.-J.; Wu, S.-G.; Hui, Q.-R.; Yang, C.-B.; Fang, R.-J.; Qi, G.-H. Intestinal Morphologic and Microbiota
Responses to Dietary Bacillus spp. in a Broiler Chicken Model. Front. Physiol. 2019, 9, 1968. [CrossRef] [PubMed]
32. Al-Khalaifa, H.; Al-Nasser, A.; Al-Surayee, T.; Al-Kandari, S.; Al-Enzi, N.; Al-Sharrah, T.; Ragheb, G.; Al-Qalaf, S.; Mohammed, A.
Effect of dietary probiotics and prebiotics on the performance of broiler chickens. Poult. Sci. 2019, 98, 4465–4479. [CrossRef]
[PubMed]
33. Zhang, L.; Zhang, R.; Jia, H.; Zhu, Z.; Li, H.; Ma, Y. Supplementation of probiotics in water beneficial growth performance, carcass
traits, immune function, and antioxidant capacity in broiler chickens. Open Life Sci. 2021, 16, 311–322. [CrossRef] [PubMed]
34. Manafi, M.; Khalaji, S.; Hedayati, M.; Pirany, N. Efficacy of Bacillus subtilis and bacitracin methylene disalicylate on growth
performance, digestibility, blood metabolites, immunity, and intestinal microbiota after intramuscular inoculation with Escherichia
coli in broilers. Poult. Sci. 2017, 96, 1174–1183. [CrossRef]
35. Bai, S.P.; Wu, A.M.; Ding, X.M.; Lei, Y.; Bai, J.; Zhang, K.Y.; Chio, J.S. Effects of probiotic-supplemented diets on growth
performance and intestinal immune characteristics of broiler chickens. Poult. Sci. 2013, 92, 663–670. [CrossRef] [PubMed]
36. Jin, L.Z.; Ho, Y.W.; Abdullah, N.; Jalaludin, S. Digestive and Bacterial Enzyme Activities in Broilers Fed Diets Supplemented with
Lactobacillus Cultures. Poult. Sci. 2000, 79, 886–891. [CrossRef]
37. Patterson, J.A.; Burkholder, K.M. Application of prebiotics and probiotics in poultry production. Poult. Sci. 2003, 82, 627–631.
[CrossRef]
38. Karimi Torshizi, M.A.; Moghaddam, A.R.; Rahimi, S.; Mojgani, N. Assessing the effect of administering probiotics in water or as a
feed supplement on broiler performance and immune response. Br. Poult. Sci. 2010, 51, 178–184. [CrossRef]
39. Donaldson, E.E.; Stanley, D.; Hughes, R.J.; Moore, R.J. The time-course of broiler intestinal microbiota development after
administration of cecal contents to incubating eggs. PeerJ 2017, 5, e3587. [CrossRef] [PubMed]
40. Lee, S.; La, T.-M.; Lee, H.-J.; Choi, I.-S.; Song, C.-S.; Park, S.-Y.; Lee, J.-B.; Lee, S.-W. Characterization of microbial communities in
the chicken oviduct and the origin of chicken embryo gut microbiota. Sci. Rep. 2019, 9, 6838. [CrossRef]
41. Guard, J.; Gast, R.K.; Guraya, R. Colonization of Avian Reproductive-Tract Tissues by Variant Subpopulations of Salmonella
Enteritidis. Avian Dis. 2010, 54, 857–861. [CrossRef]
42. Wang, C.; Pors, S.E.; Olsen, R.H.; Bojesen, A.M. Transmission and pathogenicity of Gallibacterium anatis and Escherichia coli in
embryonated eggs. Vet. Microbiol. 2018, 217, 76–81. [CrossRef] [PubMed]
43. Kabir, S.M.L.; Rahman, M.M.; Rahman, M.B.; Rahman, M.M.; Ahmed, S.U. The dynamics of probiotics on growth performance
and immune response in broilers. Int. J. Poult. Sci. 2004, 3, 361–364.
44. Denli, M.; Okan, F.; Celik, K. Effect of dietary probiotic, organic acid and antibiotic supplementation to diets on broiler performance
and carcass yield. Pak. J. Nutr. 2003, 2, 89–91.
45. Pelicano, E.; Souza, P.d.; Souza, H.d.; Oba, A.; Norkus, E.; Kodawara, L.; Lima, T.D. Effect of different probiotics on broiler carcass
and meat quality. Braz. J. Poult. Sci. 2003, 5, 207–214. [CrossRef]
46. Aksu, M.İ.; Karaoğlu, M.; Esenbuğa, N.; Kaya, M.; Macit, M.; Ockerman, H.W. Effect of a dietary probiotic on some quality
characteristics of raw broiler drumsticks and breast meat. J. Muscle Foods 2005, 16, 306–317. [CrossRef]
47. Zhou, X.; Wang, Y.; Gu, Q.; Li, W. Effect of dietary probiotic, Bacillus coagulans, on growth performance, chemical composition,
and meat quality of Guangxi Yellow chicken. Poult. Sci. 2010, 89, 588–593. [CrossRef]
48. Liu, X.; Yan, H.; Lv, L.; Xu, Q.; Yin, C.; Zhang, K.; Wang, P.; Hu, J. Growth performance and meat quality of broiler chickens
supplemented with bacillus licheniformis in drinking water. Asian-Australas. J. Anim. Sci. 2012, 25, 682–689. [CrossRef] [PubMed]
Animals 2021, 11, 3607 20 of 20

49. Bai, K.; Huang, Q.; Zhang, J.; He, J.; Zhang, L.; Wang, T. Supplemental effects of probiotic Bacillus subtilis fmbJ on growth
performance, antioxidant capacity, and meat quality of broiler chickens. Poult. Sci. 2017, 96, 74–82. [CrossRef] [PubMed]
50. Abdulla, N.R.; Mohd Zamri, A.N.; Sabow, A.B.; Kareem, K.Y.; Nurhazirah, S.; Ling, F.H.; Sazili, A.Q.; Loh, T.C. Physico-chemical
properties of breast muscle in broiler chickens fed probiotics, antibiotics or antibiotic–probiotic mix. J. Appl. Anim. Res. 2017, 45,
64–70. [CrossRef]
51. Qiao, M.; Fletcher, D.L.; Smith, D.P.; Northcutt, J. The Effect of Broiler Breast Meat Color on pH, Moisture, Water-Holding
Capacity, and Emulsification Capacity. Poult. Sci. 2001, 80, 676–680. [CrossRef] [PubMed]
52. Jha, R.; Das, R.; Oak, S.; Mishra, P. Probiotics (Direct-Fed Microbials) in Poultry Nutrition and Their Effects on Nutrient Utilization,
Growth and Laying Performance, and Gut Health: A Systematic Review. Animals 2020, 10, 1863. [CrossRef]
53. Alagawany, M.; El-Hack, M.E.A.; Farag, M.R.; Sachan, S.; Karthik, K.; Dhama, K. The use of probiotics as eco-friendly alternatives
for antibiotics in poultry nutrition. Environ. Sci. Pollut. Res. 2018, 25, 10611–10618. [CrossRef] [PubMed]
54. Smith, J.M. A Review of Avian Probiotics. J. Avian Med. Surg. 2014, 28, 87–94. [CrossRef]
55. Fontana, L.; Bermudez-Brito, M.; Plaza-Diaz, J.; Munoz-Quezada, S.; Gil, A. Sources, isolation, characterization and evaluation of
probiotics. Br. J. Nutr. 2013, 109, S35–S50. [CrossRef]
56. Diaz Carrasco, J.M.; Casanova, N.A.; Fernández Miyakawa, M.E. Microbiota, Gut Health and Chicken Productivity: What Is the
Connection? Microorganisms 2019, 7, 374. [CrossRef] [PubMed]
57. Shang, Y.; Kumar, S.; Oakley, B.; Kim, W.K. Chicken Gut Microbiota: Importance and Detection Technology. Front. Vet. Sci. 2018,
5, 254. [CrossRef] [PubMed]
58. Lu, J.; Idris, U.; Harmon, B.; Hofacre, C.; Maurer, J.J.; Lee, M.D. Diversity and Succession of the Intestinal Bacterial Community of
the Maturing Broiler Chicken. Appl. Environ. Microbiol. 2003, 69, 6816–6824. [CrossRef] [PubMed]
59. Yadav, S.; Jha, R. Strategies to modulate the intestinal microbiota and their effects on nutrient utilization, performance, and health
of poultry. J. Anim. Sci. Biotechnol. 2019, 10, 2. [CrossRef] [PubMed]
60. Zhu, C.; Gong, L.; Huang, K.; Li, F.; Tong, D.; Zhang, H. Effect of Heat-Inactivated Compound Probiotics on Growth Performance,
Plasma Biochemical Indices, and Cecal Microbiome in Yellow-Feathered Broilers. Front. Microbiol. 2020, 11, 585623. [CrossRef]
61. Dandachi, I.; Anani, H.; Hadjadj, L.; Brahimi, S.; Lagier, J.C.; Daoud, Z.; Rolain, J.M. Genome analysis of Lachnoclostridium
phocaeense isolated from a patient after kidney transplantation in Marseille. N. Microbes N. Infect. 2021, 41, 100863. [CrossRef]
62. Gao, P.; Ma, C.; Sun, Z.; Wang, L.; Huang, S.; Su, X.; Xu, J.; Zhang, H. Feed-additive probiotics accelerate yet antibiotics delay
intestinal microbiota maturation in broiler chicken. Microbiome 2017, 5, 91. [CrossRef] [PubMed]
63. Wexler, H.M. Bacteroides: The good, the bad, and the nitty-gritty. Clin. Microbiol. Rev. 2007, 20, 593–621. [CrossRef] [PubMed]
64. Garcia, G.D.; Carvalho, M.A.R.; Diniz, C.G.; Marques, J.L.; Nicoli, J.R.; Farias, L.M. Isolation, identification and antimicrobial
susceptibility of Bacteroides fragilis group strains recovered from broiler faeces. British Poult. Sci. 2012, 53, 71–76. [CrossRef]
65. Vedantam, G. Antimicrobial resistance in Bacteroides spp.: Occurrence and dissemination. Futur. Microbiol. 2009, 4, 413–423.
[CrossRef]