Growth performance and gastrointestinal responses in heavy Tom turkeys fed

antibiotic free corn−soybean meal diets supplemented with multiple doses of

a single strain Bacillus subtilis probiotic (DSM29784)1

M. Mohammadigheisar,∗ R. B. Shirley,† J. Barton,† A. Welsher,† P. Thiery,‡ and E. Kiarie ∗,2

∗
Department of Animal Biosciences, University of Guelph, Guelph, ON N1G 2W1, Canada; † Adisseo, Alpharetta,
GA 30022, USA; and ‡ Adisseo France, SAS, Antony, France

ABSTRACT Growth performance and gastrointesti- and grower-1 phases whereas bird fed mid and high SSB
nal (GIT) responses to a single-strain of Bacillus sub- had lower BWG in grower-2 and birds fed low and mid
tilis (SSB) were investigated using 960 Hybrid Con- SSB had higher BWG in finisher phase. Consequently,
verter Toms. A total of 4 iso-caloric and iso-nitrogenous birds fed low and mid SSB doses were heavier (P <
corn−soybean meal-based diets were allocated to 12 0.05) than control fed birds at the end of trial. The FCR
replicate cages/pens and fed (ad libitum) in a four-phase response to SSB was linear and non-linear (P < 0.05)
feeding program (starter; days 0 to 28, grower-1; days with birds fed low SSB showing lower FCR than con-
29 to 56, grower-2; days 57 to 84, and finisher; days 85 trol fed birds in starter, grower-1 and finisher phases.
to 126). The diets had either 0 (control), 1E+08 (low), Supplemental SSB had linear and non-linear (P < 0.05)
2E+08 (mid) or 1E+09 (high) cfu B. subtilis/kg. Feed effects on AR of components (DM, ash, crude protein,
intake (FI) and BW were recorded by phase. Excreta crude fat, neutral detergent fiber, and gross energy),
samples were collected towards the end of starter and litter moisture, GIT weight, jejunal histomorphology,
grower-1 phases for apparent retention (AR) of com- and SCFA. Relative to control, birds fed high SSB
ponents by marker method and litter moisture, respec- showed higher AR of components, villi height, day 56
tively. Selected birds were necropsied on days 28 and 56 ceca digesta total SCFA concentration, and lower litter
for GIT weight and samples for jejunal histomorphology moisture. In conclusions, under condition of the cur-
and ceca digesta short chain fatty acids (SCFA). Sup- rent study, growth performance was optimized by low
plemental SSB had linear and non-linear (P < 0.05) re- to mid SSB. Improved nutrient retention and indices
sponse on BW gain (BWG). Specifically, relative to the of gut health suggested higher SSB doses may optimize
control, birds fed low SSB had higher BWG in starter growth performance under challenging farm conditions.
Key words: gut health and function, Bacillus subtilis probiotic, heavy Tom turkeys, growth performance
2019 Poultry Science 98:5541–5550
http://dx.doi.org/10.3382/ps/pez305

INTRODUCTION However, shortly after discovery of growth promot-

ing effects of antibiotics, antibiotic-resistant bacte-
Discovery of growth promoting effects of sub- ria were isolated (Dibner and Richards, 2005; Starr
therapeutic antibiotics when offered to healthy an- and Reynolds, 1951). Increasing recovery of antibiotic-
imals in the 1950s is the basis of widespread use resistant bacteria over the past decades has resulted in
of antibiotics for growth promotion (AGP) in inten- increasing concerns on the overuse or abuse of antibi-
sive animal protein production systems (Moore et al., otics worldwide (Grant et al., 2018). In this context,
1946; Markovic et al., 2009; Kiarie et al., 2016). Al- concerns over emergence of microbes that are resistant
though not well understood, growth promoting effects to antibiotics used to treat human and animal infections
of AGP have been suggested to be linked to modula- along with increasing consumer demand for antibiotic
tion of intestinal microbiota composition and inflamma- and drug-free animal protein products have initiated a
tions for improved growth performance and nutrients search for effective non-antibiotic functional feed strate-
utilization (Batkowska et al., 2015; Kiarie et al., 2016). gies as alternatives to AGP (Kiarie et al., 2016). Con-
sidering bans and restriction on the use of AGP in the
C 2019 Poultry Science Association Inc.
poultry industry, plenty of studies have been conducted
Received January 4, 2019.
Accepted May 16, 2019.
to assess emerging alternatives (Markovic et al., 2009;
1
Presented in part at the 2018 International Poultry Scientific Fo- Mountzouris et al., 2010; Vondruskova et al., 2010;
rum, Jan. 29 to 30, Atlanta, GA. & 2018 ASAS-CSAS Annual meeting Kiarie et al., 2013; Zhang and Kim 2013; Zhang et al.,
& Trade show, July 8 to 12, Vancouver, BC. 2013; Mohammadigheisar et al., 2015, 2016). Probiotics
2
Corresponding author: ekiarie@uoguelph.ca
or direct-fed microbials (DFM) have been shown to be
5541
5542 MOHAMMADIGHEISAR ET AL.

a viable option that can be added to the feed and benefi- the different levels of inclusion a single-strain of Bacil-
cially affect the intestinal microflora, improve nutrients lus subtilis (SSB; DSM 29,784) on performance, appar-
digestibility, and enhance gut health of the host animal ent retention (AR) of components, litter moisture, and
(Waititu et al., 2014; Cai et al., 2015; Payling et al., indices of gut health.
2017).
According to the National Agricultural Statistic Ser-
vice of the United State Department of Agriculture, MATERIALS AND METHODS
turkey production in the US increased from 2.45 million The experimental protocol was reviewed and ap-
tons in 2000 to 2.71 million tons in 2016, a remarkable proved by the University of Guelph Animal Care
share of meat-type turkey production in the poultry Committee and turkeys were cared for in accordance
industry. Mortality of turkeys in commercial farming with the Canadian Council on Animal Care guidelines
system causes economic losses for farmers. Hatching (CCAC, 2009).
process, breeder flock age, genetic strain, and charac-
teristics of the farm on which birds were reared are
some of the factors that have been reported to be in- Birds and Housing
fluential on poults mortality and grow-out performance
(Carver et al., 2000, 2002; Batkowska et al., 2015). In A total of 960-day-old Tom turkeys (Hybrid con-
addition to the rearing challenges, in-feed antibiotic re- verter, Hendrix Genetics, Kitchener, ON, Canada) with
strictions have also increased the risk of turkey pro- an average initial BW of 69 g were obtained from a lo-
duction. Many researchers have studied application of cal commercial hatchery (Cuddy Farms Ltd., Strathroy,
probiotics in poultry production. However, relative to Ontario, Canada) and used in a 126-day feeding trial.
broilers and laying hens, there are limited studies in The poults were housed in metabolic cages (105 ×
turkeys but with variable responses. For example, ap- 62 cm) in an environmentally controlled room (32 to
plication of probiotics in feed and/or water improved 24◦ C; 60% relative humidity) during the first 28 D of
growth performance and feed efficiency in turkeys life (starter phase) and subsequently transferred to floor
(Torres-Rodriguez et al., 2007; Grimes et al., 2008; Rus- pens (200 × 213 cm) with fresh wood shavings for the
sell and Grimes, 2009; Batkowska et al., 2015). In con- rest of the trial. The starter phase was in metabolism
trast, probiotic supplement increased BW at 8, 10, and cages to enable collection of excreta samples as ex-
12 wk, but not at 16 wk, and had no effect on feed effi- plained later. There were 20 birds per cage in starter
ciency (Potter et al., 1979). Blair et al. (2004) did not phase, 15 birds per pen in grower-1 and 12 birds per
observe growth performance effects in turkeys fed pro- pen in grower-2 and finisher phases. Floor pens were lo-
biotics for 12. Francis et al. (1978) fed probiotics alone cated in rooms with automated environment controlling
or in combination with zinc bacitracin but did not ob- equipment to control and record temperature, relative
serve effects on growth performance. Although varied humidity, ventilation, and light intensity. After mov-
responses to supplemental probiotics in turkeys may be ing Tom turkeys to the floor pens the temperature was
related to many factors, there are few experiments that set at 24◦ C for adaptation and then it was decreased
evaluated dose response of probiotics throughout the gradually to 18◦ C for the rest of the trial. The lighting
growth cycle. program was according the recommendation of Hendrix
Considering the challenges in turkey production, the genetics (days 0 to 2: 1 h, days 3 to 5: 4 h, days 6 to 9:
adaptation of the gastrointestinal tract (GIT) to envi- 6 h, and days 10 to 126: 8 h of darkness). All the pens
ronmental stress is a critical characteristic for achieving were equipped with a bucket feeder and a 4 nipples
optimal performance. Due to the high requirements for water line.
AA, commercial turkey diets contain high level of pro-
tein feedstuffs. Feeding turkeys with high crude protein Probiotic, Diets, and Management
(CP) diets increases the risk of passing the undigested
to the hindgut. It has been reported that high level of The probiotic preparation was a single strain of
dietary CP increases the activity of putrefactive bac- B. Subtilis DSM29784 prepared on calcium car-
teria resulting in the production of toxic fermentation bonate carrier (Alterion R
-NE50, Adisseo USA Inc.,
components such as H2 S, NH3 , amines, phenols, and Alpharetta, GA, USA). Four iso-caloric and iso-
indoles (Searle et al., 2009; Apajalahti and Vienola, nitrogenous corn−soybean meal basal diets were for-
2016; Ghasemian and Jahanian, 2016; Classen et al., mulated to meet the specifications (Hybrid converter,
2017). There is some evidence showing that indole tox- Hendrix Genetic, Kitchener, ON, Canada; Table 1) in a
icity uncouples the protein gradient across biological four-phase feed program: starter; days 0 to 28, grower-1;
membranes and inhibits ATP production (Chimerel et days 29 to 56, grower-2; days 57 to 84, and finisher; days
al., 2013). Activity of putrefactive bacteria, absence of 85 to 126. In each phase, 4 experimental diets were cre-
in-feed antibiotics, and insufficient biosecurity in com- ated by adding 0 (control), 1E+08 (low), 2E+08 (mid),
mercial farms increase the incidence of pathogens which and 1E+09 (high) cfu SSB/kg corresponding to 0.0,
can cause losses in turkey production. Therefore, this 0.5, 1.0, and 5.0 kg of AlterionR
-NE50 per metric ton
experiment was conducted to determine the influence of of feed. The diets were created by adjusting limestone
 PROBIOTIC SUPPLEMENT FOR HEAVY TOM TURKEYS 5543
Table 1. Composition of the basal diets, as fed basis.1

Starter Grower-1 Grower-2 Finisher

Item (days 0 to 28) (days 29 to 56) (days 57 to 84) (days 85 to 126)

Ingredient, %
Corn 38.4 46.4 50.1 63.2
Soybean meal-46% 34.1 28.4 25.0 16.2
Wheat 7.00 7.00 7.00 5.37
Corn gluten meal-60% 7.00 4.00 3.93 3.00
Blood meal 2.00 1.00 0.55 0.01
Pork meal-60% 6.00 7.00 4.19 4.80
Soybean oil 1.21 2.78 5.18 4.50
Mono calcium phosphate 0.63 0.32 0.61 0.00
Limestone 1.16 0.82 1.11 0.63
NaCl 0.17 0.24 0.28 0.27
NaHCO3 0.21 0.00 0.00 0.00
Na2 SO4 2 0.00 0.13 0.14 0.11
DL-methionine 0.34 0.29 0.24 0.19
L-lysine HCl, 78% 0.43 0.42 0.39 0.37
L-threonine, 98% 0.15 0.13 0.13 0.11
L-tryptophan 0.00 0.00 0.00 0.01
Choline chloride-60% 0.13 0.07 0.07 0.05
Vitamins and trace premix3 1.00 1.00 1.00 1.00
25-hydroxyvitamin D34 0.00 0.00 0.10 0.10
Phytase5 0.01 0.01 0.01 0.01
Calculated composition
AME, mcal/kg 2.85 3.00 3.17 3.25
Crude protein, % 30.1 25.9 22.5 18.5
SID Lys, % 1.65 1.44 1.24 0.98
SID Met + Cys, % 1.11 0.94 0.83 0.69
SID Thr, % 1.07 0.91 0.81 0.64
SID Trp, % 0.30 0.26 0.22 0.18
Ca, % 1.38 1.29 1.15 0.92
Available P, % 0.69 0.66 0.57 0.46
Ca: available P ratio 2.00 1.95 2.02 2.00
Na, % 0.17 0.18 0.18 0.17
Cl, % 0.23 0.27 0.27 0.26
Analyzed nutrient contents
Crude protein, % 29.29 28.45 23.30 19.31
Ca, % 1.39 0.90 1.10 0.68
P, % 0.71 0.67 0.66 0.53
Na, % 0.18 0.19 0.19 0.19
Starch, % 30.93 31.11 35.23 42.71
1
In each phase, 4 experimental diets were created by adding 0 (control), 1E+08 (Low), 2E+08 (Med), and 1E+09 (High) cfu SSB/kg feed,
corresponding to 0, 0.5, 1.0, and 5.0 kg/metric ton inclusion of Alterion-NE50 product (a single strain of B. Subtilis DSM29784, Adisseo USA Inc.,
Alpharetta, GA).
2
AdiSodium, (Adisseo USA Inc., Alpharetta, GA).
3
Provided per kilogram of diet: vitamin A, 8800.0 IU; vitamin D3 , 3300.0 IU; vitamin E, 40.0 IU; vitamin B12 , 12.0 mg; vitamin K3 , 3.3 mg; niacin,
50.0 mg; choline, 1200.0 mg; folic acid, 1.0 mg; biotin, 0.22 mg; pyridoxine, 3.3 mg; thiamine, 4.0 mg; calcium pantothenic acid, 15.0 mg; riboflavin,
8.0 mg; manganese, 70.0 mg; zinc, 70.0 mg; iron, 60.0 mg; iodine, 1.0 mg; copper, 10 mg; and selenium, 0.3 mg.
4
Provided 69 μ g hydroxyvitamin D3 per kg feed (Rovimix HyD, DSM Nutritional Products Ayr, ON, Canada).
5
Provided 500 FTU of phytase per kg of feed supplying provided 0.15% available P and 0.16% Ca (Quantum Blue, AB Vista, Marlborough, UK).

in accord with SSB inclusion. The diets were free of an- feed conversion ratio (FCR). Mortality was recorded
tibiotics and coccidiostats. Starter diets contained 0.3% daily to allow the further correction of FCR.
TiO2 as indigestible marker and were fed in crumble
form. Grower-1, grower-2, and finisher diets were fed in
pelleted form. To accomplish proper mixing, the SSB Sampling
was initially pre-mixed with 10 kg of ground corn (a On day 23, collection trays were installed in all cages
portion of the diet formulation) in a stepwise manner. and fresh grab excreta samples were collected from days
First, the supplement was pre-mixed with 5 kg of corn 24 to 28 and stored at −20◦ C until further analyses.
for 3 min in a small mixer. The resulting mixture was Immediately after weighing on day 28, 5 birds per cage
added to the remaining 5 kg of corn and mixed for 2 min were randomly selected for necropsy and the rest of the
to give a 10 kg SSB−corn premix. The treatments were birds reweighed on cage basis and transferred to floor
allocated to 12 replicate cages based on hatch BW in a pens for the rest of the trial. For the necropsied birds,
completely randomized design. Birds had free access to empty gizzard, small intestine, and ceca were weighed
feed and water throughout the trial. BW and feed disap- and the samples of jejunal tissue and ceca digesta col-
pearance were monitored on days 0, 28, 56, 84, and 126 lected for histomorphology and short chain fatty acids
to calculate BW gain (BWG), feed intake (FI), and (SCFA) concentration, respectively. Jejunal segments
5544 MOHAMMADIGHEISAR ET AL.

(∼3 cm) were placed in buffered formalin for histomor- Germany). A total of 20 μL of the resulting sample
phology analysis (Kiarie et al., 2009). Ceca digesta sam- was injected into the column, with a column tempera-
ples were placed on ice and transported to the labora- ture of 60◦ C and mobile phase of 0.005 N H2 SO4 buffer
tory immediately upon collection and stored at −20◦ C at 0.5 mL/min isocratic for 35 min. The detector was
until required for analysis. At the end of the grower-1 heated to 40◦ C.
phase (day 56), 3 birds were randomly selected, weighed
and necropsied for similar measurements and samples
as described for day 28 necropsy. Litter samples for lit-
Calculations and Statistical Analysis
ter moisture content analyses were collected from the The AR of components was calculated according to
centre and mid-way between centre and 4 corners of Kiarie et al. (2014) as follows:
each pen (Leung et al., 2019).
Apparent retention%
= [((NT/Ti)diet × (NT/Ti)excreta ) /(NT/Ti)diet ] × 100,
Sample Processing and Analyses
The litter samples were pooled and homogenized in where (NT/Ti)diet is ratio of component and Ti in the
plastic bags and the moisture content determined by diet, and (NT/Ti)excreta = ratio of component and Ti
placement in an oven at 80◦ C for 48 h. Diet sam- in excreta. Component can be DM, CP, crude fat, NDF
ples and air-dried excreta samples were finely ground. or GE.
All the samples were analyzed for DM, N, crude fat, The organ weight (gizzard, small intestine and ceca)
neutral detergent fiber (NDF), gross energy, and Ti. data were reported as g/kg BW. Data were analyzed
The DM was determined according to standard proce- using the GLM procedure of SAS, with the pen being
dures (AOAC International, 2005; method 930.15). The defined as the experimental unit. Initial BW at the start
NDF content was determined according to Van Soest of the phase was used as covariate in statistical analyses
et al. (1991) using Ankom 200 Fiber Analyzer (Ankom for grower-1, grower-2, and finisher phase parameters.
Technology, Fairport, NY). Crude fat content was de- The linear and quadratic effects of SSB among treat-
termined using ANKOM XT 20 Extractor (Ankom ments were analysed by using a polynomial regression
Technology, Fairport, NY). Gross energy was mea- to describe the shape of the response to increasing con-
sured using IKA bomb calorimeter (C5000; IKA Works, centrations of SSB in the diet. Proc IML procedures of
Wilmington, NC). Determination of N was carried out SAS was used to generate the polynomial coefficients
by using Leco N analyzer (FP-528; Leco, Saint Joseph, for unequally spaced treatments. Significance was set
MI). Titanium content was measured on a UV spec- at P < 0.05.
trophotometer according to the method described by
Myers et al. (2004). Fixed jejunal tissues were embed- RESULTS
ded in paraffin, sectioned (5 μm), and stained with
hematoxylin and eosin for morphological examinations Addition of SSB to the diet of Tom turkeys had a
at the University of Guelph Animal Health Laboratory. quadratic response on BW, BWG, and FI (P < 0.01)
In each cross-sectioned tissue, at least 4 to 5 complete during the starter phase (days 0 to 28; Table 2). Birds
villus-crypt structures were examined under a Leica fed diets containing SSB showed higher BW and BWG
DMR microscope (Leica Microsystems, Wetzlay, Ger- compared with the birds fed with control diet, however,
many) and villous height (VH) and crypt depth (CD) these parameters were not (P > 0.05) different between
measured using a calibrated micrometer and the ra- birds receiving SSB. The quadratic response of SSB
tion of VH:CD was calculated (Kim et al., 2017). The on FI was such that only mid dose fed birds differed
concentration of SCFA was analyzed as described by (P < 0.01) with the control. The FCR response was cu-
Lueng et al. (2018). Briefly, the digesta was thawed bic (P = 0.05) during starter phase with birds fed low
and approximately 0.1 g of the digesta was resuspended SSB showing better FCR than the birds fed with the
with 1 mL 0.005 N H2 SO4 (1:10, wt/vol) in a microcen- control. In grower-1 (days 29 to 56), linear and cubic
trifuge tube. The tube was vortexed vigorously until (P = 0.01) effects were observed for BW and BWG, lin-
the sample was completely dissolved. Then the samples ear effect (P = 0.01) was observed for FI and quadratic
were centrifuged at 11,000 × g for 15 min. After cen- (P = 0.05), and cubic (P = 0.05) effect was observed for
trifuging, 400 μL of supernatant was transferred into a FCR. The shape of responses to various levels of SSB
high-pressure liquid chromatography (HPLC) vial and indicated that feeding the birds with the diet contain-
400 μL of 0.005 N H2 SO4 buffer was added. The result- ing the lowest level of SSB resulted in an improvement
ing digesta fluid was then assayed for SCFA by using in these parameters. During grower-2 phase (days 57
HPLC (Hewlett Packard 1100, Germany) with Rezex to 84), the shape of response to various levels of SSB
ROA-Organic Acid LC column, 300 × 7.8 mm from was different from grower-1 phase. The results showed
Phenomenex and Refractive Index detector at 40◦ C that feeding Toms with the diets supplemented with
(Agilent 1260 Infinity RID from Agilent Technologies, the increasing levels of SSB linearly and quadratically
 PROBIOTIC SUPPLEMENT FOR HEAVY TOM TURKEYS 5545
Table 2. Effect of different levels of single strain B. subtilis on body weight, body weight gain, feed intake, and feed conversion ratio
of Tom turkeys.

Inclusion level of SSB1 P-Value

Item 0 Low Med High SE Lin. Quad. Cub.

Starter (days 0 to 28)

Initial BW, kg/bird 0.07 0.07 0.07 0.07 0.000 0.10 0.97 0.88
BW, kg/bird 1.25 1.30 1.31 1.30 0.010 0.12 < 0.01 0.12
BWG, kg/bird 1.18 1.23 1.24 1.23 0.010 0.11 < 0.01 0.12
FI, kg/bird 1.45 1.48 1.52 1.49 0.012 0.25 < 0.01 0.81
FCR 1.23 1.20 1.22 1.21 0.009 0.54 0.75 0.05
Grower 1 (days 29 to 56)2
BW, kg/bird 4.84 4.98 4.84 4.71 0.052 0.01 0.55 0.03
BWG, kg/bird 3.54 3.68 3.54 3.41 0.052 0.01 0.55 0.03
FI, kg/bird 5.56 5.51 5.39 5.31 0.065 0.01 0.19 0.54
FCR 1.58 1.50 1.52 1.56 0.020 0.40 0.05 0.05
Grower 2 (days 57 to 84)2
BW, kg/bird 10.65 10.61 10.24 10.22 0.116 0.01 0.05 0.19
BWG, kg/bird 5.80 5.76 5.38 5.37 0.116 0.01 0.05 0.19
FI, kg/bird 11.39 11.37 10.76 11.03 0.155 0.19 0.01 0.10
FCR 1.97 1.99 2.00 2.05 0.022 0.01 0.61 0.99
Finisher (days 85 to 126)2
BW, kg/bird 18.93 19.44 19.48 18.70 0.132 < 0.01 < 0.01 0.23
BWG, kg/bird 8.50 9.01 9.05 8.27 0.132 < 0.01 < 0.01 0.23
FI, kg/bird 27.92 27.85 28.63 26.35 0.416 < 0.01 0.08 0.35
FCR 3.29 3.09 3.17 3.19 0.050 0.89 0.08 0.04
1
Abbreviation: 0 (control), 1E+08 (Low), 2E+08 (Med), and 1E+09 (High) cfu SSB/kg feed.
2
At the end of the starter (day 28) and grower-1 (day 56), 5 and 3 birds, respectively, were euthanized for sampling, so the initial BW were used
as covariate in statistical analyses of grower-1 and grower-2 phase parameters.

(P ≤ 0.05) reduced BW and BWG. Specifically, birds (P ≤ 0.05) were observed on the relative weights of giz-
receiving high SSB showed the lowest BW and BWG. zard, small intestine, and ceca. The gizzard weight was
The FI response was quadratic (P = 0.01) and FCR higher for birds fed mid-level of SSB whereas the small
showed a linear (P = 0.01) response in grower-2. Dur- intestine weight was lower for birds fed low and mid
ing the finisher phase (days 85 to 126) birds fed low SSB. Supplementing SSB led to a linear (P < 0.01) and
and mid-levels of SSB showed higher (P < 0.01) BW quadratic (P = 0.05) increase in the relative weight of
(+2.60 and +2.82%, respectively) and BWG (+5.7 and ceca.
+6.1%, respectively) compared with the birds receiv- Addition of different levels of SSB in the diets of
ing control or high level of SSB. During this phase FI Tom turkeys linearly (P = 0.03) increased jejunal VH in
was linearly affected (P < 0.01) such that increasing starter phase (Table 4). However, the increase was not
level of SSB resulted in a linear decrease in FI. The step-wise, thus resulting also in a quadratic (P = 0.01)
results showed a tendency for quadratic (P = 0.08) re- response of jejunal VH to SSB supplementation. How-
sponse of SSB on FCR such that birds fed low SSB ever, a linear decrease (P < 0.01) was observed for CD
had 6.1% lower FCR compared with the control fed in response to SSB consequently leading to linear (P <
birds. 0.01) increase in jejunal VH:CD ratio. Comparatively,
Feeding the birds with the diets supplemented with addition of low, med, and high level of SSB to the diet
various levels of SSB during starter phase resulted in of Tom turkeys increased VH:CD ratio by 10.8, 17.6,
a linear response (P < 0.01) in AR of DM, Ash, CP, and 41.9%, respectively relative to the control. Sup-
crude fat, NDF, and GE, and a quadratic response (P plemental SSB decreased (P < 0.01) CD and increased
< 0.01) for AR of CP, crude fat, and NDF (Table 3). (P = 0.01) VH:CD ratio in quadratic fashion in grower-
A linear and cubic effect in response to SSB (P < 1 phase (day 56). Specifically, relative to birds fed con-
0.04) was observed for litter moisture content on day 56 trol, the VH:CD ratio was 8.0, 35.7, and 19.6% for birds
(Table 3). In this context, birds fed high SSB showed fed low, medium, and high SSB, respectively. Addition
the lowest litter moisture relative to control. Supple- of SSB linearly and quadratically decreased (P = 0.02)
menting the diets with SSB did not (P > 0.05) affect concentration of lactic acid of ceca digesta in starter
relative weight of gizzard in starter phase (Table 4), phase (Table 5). The total SCFA (summation of lac-
but the relative weight of small intestine was affected tic, acetic, propionic, and butyric acids) quadratically
non-linearly (P = 0.01). The results showed that the reduced (P = 0.04) in response to supplemental SSB
relative weight of small intestine of the birds fed the in starter phase. A linear (P ≤ 0.05) effect on the con-
diet with mid-level of SSB was higher (P < 0.05) than centration of acetic acid, butyric acid, and total SCFA
other treatment groups on day 28. A linear effect (P = was observed in grower-1. The high SSB increased con-
0.02) was observed on the relative weight of ceca on day centration of total SCFA by 15.8% compared with the
28. In grower-1 (day 56) linear and quadratic responses control group (Table 5).
5546 MOHAMMADIGHEISAR ET AL.

Table 3. Effect of different levels of single strain B. subtilis on apparent retention (%) of components and litter moisture in Tom
turkeys fed corn soybean meal diets.1

Inclusion level of SSB1 P-Value

Items 0 Low Medium High SE Lin. Quad. Cub.

Dry matter 75.22 73.12 74.77 80.24 0.991 < 0.01 0.19 0.15
Ash 40.29 35.41 40.50 55.11 2.560 < 0.01 0.31 0.13
Crude protein 62.42 55.86 58.25 67.22 1.630 < 0.01 0.01 0.05
Crude fat 89.71 88.15 88.33 91.83 0.604 < 0.01 0.02 0.31
Neutral detergent fiber 42.78 37.58 37.98 53.18 2.679 < 0.01 0.04 0.48
Gross energy 80.30 79.00 79.93 83.86 0.803 < 0.01 0.24 0.30
Day 56
Litter moisture, % 31.42 35.79 30.97 27.96 1.75 0.02 0.70 0.04
1
Abbreviation: 0 (control), 1E+08 (Low), 2E+08 (Med), and 1E+09 (High) cfu SSB/kg feed.

Table 4. Effect of different levels of single strain B. subtilis on weight of gut organs g/kg BW, organs weight growth, and intestinal
histomorphology of Tom turkeys.

Inclusion level of SSB1 P-Value

Items 0 Low Medium High SE Lin. Quad. Cub.

Day 28
Gizzard 17.01 17.03 17.07 17.48 0.23 0.11 0.92 0.98
Small intestine 21.96 20.69 24.95 21.48 0.73 0.48 0.01 < 0.01
Ceca 6.62 6.23 6.55 5.70 0.26 0.01 0.86 0.26
VH, μ m 1088.58 1271.77 1319.22 1331.66 52.48 0.03 0.01 0.39
CD, μ m 147.08 155.26 152.42 127.54 4.75 < 0.01 0.11 0.40
VH:CD ratio 7.37 8.24 8.74 10.50 0.38 < 0.01 0.13 0.78
Day 56
Gizzard 16.57 17.48 18.51 18.02 0.55 0.23 0.03 0.82
Small intestine 19.36 17.78 17.31 17.86 0.40 0.21 < 0.01 0.37
Ceca 4.78 4.97 5.24 5.42 0.13 < 0.01 0.05 0.69
VH, μ m 3826.20 3889.25 4127.81 3959.09 294.1 0.89 0.47 0.76
CD, μ m 218.75 202.25 172.21 186.23 10.1 0.17 < 0.01 0.43
VH:CD ratio 17.83 19.25 24.20 21.3 1.56 0.37 0.01 0.25
1
Abbreviation: 0 (control), 1E+08 (Low), 2E+08 (Med), and 1E+09 (High) cfu SSB/kg feed. VH, villus height; CD, crypt depth.

Table 5. Effect of different levels of single strain B. subtilis on short chain fatty acids concentration (μmol/g) of Tom turkeys.

Inclusion level of SSB1 P-Value

Items 0 Low Medium High SE Lin. Quad. Cub.

Day 28
Lactic 34.96 31.96 23.76 23.72 2.66 0.02 0.02 0.35
Acetic 46.11 46.49 42.34 49.15 2.82 0.29 0.28 0.47
Propionic 2.95 1.48 1.99 3.03 0.79 0.43 0.29 0.35
Butyric 14.41 16.35 11.75 14.28 1.39 0.86 0.24 0.06
Total SCFA 98.42 96.29 79.83 90.17 5.53 0.54 0.04 0.24
Day 56
Lactic 13.03 10.47 12.00 10.65 2.07 0.59 0.76 0.44
Acetic 69.43 63.69 73.98 81.43 5.40 0.05 0.88 0.23
Propionic 10.34 12.52 10.29 11.15 0.95 0.93 0.94 0.07
Butyric 11.86 10.97 22.97 21.29 1.74 < 0.01 < 0.01 < 0.01
Valeric 2.51 1.97 2.37 2.75 0.61 0.53 0.75 0.54
Total SCFA 107.17 99.62 121.61 127.27 7.15 0.03 0.38 0.10
1
Abbreviation: 0 (control), 1E+08 (Low), 2E+08 (Med), and 1E+09 (High) cfu SSB/kg feed.

DISCUSSION 2006; Kiarie et al., 2016). Bacillus, a spore-forming fac-

ultative anaerobe, have higher resistance to temper-
The World Health Organization defines probiotics atures of up to 113◦ C. High temperature resistance
as “live microorganisms which when administered in helps Bacillus spp. to survive during feed processing
adequate amounts confer health benefit on the host” and also, they can tolerate low pH of GI tract (Grant
(FAO/WHO, 2001). Commercially produced probiotics et al., 2018). However, literature shows that the ef-
include different microorganisms, such as: Bacillus fects of Bacillus spp. on BWG, FI, and FCR are
(Gram-positive spore forming bacteria), lactic-acid pro- variable. Most of the researchers have reported that
ducing bacteria (Lactobacillus, Bifidobacterium, Ente- supplementing the diets with Bacillus probiotic im-
rococcus), and yeast (Roselli et al., 2005; Stein and Kil, proved performance of broiler chickens and laying hens
 PROBIOTIC SUPPLEMENT FOR HEAVY TOM TURKEYS 5547
(Fritts et al., 2000; Knap et al., 2010; Jeong and Kim, feeding turkey poults with a diet supplemented with B.
2014; Waititu et al., 2014; Lee et al., 2015; Gadde et al., subtilis increased villi height and villi:crypt ratio (Ag-
2017; Rhayat et al, 2017; Neijat et al., 2018, 2019), while boola et al., 2014; Omidiwura et al., 2018). Laudadio et
others reported that Bacillus probiotic either had no ef- al. (2012) suggested that a higher VH:CD ratio results
fect on growth performance or even caused a decrease in a decreased turnover of the intestinal epithelium and
in BW (Teo and Tan, 2007). consequently it decreases the maintenance requirement,
The impact of application of probiotics over the which possibly results in a higher growth rate or effi-
long-term growth cycle of turkeys has been variable ciency of the animal.
ranging from positive effects (Torres-Rodriguez et al., It has been reported that the typical corn soybean-
2007; Grimes et al., 2008; Russell and Grimes, 2009; based diets given to commercial poultry are mostly rich
Batkowska et al., 2015) to mixed effects (Potter et al., in carbohydrates and provide substrates for ferment-
1979; Blair et al., 2004). The current study focused on ing bacteria in the hindgut and those bacteria pro-
evaluation of dose response in view of limited studies duce fermentation metabolites including lactate, suc-
on application of probiotics in turkeys. The shape cinate, and fumarate which can be either absorbed by
of responses to various levels of SSB in the current the host or consumed by other microbes to produce
study indicated that feeding diets containing lowest other products (Kiarie et al., 2014, 2017; Grant et al.,
to mid-level of SSB was most effective in improving 2018). Manipulation of gut microflora has been shown
growth performance relative to control. Other studies to be effective on the reduction of the amounts of lac-
have shown similar dose responses. For example, tate in the cecum, suggesting either the absorption of
dose response investigations of a dry Lactobacillus the lactate by the host or consumption by other bac-
acidophilus culture (0, 0.025, 0.05, and 0.075% dietary teria (Chaveerach et al., 2004). Our findings indicated
inclusion) in turkeys from 0 to 16 wk of age showed that that addition of SSB to the diet of Tom turkeys resulted
only the lowest level (0.025%) improved BW (Potter et in changes in the gut microflora over time. During the
al., 1979). Owings (1992) fed 5 levels of Streptococcus starter phase, increasing levels of probiotic in the diet
spp probiotic (0, 100, 1,000, 10,000, or 100,000 cfu/g of of Tom turkeys resulted in the reduced amount of lac-
feed) to Tom turkeys from 1 to 126 D. Although there tic acid, but the results showed that total concentration
was no effect of diet on final BW, birds fed 10,000 cfu/g of SCFA was affected quadratically. The results of the
dose showed improved FCR compared with control grower-1 phase showed that in contrast with the starter
birds whereas the other doses were intermediate. phase, lactic acid concentration did not differ between
Taken together, it appears low to mid doses are the treatments, but a significant difference was observed
most effective in improving growth performance in between the concentration of butyric acid in different
turkeys. Our results indicated that the AR of nutrients treatment groups. These results indicated that there
was correlated with the inclusion level of SSB and was a correlation between the inclusion level of SSB,
with increasing levels of SSB the AR of nutrients was the relative weight of ceca, and the total SCFA concen-
increased linearly during the starter phase. However, tration at the end of grower-1 phase.
this improvement was not reflected in performance, It should be mentioned that the influence of supple-
especially feed efficiency. We assumed that addition of mental Bacillus on the production of SCFA and nu-
higher levels of probiotic to the diet resulted in a com- trients varies from study to study. Murugesan et al.
petition between bacteria and the host for nutrients, (2014) reported that feeding chickens with the diet
and therefore, it led to a higher FI during the starter supplemented with Bacillus licheniformis resulted in a
phase (days 0 to 28). The results of previous studies significant increase in the concentration of butyrate.
suggested that the use of B. subtilis bacteria as a pro- Novak et al. (2011) reported reduced production of bu-
biotic reduced colonization of Salmonella typhimurium tyrate and other SCFA in broiler chickens fed probiotic
and enteritidis (Vincente et al., 2007; Menconi et al., containing B. subtilis and B. licheniformis. Fujiwara
2011; Shivaramaiah et al., 2011), and consequently et al. (2009) showed that feeding broiler chickens with
reduced detrimental pathogenic impacts on gut health. diets supplemented with Bacillus spp. increased acetic
Reducing the negative impact of pathogens on in- acid and total SCFA concentration in the ceca. Muruge-
testinal epithelium optimizes gut health and function san et al. (2014) reported that feeding broiler chick-
(Kiarie et al., 2013; Kiarie and Mills, 2019). Similar ens with the diets containing Bacillus DFM increased
to the results of the study conducted by Hutsko et al. total SCFA, acetic acid, and butyric acid concentra-
(2016), the results of the present study showed that tion which support the findings of the current study.
feeding the turkeys with the diets containing Bacil- The results of the present research were consistent with
lus DFM improved villi height. It has been suggested the results reported by Murugesan et al. (2014) and
that longer villi increase the intestinal surface area and there was linear and non-linear increase of acetic, bu-
subsequently provide a better opportunity for nutri- tyrate and as a result total SCFA with mid and high
ents to be absorbed (Samanya and Yamauchi 2002), SSB in grower 1, however, the same phenomenon was
while shorter villi and deeper crypts lead to lower ab- not observed in starter phase. Cherbut (2003) suggested
sorption of nutrients and as a result lower performance that butyrate, has been shown to be the most effec-
(Xu et al., 2003). Previous researchers reported that tive SCFA to promote the proliferation and functional
5548 MOHAMMADIGHEISAR ET AL.

maturation of intestinal epithelial cells resulting in in- electrical properties of lipid membranes. ChemPhysChem 14:417–
creased arterial blood flow linked to increased nutrient 423.
Classen, H. L., A. Deep, D. D. L. S. Bryan, R. K. Savary, E. Herwig,
absorption. These physiological functions of butyrate and K. Schwean-Lardner. 2017. Does location and extent of starch
in increasing nutrient digestion and absorption could and protein digestibility affect poultry productivity and health?
be responsible for the increased AR of nutrients and Proc. 11th Turkey Science and Production Conf., Turkeytimes,
GE by the addition of SSB. Woodbank, john Street, Utkinton, Cheshire CW6 0LU, Chester,
UK. http://jsbentley.co.uk/77842˙Turkey˙times˙inner˙2017˙4.pdf.
In conclusion, the results of the present study indi- Dibner, J. J., and J. D. Richards. 2005. Antibiotic growth promoters
cated that supplementing the diets of Tom turkeys with in agriculture: History and mode of action. Poult. Sci. 84:634–
a single B. subtilis strain improves growth performance 643.
by stimulating FI during the starter phase. Feeding FAO/WHO. 2001. Health and nutritional properties of pro-
biotics in food including powder milk with live lactic acid
turkeys with the diets supplemented with low (1E+08 bacteria, a joint FAO/WHO expert consultation. Cordoba,
cfu SSB/kg) and mid (2E+08 cfu/kg) level of SSB im- Argentina. 1–4 October 2001. Accessed December 2018. http://
proved BW, BWG, and FCR. The data also indicated isappscience.org/wp-content/uploads/2015/12/FAO-WHO-2001-
that mid (2E+08 cfu/kg) to high (1E+09 cfu/kg) doses Probiotics-Report.pdf.
Francis, C., D. M. Janky, A. S. Arafa, and R. H. Harms. 1978. In-
supported gut health and development. Given the cur- terrelationship of Lactobacillus and zinc bacitracin in the diets of
rent study was conducted in a highly controlled facility, turkey poults. Poult. Sci. 57:1687–1689.
perhaps mid to higher SSB may be applicable in opti- Fritts, C. A., J. H. Kersey, M. A. Motl, E. C. Kroger, F. Yan, J. Si,
Q. Jiang, M. M. Campos, A. L. Waldroup, and P. W. Waldroup.
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Fujiwara, K., M. Yamazaki, H. Abe, K. Nakashima, Y. Yakabe,
ACKNOWLEDGMENTS M. Otuska, Y. Ohbayashi, Y. Kato, K. Namai, A. Toyoda, Y.
Miyaguchi, and Y. Nakamura. 2009. Effect of Bacillus subtilis
The research has been funded by the Ontario Agri- var. Natto fermented soybean on growth performance, microbial
Food Innovation Alliance (ON, Canada) and the Adis- activity in the caeca and cytokine gene expression of domestic
seo USA Inc. Alpharetta, GA (USA). meat type chickens. J. Poult. Sci. 46:116–122.
Gadde, U., S. T. Oh, Y. S. Lee, E. Davis, N. Zimmerman, T. Re-
hberger, and H. S. Lillehoj. 2017. The effects of direct-fed micro-
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