Evaluation of effects of EarlyBird associated with FloraMax-B11

on Salmonella Enteritidis, intestinal morphology,

and performance of broiler chickens

A. Biloni,*1 C. F. Quintana,* A. Menconi,† G. Kallapura,† J. Latorre,† C. Pixley,‡ S. Layton,§

M. Dalmagro,§ X. Hernandez-Velasco,# A. Wolfenden,† B. M. Hargis,† and G. Tellez†2

*Universidad Nacional del Nordeste, Facultad de Ciencias Veterinarias, Corrientes Argentina, C.P. 3400,
Argentina; †Department of Poultry Science, University of Arkansas, Fayetteville 72701;
‡Pacific Vet Group USA Inc., Fayetteville, AR 72703; §Argentina Vetanco S.A. Chile 33 (B1603CMA)
Vicente López, Buenos Aires, Argentina; and #Facultad de Medicina Veterinaria y Zootecnia,
Universidad Nacional Autonoma de Mexico, 04510, Mexico

ABSTRACT A posthatch fasting period of 24 to 72
h is a common and inevitable practice in commercial
poultry production. This delay in start of feed intake
has been reported to negatively affect yolk utilization,
gastrointestinal development, slaughter weight, breast
meat yield, performance, and to also depress immuno-
logical development, making the birds more susceptible
to infection from pathogens such as Salmonella. Fur-
thermore, public concerns regarding the considerable
human rates of illness reported and the emergence of
antibiotic-resistant strains of Salmonella have doubled
the challenge on the poultry industry to find alternative
means of Salmonella control. In the present study, we
evaluated the effects of a combination of early feeding
with probiotic supplementation on morphological de-
velopment of mucosa, control of Salmonella, and overall
performance in broiler chickens. We used a blend of a
commercially available perinatal supplement, EarlyBird
(EB; Pacific Vet Group USA Inc., Fayetteville, AR),
and a successful probiotic supplement, FloraMax-B11
(FM; Pacific Vet Group USA Inc.), to evaluate the ef-
fects on gut morphology, Salmonella intestinal coloniza-
Key words: early feeding, Salmonella Enteritidis, broiler, gut morphology, performance
tion, and horizontal transmission, along with its effects
on BW and related performance in broiler chickens un-
der simulated commercial hatching management and
shipping conditions. Morphometric analysis showed in-
creased villus height, villus width, villus to crypt ratio,
and villus surface area index in chickens treated with
EB + FM groups. Significant reductions in Salmonella
recovery, incidence, and horizontal transmission were
also observed among the same groups, suggesting ben-
eficial effects of early feeding and competitive exclusion
by probiotic bacteria. Improved gut morphology and
Salmonella exclusion was very well supported by BW
data with significantly lower early BW loss and overall
BW gains in birds treated with EB + FM mixture. The
results of this study demonstrated that the combina-
tion of EB and FM improved gut morphology, reduced
the amount of Salmonella that could be recovered, as
well as improved BW when compared with controls and
each product individually. These data address both ani-
mal welfare and food safety concerns faced by the poul-
try industry.
2013 Poultry Science 92:2337–2346
http://dx.doi.org/10.3382/ps.2013-03279

INTRODUCTION ulation has hatched. During this period, hatched chicks

In a commercial hatchery, chicks hatch over a 24- to
36-h period of time. Chicks that hatch early usually re-
main in the hatcher until a large portion of the egg pop-
©2013 Poultry Science Association Inc.
Received May 2, 2013.
Accepted May 28, 2013.
1 Part of the work of the thesis of the first author: Maestría en Pro-
ducción Animal Subtropical, FCV, Universidad Nacional del Nordeste,
Argentina.
2 Corresponding author: gtellez@uark.edu
are kept without food and water, and the chicks that
hatch early are at a disadvantage because of the pro-
longed fasting period and potential dehydration (Bigot
et al., 2003; Careghi et al., 2005). After hatch, sexing,
vaccinations, beak trimming, comb dubbing, and ship-
ping are performed, which means an additional increase
to the fasting period (Casteel et al., 1994; Juul-Madsen
et al., 2004). A direct correlation has been reported
between the time involved hatching and transportation
and BW loss (BWL) in broilers (Noy and Sklan, 1998,
2002; Hooshmand, 2006; Henderson et al., 2008). In

2337
2338 Biloni et al.
the absence of feed and water, a linear reduction in
BW has been reported at 0.17 g/h in hatched chickens
(Sklan and Noy, 2000). Delay of placement exacerbates
this condition with further decreases in BW (Casteel
et al., 1994; Careghi et al., 2005). This early weight
loss has a profound adverse effect on final performance
of chickens and poults, and it is mainly attributed to
the metabolism and dehydration that occurs during the
holding time in the incubator and to transportation
conditions (Noy and Sklan, 1999).
Yolk is the main energy supplier of avian embryos
(Noy and Sklan, 2002). About 20% of the BW of newly
hatched commercial poultry is yolk sac, which provides
energy immediately posthatch (Noy and Sklan, 1998).
Literature reports that the basal metabolic rate for a
45-g broiler is 11 kcal per day calculated using oxygen
consumption data (Kuenzel and Kuenzel, 1977; Meltzer,
1983), but the yolk can only supply about 5.3 kcal/d
in chicks with no access to feed, resulting in a deficit
of almost 6 kcal/d. This value represents feed-deprived
metabolism and tissue growth (Kuenzel and Kuenzel,
1977; Noy and Sklan, 2002; Noy and Uni, 2010). The
result of this metabolic imbalance is ketosis and keto-
acid production from gluconeogenesis from fat and pro-
tein of the yolk, which depresses appetite and causes
lethargy in chicks (Erlanson-Albertsson and Mei, 2005;
Noy and Uni, 2010). Normally, these ketones and keto-
acids are removed by kidney filtration and are excreted
in the urine. However, because these chicks are also wa-
ter deprived, the kidneys are not able to remove these
by-products in an efficient way. Our research has shown
that this buildup of ketones and keto-acids (ketosis and
ketoacidosis) is the primary cause of slow-starting or
poor-starting chicks (Henderson et al., 2008). In the
same way, early feed deprival has been linked with
delayed gut maturation (Noy et al., 2001; Uni et al.,
2003a; Yi et al., 2005), which in turn causes inefficient
absorption of nutrients at later feeding stages.
In addition to reduction in overall performance (Noy
and Sklan, 1999; Halevy et al., 2000; Hooshmand 2006;
Henderson et al., 2008) resulting from delayed early
feeding, reduced immunity, and increased susceptibility
to diseases have also been reported by several investiga-
tors (Dibner et al., 1998; Juul-Madsen et al., 2004; Yi
et al., 2005; van den Brand et al., 2010). Forcing chicks
to use their maternal antibody reserves for energy is
highly inefficient and harmful to production because
these birds then have increased risk for infectious dis-
eases (Dibner et al., 1998; Batal and Parsons, 2002).
Some of the lipids used have a specific function in early
growth and some fatty acids are important as precur-
sors for secondary messengers. For these reasons, utili-
zation of yolk as the sole source of energy for prolonged
time periods before placement is a very damaging
and expensive proposition. The costs associated with
restriction of feed and water after hatching has been
well documented in many studies (Kuenzel and Kuen-
zel, 1977; Dibner et al., 1998; Uni et al., 1999a; Bigot
et al., 2003; Henderson et al., 2008; Chearskul et al.,
2008; Noy and Uni, 2010; van den Brand et al., 2010).
For all these reasons, provision of early access to feed
and water to hatchlings has become a necessary part of
poultry production, which will result in enhanced ini-
tial growth, maintained through market age. EarlyBird
(EB; Pacific Vet Group USA Inc., Fayetteville, AR) is
a commercially available natural hydration and nutri-
tion supplement for neonatal broilers and poults, which
promotes instinctive feeding that leads to a rapid onset
and increased early weight gains that will eventually
be maintained throughout the bird’s lifetime (Hender-
son et al., 2008). In addition to providing a reliable
source of protein and carbohydrates, after hydration,
EB contains 64% water, thus helping to initiate kidney
function and removal of previously mentioned harmful
metabolic by-products.
Increasing sociopolitical concerns with antibiotic us-
age have led to investigations of potential alternatives
for food safety and growth promotion. Both live and
spore-based probiotics have earned tremendous atten-
tion as a viable control of enteric pathogens. Probiot-
ics or direct-fed microbials are composed of a variable
number of species and strains of beneficial bacteria
known to have positive implications on poultry health
and performance. Chickens and poults for commercial
production are hatched in a clean environment, hence
delaying their colonization by healthy microflora. In
this near-sterile environment, the intestinal tract of
these newly hatched chickens and poults provides a
suitable ecological niche for any pathogen (Methner et
al., 1997; Crhanova et al., 2011). Colonization of mu-
cosal surfaces of newly hatched chickens with beneficial
gut microflora is therefore a matter of significance. In
this regard, the use of competitive exclusion probiotic
products enabling early rapid colonization of chickens
with healthy adult gut microbiota has been suggested
(Alvarez-Olmos and Oberhelman, 2001; Flint and Gar-
ner, 2009).
Extensive laboratory and field research conducted
by our laboratory with a defined lactic acid bacteria
probiotic has demonstrated accelerated development of
normal microflora in chickens and turkeys. FloraMax-
B11 (FM; Pacific Vet Group USA Inc.) is a unique
probiotic for poultry developed in our laboratory at the
University of Arkansas after years of research. FloraM-
ax-B11 is specifically formulated to address economi-
cally important factors affecting the poultry industry.
The benefits of FM have been documented in more
than 110 published, refereed manuscripts, abstracts,
and proceedings (Tellez et al., 2001, 2006; Farnell et
al., 2006; Higgins et al., 2007, 2008, 2011; Vicente et al.,
2007, 2008; Wolfenden et al., 2007a,b).
In the present study, we have evaluated the effect of
a combination of the above-mentioned 2 commercially
available products: EB, the neonatal nutritional sup-
plement associated with FM, a probiotic. Their effects
on intestinal morphology of broiler chickens, Salmonella
 EFFECTS OF EARLY FEEDING IN COMBINATION WITH PROBIOTICS
Enteritidis cecal colonization, and overall performance
in broiler chickens were evaluated under simulated com-
mercial hatching management and shipping conditions.
MATERIALS AND METHODS
Source of Birds
Day-of-hatch, off-sex broiler chickens were obtained
from Cobb-Vantress (Siloam Springs, AR) for all the
trials mentioned below. All animal handling procedures
were in compliance with Institutional Animal Care and
Use Committee at the University of Arkansas.
Perinatal Supplement and Probiotic Culture
EarlyBird is an all-natural hydration and nutrition
supplement for young birds. One gram of EB contains
64% of water, 22.0% of protein, 10% of fiber, 20%
carbohydrate, and less than 2.2% of fat. EarlyBird is
shipped as a dry material, and hence, according to the
manufacturer’s instructions, it should be mixed with
warm water in a ratio of 1 part EB to 1 part water (wt/
vol). For experiment 3, where one of the groups received
only hydrated EB, the following procedure was followed
to prepare the product. Take required volume of warm
water in a plastic pail and start adding an equal weight
of dry EB powder, thoroughly mixing while doing so.
The appropriately mixed product was allowed to stand
for 5 min and 200 g of this hydrated EB was placed
over the top of 100 chicks in each chick box. Care was
taken to provide normal to bright lighting to allow the
chicks to see the product and initiate rapid consump-
tion upon placement of birds in the box.
FloraMax-B11 is a probiotic culture derived from
poultry, consisting of 2 strains of lactic acid bacterial
isolates: Lactobacillus salivarius and Pediococcus par-
vulus of poultry gastrointestinal origin. The mixture of
EB + FM in experiments 1, 2, and 3 was done as fol-
lows. A bottle of 140 g of FM, provided by the manu-
facturer, is used to treat 20,000 chickens (at 106 cfu/
chick). Hence, the amount required to treat 100 chick-
ens was calculated to be approximately be 1 g (final
dose 1 g/100 birds). The EB should be mixed with
warm water in a ratio of 1 part EB to 1 part water (wt/
vol). For all experiments, where the groups received
hydrated EB+FM mix, the following procedure was fol-
lowed to prepare the product. For 100 chickens, take
200 mL of warm water in a plastic pail and start adding
200 g of dry EB powder, thoroughly mixing while doing
so. With continued stirring, add 1 g of FM dry powder
to make a hydrated mixture of EB+FM at 200:1 ratio,
respectively. Appropriately mixed product was allowed
to stand for 5 min, and 200 g of this hydrated EB was
placed over the top of 100 chicks in each chick box.
Care was taken to provide normal to bright lighting to
allow the chicks to see the product and initiate rapid
consumption upon placement of birds in the box.
2339
For FM spray application in experiment 3, FM was
diluted in reconstituted powdered skim milk to an ex-
pected concentration of 107 cfu/mL. Actual cfu admin-
istered per chick, for each experiment, was determined
retrospectively by spread plating the administered sam-
ple on DeMan, Rogosa, Sharpe agar (Becton Dickinson,
Sparks, MD). Briefly, chicks were sprayed (25 mL/100
chicks) using a handheld garden sprayer (catalog no.
013622667, Walmart, Bentonville, AR), adjusted to a
coarse spray, with the probiotic containing green dye
(catalog no. 065031001, Sensient Food Colors, St. Lou-
is, MO). Chicks were then placed under a halogen light
for 2.5 min to stimulate preening (Wolfenden et al.,
2007a).
Bacterial Strain and Culture Conditions
The challenge organism used in all experiments was
a poultry isolate of Salmonella enterica subspecies en-
terica serovar Enteritidis, bacteriophage type 13A, ob-
tained from the USDA National Veterinary Services
Laboratory (Ames, IA). This isolate was resistant to
25 µg/mL of novobiocin (NO, catalog no. N-1628, Sig-
ma, St. Louis, MO) and was selected for resistance to
20 µg/mL of nalidixic acid (NA, catalog no. N-4382,
Sigma) in our laboratory. For the present studies, 100
µL of Salmonella Enteritidis from a frozen aliquot was
added to 10 mL of tryptic soy broth (catalog no. 22092,
Sigma) and incubated at 37°C for 8 h, and passed ev-
ery 8 h to ensure that all bacteria were in log phase.
Postincubation, bacterial cells were washed 3 times in
sterile 0.9% saline by centrifugation at 1,864 × g at 4°C
for 15 min, quantified with a spectrophotometer (Spec-
tronic 20D+, Spectronic Instruments Thermo Scientif-
ic, Little Rock, AR), and diluted in sterile 0.9% saline
to a concentration of approximately 108 cfu/mL. Con-
centrations of Salmonella Enteritidis were determined
retrospectively by serial dilution and further plating on
brilliant green agar (BGA, catalog no. 70134, Sigma)
with NO and NA for enumeration of actual cfu used to
challenge the chickens.
Experimental Design
Experiment 1. Experiment 1 consisted of 3 inde-
pendent trials evaluating the effects of EB + FM on
Salmonella colonization in ceca (including cecal ton-
sils) under simulated commercial hatching manage-
ment and shipping conditions of 72 h. For each trial,
day-of-hatch off-sex broiler chickens were obtained and
randomly distributed into 2 separated groups in plastic
poultry transport crates with 100 birds per group. A
small number of chickens (n = 20) for each trial were
humanely killed on arrival, and ceca (including cecal
tonsils), liver, and spleen were aseptically removed, cul-
tured in tetrathionate enrichment broth (Tet, catalog
no. 210420, Becton Dickinson, Sparks, MD) and con-
firmed for Salmonella by plating the samples on selec-
2340 Biloni et al.
tive BGA with NO. All groups were challenged with
Salmonella Enteritidis at 104 cfu/bird. One hour post-
challenge, one group received EB + FM mix, whereas
the other group acted as the control. No feed or water
was provided during this period, and all chicks were
kept in crates with proper heating arrangements for
72 h. Twenty chickens from control or treated groups
were humanely killed and cultured at 24 and 72 h, re-
spectively, for Salmonella Enteritidis recovery in ceca
(including cecal tonsils) and Salmonella incidence (pos-
itive/total birds).
Experiment 2. This experiment consisted of a single
trial evaluating effect of EB + FM on Salmonella colo-
nization and horizontal transmission in simulated com-
mercial hatching management and shipping conditions
of 24 h. A total of 220 day-of-hatch broiler chickens
were obtained, and 20 birds tested negative for Salmo-
nella. The rest of the chicks were randomly distributed
in 2 separate groups in plastic poultry transport crates
with 100 birds per group. All chickens were identified
through neck tags. Twenty birds in each group were
challenged with Salmonella Enteritidis at 104 cfu/bird
acting as “seeders,” whereas the other 80 birds/group
acted as “contacts.” One hour postchallenge, treated
chickens received EB + FM mix, whereas the other
group acted as control. No feed or water was provided
during this period, and all chicks were kept in crates
with proper heating arrangements for 24 h. All chicks
from both groups were humanely killed and cultured at
24 h for Salmonella Enteritidis recovery in ceca (includ-
ing cecal tonsils), and Salmonella Enteritidis incidence
(positive/total birds) was enumerated as positive/total
(%).
Experiment 3. Four hundred twenty off-sex broiler
chicks were obtained and transported to the University
of Arkansas facility where they were randomly distrib-
uted in 4 groups of 100 birds each, and placed into
commercial plastic poultry transport crates: group 1,
control, no treatment; group 2, FM only; group 3, EB
only; and group 4 EB + FM. A small number (n = 20)
were tested negative for Salmonella. All birds were neck
tagged for identification for the remainder of the exper-
iment and were individually weighed. Twenty chickens
per group were challenged at 1 d of age with Salmo-
nella Enteritidis at 104 cfu/bird (seeders). Seeders were
then placed back into their respective crates to evaluate
horizontal transmission of Salmonella Enteritidis, as de-
scribed in experiment 2. One hour postchallenge, treat-
ments were administered according to groups: group 1
received no treatment; group 2 received FM only; group
3 received 200 g of EB only; and group 4 received the
combination of EB + FM (2:1 wt/wt). All products
were administered according to manufacturer’s instruc-
tions. All birds were kept fasted, with no feed or water,
for 48 h under simulated shipping conditions, at room
temperature (25.5°C) maintained with constant air flow
to ensure the chicks were comfortable.
After 48 h under simulated shipping conditions, chicks
were placed onto floor pens with fresh wood shavings,
with a stocking density of 0.15 m2/chick. Age-appropri-
ate environmental temperatures were maintained and
supplemental heat lamps were provided for each pen.
Chicks were provided ad libitum access to water and a
balanced, unmedicated corn-soybean diet meeting or
exceeding the nutrition requirements of poultry recom-
mended by the NRC (1994).
All birds were weighed at 24 h, 48 h, 7 d, and 14
d of age. Recorded BW were then used to determine
BWL at 24 and 48 h or BW gain (BWG) at 7 and 14
d of age. Five ileum and duodenum samples from each
group were collected for enteric morphometric analy-
sis of mucosal development at each point, processed,
and analyzed further as explained below. Furthermore,
Salmonella Enteritidis recovery and incidence of Salmo-
nella (positive birds/total birds) were enumerated from
the contact birds by sampling ceca (including cecal ton-
sils) at 48 h, 7 d, and 14 d, as explained below.
Salmonella Recovery
In experiment 1 and 2, chickens were humanely killed
by CO2 asphyxiation; ceca-ceca tonsils were aseptically
removed to culture and enumerate Salmonella. Briefly,
samples were placed in 10 mL of Tet for enrichment and
incubated at 37°C for 24 h. Samples were then plated
on BGA NO and NA plates and incubated at 37°C for
24 h to confirm presence/absence of typical lactose-
negative colonies of Salmonella. In experiment 3, ceca
were homogenized and diluted with saline (1:4 by wt/
vol) and 10-fold dilutions were plated on BGA with NO
and NA, incubated at 37°C for 24 h to enumerate total
Salmonella cfu. Later, the cecal samples were enriched
in double-strength Tet and further incubated at 37°C
for 24 h. Following this, ceca enrichment samples were
plated on BGA NO and NA plates and incubated at
37°C for 24 h to confirm presence/absence of typical
lactose-negative colonies of Salmonella.
Intestinal Morphological Analysis
For enteric morphometric analysis, birds on the des-
ignated evaluation day were euthanized, and ileum and
duodenum samples were collected (n = 5). A 1-cm seg-
ment of the midpoint of the duodenum and the distal
end of the lower ileum from each bird was removed
and fixed in 10% buffered formaldehyde for 48 h. Each
of these intestinal segments was embedded in paraffin,
and a 5-μm section of each sample was placed on a
glass slide and stained with hematoxylin and eosin for
examination under a light microscope. All morphologi-
cal parameters were measured using the ImageJ soft-
ware package (http://rsb.info.nih.gov/ij/). Ten repli-
cate measurements for each variable studied were taken
from each sample, and the average values were used in
statistical analysis. Villus length was measured from
the top of the villus to the top of the lamina propria.
Crypt depth was measured from the base upward to the
region of transition between the crypt and villus (Ap-
 EFFECTS OF EARLY FEEDING IN COMBINATION WITH PROBIOTICS 2341
Table 1. Evaluation of EarlyBird (EB; Pacific Vet Group USA Inc., Fayetteville, AR) associated with FloraMax B-11 (FM; Pacific
Vet Group USA Inc.) on Salmonella Enteritidis intestinal colonization at 24 and 72 h in broiler chickens from experiment 11
Trial 1 Trial 2 Trial 3

Group 24 h 72 h 24 h 72 h 24 h 72 h

Control 14/20 (70) 13/20 (65) 13/20 (65) 13/20 (65) 17/20 (85) 17/20 (85)
EB and FM 1/20 (5)* 5/20 (25)* 4/20 (20)* 4/20 (20)* 1/20 (5)* 6/20 (30)*
1For each trial, day-of-hatch off-sex broiler chickens were obtained and randomly distributed into 2 separate groups (n = 100). Chickens were kept
under simulated commercial hatching management and shipping conditions of 72 h. In each trial, both groups were challenged with Salmonella Enter-
itidis at a dose of 104 cfu/bird. One hour postchallenge, the treatment groups received an EB + FM mixture. Twenty chickens from each group were
humanely killed and cultured at 24 and 72 h, respectively, for Salmonella Enteritidis recovery in ceca (including cecal tonsils). Salmonella incidence
was expressed as positive/total birds (% in parentheses).
*Within a column, an asterisk indicates a significant difference at P < 0.05.

tekmann et al., 2001). Villus width was measured at the
widest area of each villus, whereas the villus:crypt ratio
was determined as the ratio of villus height to crypt
depth. Villus surface area was calculated using the for-
mula (2π)(VW/2)(VL), where VW = villus width and
VL = villus length (Sakamoto et al., 2000).
Statistical Analysis
Any statistical differences in BW, BWL, BWG, log10
Salmonella Enteritidis cfu/g of ceca, and morphomet-
ric measurements were determined by ANOVA using
PROC GLM using commercial SAS statistical software
(SAS Institute, 2002). Significant differences, set at P
< 0.05, were further separated using Duncan’s multi-
ple-range test. The percent recovery of Salmonella was
compared using the chi-squared test of independence
testing all possible group combinations to determine
significance for these studies (Zar, 1984).
RESULTS
The results of experiment 1 (3 trials), evaluating the
effects of EB associated with FM against Salmonella
Enteritidis intestinal colonization at 24 and 72 h in
broiler chickens, are summarized in Table 1. Significant
differences (P < 0.05) in the rate of intestinal coloni-
zation of Salmonella Enteritidis at 24 and 72 h were
observed in the groups that received EB + FM treat-
ments. The Salmonella Enteritidis incidence (positive/
total chickens) results analyzed from treated groups
were consistently significant in all 3 independent trials
compared with the control groups.
Table 2 summarizes the results of experiment 2, eval-
uating the effects of EB + FM treatments on intesti-
nal colonization of Salmonella Enteritidis followed by
its effects on horizontal transmission at 24 h in broiler
was observed between control and EB + FM treated
groups.
Table 3 summarizes the results of the evaluation of
effects of EB associated with FM on BW and perfor-
mance of broiler chickens in experiment 3, and it is
explained below as per sampling time points. At 24
and 48 h, BW of EB + FM groups were significantly
higher compared with those of nontreated controls and
FM-only groups. Significant BWL were observed with
control and FM-only groups compared with the groups
that received the perinatal supplements (EB, and EB +
FM). These differences were maintained at 7 and 14 d
of evaluation, with significantly higher BWG seen with
groups treated with EB only and EB + FM. The BW
of the EB + FM treated group was, on average, about
15 g heavier than that of nontreated control groups.
The results of effects of EB associated with FM on
morphological development of mucosa in the duode-
num of broiler chickens, evaluated in experiment 3,
are summarized in Table 4. Significantly and in some
cases numerically increased villus height, villus width,
villus:crypt ratio, and villus surface area index were ob-
served with groups treated with EB only and EB + FM
groups, when compared with nontreated control groups
at 24 h. It was interesting to note that significant dif-
Table 2. Evaluation of EarlyBird (EB; Pacific Vet Group USA
Inc., Fayetteville, AR) associated with FloraMax B-11 (FM; Pa-
cific Vet Group USA Inc.) on horizontal transmission of Salmo-
nella Enteritidis intestinal colonization at 24 h in broiler chickens
from experiment 21
Ceca Ceca
Treatment seeders contacts
Control 5/20 (25) 7/20 (35)
EB and FM 3/20 (15) 2/20 (10)*
1Day-of hatch off-sex broiler chickens were obtained and randomly

chickens. The Salmonella Enteritidis incidence (posi-
tive/total chickens) results analyzed between control
and treated groups showed no significant differences in
the rate of intestinal colonization of Salmonella Enter-
itidis in seeder chickens. However, a significant differ-
ence (P < 0.01) in the rate of horizontal transmission of
Salmonella Enteritidis from seeders to contact chickens
distributed into 2 separate groups (n = 100). Twenty birds in each group
were challenged with Salmonella Enteritidis at a dose of 104 cfu/bird
(seeders). One hour postchallenge, the treatment group received EB +
FM mix. All birds were humanely killed and cultured at 24 h postchal-
lenge for Salmonella Enteritidis recovery in ceca (including the cecal
tonsils). Salmonella incidence was expressed as positive/total birds (%
in parentheses).
*Within a column, an asterisk indicates a significant difference at P
< 0.05.
2342 Biloni et al.

ferences in villus surface area index were observed with

at a dose of 104 cfu/bird (seeders). One hour postchallenge, treatments were administered according to groups: 1, received no treatment; 2, received 200 g of FM only; 3, received 200 g of EB only; and
Table 3. Evaluation of EarlyBird (EB; Pacific Vet Group USA Inc., Fayetteville, AR) associated with FloraMax B-11 (FM; Pacific Vet Group USA Inc.) on BW and BW loss

hundred day-of-hatch off-sex broiler chickens were obtained and randomly distributed into 4 separate groups (n = 100). Twenty birds in each group were challenged with Salmonella Enteritidis

4 received the combination of EB + FM (2:1 wt/wt). All birds were weighed at 24 h, 48 h, 7 d, and 14 d of age. Recorded BW were then used to determine BWL at 24 and 48 h or BW gain (BWG) at
FM-only group when compared with controls, but these

5.89b
5.02b
3.61a
3.48a
BWG (g)
were numerically, if not significantly, less from that of

at 14 d

±
±
±
±
EB-only and EB + FM groups. The trend of significant

183.68
188.60
215.12
230.43
morphometric changes were observed throughout the
study in both duodenum and distal ileum samples, as
summarized in Tables 4 and 5.
Table 6 summarizes the results of the effects of EB

5.06b
3.86b
5.16a
5.40a
associated with FM against Salmonella Enteritidis in-
BW (g)
at 14 d

±
±
±
±
303.29 testinal colonization at 48 h, 7 d, and 14 d, evaluated in
307.34
315.65
322.57
experiment 3. A significant reduction in the Salmonella
Enteritidis recovery, evaluated as log10 Salmonella En-
teritidis per gram of ceca content, was observed at 48 h,
7 d, and 14 d of evaluation, in groups that received the
2.48b
3.36b
2.56a
2.96a
BWG (g)

probiotic treatments compared with the nonprobiotic

at 7 d

±
±
±
±

groups. However, significant reduction in the incidence

73.20
74.10
79.28
80.39

and total number of positive chickens to Salmonella En-

teritidis in the enriched cecal samples were observed
only at 48 h and 14 d in groups that received probiotic
1.78b
1.78b
1.84a
3.58a

treatment (FM only and EB + FM) compared with

BW (g)

nontreated control or EB-only groups.

at 7 d

±
±
±
±
118.79
119.08
124.52
125.92

DISCUSSION
A fasting period of 24 to 72 h after hatch is a com-
0.68b
1.77b
0.97a
0.27a

mon practice in commercial poultry operations (Dib-

BWL (g)
at 48 h

ner et al., 1998) due to variation in hatching time and

±
±
±
±

management in the hatchery. This delay in start of

−7.99
−7.53
−5.12
−3.93

feed intake has been shown to negatively affect yolk

utilization (Noy and Sklan, 1999), gastrointestinal de-
velopment (Noy and Sklan, 1998; Uni et al., 1999b),
0.60b
0.20b
0.20a
0.35a

slaughter weight (Halevy et al., 2000), and breast meat

BW (g)
at 48 h

yield (Noy and Sklan, 1999; Halevy et al., 2000). In

±
±
±
±

addition, delayed feeding seems to depress immunologi-

37.88
38.18
40.20
41.88
within columns with no common superscript differ significantly at P < 0.05.

7 and 14 d of age. The BW, BWL, and BWG (g) data were expressed as mean ± SE.

cal development (Dibner et al., 1998; Juul-Madsen et

al., 2004). The immediate posthatch period is critical
for intestinal morphological development to digest feed
0.13b
0.11b
0.11a
0.24a

and assimilate nutrients (Noy and Sklan, 1999; Bigot

BWL (g)
at 24 h

et al., 2003).
±
±
±
±
−3.91
−3.41
−2.32
−1.91

Our overall objective for this series of experiment was

to evaluate if the combination of neonatal feeding (EB)
and neonatal probiotic exposure (FM) would work in
combination and affect poultry health and performance
0.21ab
0.17b
0.21b

0.30a

significantly.
BW (g)
at 24 h
(BWL) of broiler chickens from experiment 31

The primary objective of experiment 1 was to confirm

±
±
±
±
41.96
42.13
43.40
43.70

if hydrated EB can be used as a delivery method for

FM probiotic, instead of the manufacturer-suggested
drinking water application. As a secondary objective,
we also evaluated the effect of such a combination on
0.23a
0.19a
0.23a
0.33a
Initial BW

cecal colonization of Salmonella on neonatal chicks with

(g)

±
±
±
±

just incidence studies. So in experiment 1, we evaluated

45.87
45.53
45.72
45.61

the effects of the combination of FM with EB, admin-

istered once to chickens under simulated commercial
hatching management and shipping conditions. The
products were effective in reducing Salmonella Enter-
EB and FM

itidis cecal colonization in 3 independent experiments

a,bValues
Treatment

(Table 1), demonstrating that a combination of early

Control

1Four

feeding along with probiotic for early microbial coloni-

FM
EB

zation can be beneficial in more than one aspect. Our

1.
2.
3.
4.
 EFFECTS OF EARLY FEEDING IN COMBINATION WITH PROBIOTICS 2343
Table 4. Evaluation of EarlyBird (EB; Pacific Vet Group USA Inc., Fayetteville, AR) associated with FloraMax B-11 (FM; Pacific
Vet Group USA Inc.) on morphometric analysis of the duodenal mucosa of broiler chickens from experiment 31
Villus height Crypt depth Villus width Villus:crypt Villus surface area
Treatment (µm) (µm) (µm) ratio index (mm2)

24 h
  1. Positive control 632.4 ± 14.2c 62.7 ± 80.9a 160.2 ± 12.8c 9.8 ± 0.1b 142.5 ± 82.81c
  2. FM 713.9 ± 11.4b 61.2 ± 90.3a 174.9 ± 10.9b 12.1 ± 0.2a 182.8 ± 10.8b
  3. EB 791.2 ± 15.6a 61.7 ± 12.1a 200.2 ± 16.7a 11.5 ± 0.3a 236.3 ± 14.3a
  4. EB and FM 773.2 ± 73.62a 62.1 ± 77.6a 207.9 ± 18.2a 11.4 ± 0.5a 190.5 ± 16.0b
48 h
  1. Positive control 616.5 ± 15.8c 87.0 ± 75.2a 145.7 ± 36.9c 6.7 ± 0.7b 202.8 ± 14.4a
  2. FM 681.9 ± 94.3b 91.9 ± 50.7a 149.8 ± 63.8c 7.4 ± 0.6b 191.6 ± 10.5a
  3. EB 724.4 ± 18.8a 95.6 ± 53.2a 158.2 ± 80.3b 7.9 ± 0.4ab 195.9 ± 11.9a
  4. EB and FM 757.5 ± 72.5a 93.9 ± 79.5a 167.8 ± 13.0a 8.2 ± 0.8a 210.7 ± 19.3a
7d
  1. Positive control 679.9 ± 14.5c 126.5 ± 51.6b 139.0 ± 12.5b 5.6 ± 0.9a 219.1 ± 18.6b
  2. FM 723.3 ± 12.8b 130.9 ± 40.9a 149.8 ± 86.6a 5.4 ± 0.7a 260.5 ± 21.1a
  3. EB 825.6 ± 99.1a 135.2 ± 23.5a 158.2 ± 17.3a 6.2 ± 0.6a 318.4 ± 15.4a
  4. EB and FM 812.4 ± 56.9a 132.9 ± 50.1a 167.3 ± 86.0a 6.4 ± 0.4a 330.9 ± 24.5a
14 d
  1. Positive control 876.7 ± 17.0c 147.5 ± 70.4c 140.8 ± 12.5c 5.9 ± 0.8a 338.4 ± 20.1c
  2. FM 896.8 ± 40.6b 155.0 ± 70.7b 149.1 ± 24.5bc 5.8 ± 0.6a 381.8 ± 21.5b
  3. EB 903.8 ± 53.7b 160.5 ± 32.7a 158.2 ± 90.3a 6.1 ± 0.5a 405.6 ± 23.0ab
  4. EB and FM 948.7 ± 83.9a 160.1 ± 37.8a 161.8 ± 19.2a 6.4 ± 0.9a 438.5 ± 24.5a
a–cValues within columns with no common superscript differ significantly at P < 0.05.
1Four hundred day-of-hatch off-sex broiler chickens were obtained and randomly distributed into 4 separate groups (n = 100). Twenty birds in each
group were challenged with Salmonella Enteritidis at a dose of 104 cfu/bird (seeders). One hour postchallenge, treatments were administered according
to groups: 1, received no treatment; 2, received 200 g of FM only; 3, received 200 g of EB only; and 4, received the combination of EB + FM (2:1 wt/
wt) mix. Five duodenum samples from each group were collected for enteric morphometric analysis at all time points. Values were expressed as means
± SEM representing 5 birds/group and 10 measurements/parameter per bird.

laboratory has published numerous studies that have
confirmed the prophylactic and therapeutic properties
of FM against Salmonella infections in poultry under
both, laboratory or commercial conditions, with gavage
and drinking water applications (Higgins et al., 2007,
2008, 2011; Vicente et al., 2007, 2008; Wolfenden et
al., 2007a; Menconi et al., 2011; Tellez et al., 2012). To
conclusively prove the mode of delivery, at least with
respect to neonatal feeding, we considered 3 repetitions
for experiment 1. The significant reduction on ceca

Table 5. Evaluation of EarlyBird (EB; Pacific Vet Group USA Inc., Fayetteville, AR) associated with FloraMax B-11 (FM; Pacific
Vet Group USA Inc.) on morphometric analysis of the ileum mucosa of broiler chickens from experiment 31
Villus height Crypt depth Villus width Villus:crypt Villus surface area
Treatment (µm) (µm) (µm) ratio index (mm2)

24 h
  1. Positive control 170.4 ± 20.3d 55.6 ± 37.6b 89.4 ± 27.2b 3.1 ± 0.5b 20.4 ± 1.3b
  2. FM 218.9 ± 54.5c 59.6 ± 15.9b 87.9 ± 12.5b 3.6 ± 0.4b 25.7 ± 3.3b
  3. EB 340.7 ± 36.9a 60.9 ± 26.3a 90.8 ± 28.6a 5.5 ± 0.6a 41.4 ± 7.8a
  4. EB and FM 308.2 ± 17.7b 62.4 ± 22.2a 84.7 ± 14.7b 4.9 ± 0.8a 34.9 ± 5.1a
48 h
  1. Positive control 244.5 ± 59.7c 76.1 ± 30.5c 72.1 ± 74.6a 3.8 ± 0.3a 23.6 ± 7.1b
  2. FM 363.5 ± 27.3b 84.7 ± 21.0a 45.8 ± 92.8b 4.2 ± 0.5a 22.3 ± 5.2b
  3. EB 332.7 ± 69.5a 82.0 ± 26.9b 54.5 ± 98.1b 4.1 ± 0.7a 24.2 ± 8.1b
  4. EB and FM 333.1 ± 62.0b 82.1 ± 29.0b 69.8 ± 98.7a 4.2 ± 0.9a 31.1 ± 7.7a
7d
  1. Positive control 270.9 ± 30.3c 89.8 ± 22.2d 47.0 ± 96.6a 3.1 ± 0.1a 16.1 ± 6.1b
  2. FM 376.1 ± 21.4a 97.9 ± 38.1c 42.8 ± 98.4a 3.8 ± 0.8a 21.5 ± 4.9a
  3. EB 347.7 ± 62.1b 100.0 ± 43.0b 32.8 ± 87.6a 3.5 ± 0.7a 15.2 ± 7.1b
  4. EB and FM 359.8 ± 57.9b 105.5 ± 40.4a 21.7 ± 60.8a 3.4 ± 0.5a 14.4 ± 8.2b
14 d
  1. Positive control 351.3 ± 52.4b 106.6 ± 40.5c 28.5 ± 81.7a 3.2 ± 0.5a 12.4 ± 7.2b
  2. FM 434.1 ± 94.1a 116.7 ± 52.4b 27.0 ± 81.0a 3.7 ± 0.5a 15.7 ± 8.1a
  3. EB 431.9 ± 93.2a 117.4 ± 33.5a 29.1 ± 61.3a 3.6 ± 0.5a 16.8 ± 7.5a
  4. EB and FM 425.2 ± 94.2a 117.4 ± 38.6a 33.2 ± 30.4a 3.8 ± 0.5a 18.9 ± 9.0a
a–dValues within columns with no common superscript differ significantly (P < 0.05).
1Four hundred day-of-hatch off-sex broiler chickens were obtained and randomly distributed into 4 separate groups (n = 100). Twenty birds in each
group were challenged with Salmonella Enteritidis at a dose of 104 cfu/bird (seeders). One hour postchallenge, treatments were administered according
to groups: 1, received no treatment; 2, received 200 g of FM only; 3, received 200 g of EB only; and 4, received the combination of EB + FM (2:1 wt/
wt) mix. Five ileum samples from each group were collected for enteric morphometric analysis, at all time points. Values were expressed as means ±
SEM representing 5 birds/group and 10 measurements/parameter per bird.
2344 Biloni et al.
Table 6. Evaluation of EarlyBird (EB; Pacific Vet Group USA Inc., Fayetteville, AR) associated with FloraMax B-11 (FM; Pacific
Vet Group USA Inc.) on Salmonella Enteritidis intestinal colonization at 48 h, 7 d, and 14 d in contact broiler chickens from experi-
ment 31
48 h 7d 14 d

Log10 Salmonella Log10 Salmonella Log10 Salmonella

Enteritidis/g of Enteritidis/g of Enteritidis/g of
Treatment Ceca ceca content Ceca ceca content Ceca ceca content

1. Positive control 12/12 (100) 4.60 ± 0.22a 12/12 (100) 4.72 ± 0.14a 12/12 (100) 4.42 ± 0.13a
2. FM 4/12 (33.3)* 0.69 ± 0.47b 12/12 (100) 2.68 ± 0.07b 2/12 (16.6)* 1.93 ± 0.36b
3. EB 12/12 (100) 4.53 ± 0.42a 12/12 (100) 4.52 ± 0.10a 11/12 (92) 4.11 ± 0.16a
4. EB and FM 5/12 (41.6)* 1.79 ± 0.38b 12/12 (100) 2.58 ± 0.06b 4/12 (33.3)* 1.78 ± 0.32b
a,bValues
within columns with no common superscript differ significantly (P < 0.05).
1Four
hundred day-of-hatch off-sex broiler chickens were obtained and randomly distributed into 4 separate groups (n = 100). Twenty birds in each
group were challenged with Salmonella Enteritidis at a dose of 104 cfu/bird (seeders). One hour postchallenge, treatments were administered according
to groups: 1, received no treatment; 2, received 200 g of FM only; 3, received 200 g of EB only; and 4, received the combination of EB + FM (2:1 wt/
wt) mix. Salmonella incidence in ceca (including cecal tonsils) from contact birds was expressed as positive chickens to Salmonella Enteritidis/total
chickens cultured (% in parentheses). Salmonella Enteritidis recovery, at 48 h, 7 d, and 14 d was expressed as log10 Salmonella Enteritidis/g of ceca
content, as mean ± SE.
*Within a column, an asterisk indicates a significant difference at P < 0.05.

colonization rate of Salmonella Enteritidis observed in
experiment 1 confirmed that EB can indeed be a good
delivery method for FM with respect to neonatal chick-
ens.
Because experiment 1 proved the efficacy of this de-
livery method (EB + FM), in experiment 2 we evalu-
ated the effect of same mode of delivery on horizontal
transmission of Salmonella. Similar positive effects were
also observed on the horizontal transmission of Salmo-
nella Enteritidis with significant reductions (Table 2).
However, the effect of this combination on horizontal
transmission of Salmonella has already been tested in
our laboratory by Wolfenden et al. (2007b) and Men-
coni et al. (2011). The results of experiment 2 in this
study were very similar to our previous studies men-
tioned. However, the aim of published previous studies
was to evaluate the effect of the combination of EB +
FM only on horizontal transfer of the Salmonella, but
not on the cecal colonization in neonatals. Hence, we
did not consider the necessity of conducting another
trial in this regard.
Instead, we decided to repeat and extend the evalu-
ation of the effects of EB + FM on cecal colonization
of Salmonella, which was just a secondary objective
of experiment 1. So, experiment 3 included the same
challenge model used for horizontal transmission ex-
periment, with an Salmonella Enteritidis dose of 104/
chick in seeder birds, to evaluate any significant reduc-
tion on ceca colonization rate of Salmonella Enteritidis.
A significant log10 reduction in Salmonella Enteritidis
cfu/g of ceca content was observed at 48 h, 7 d, and 14
d (experiment 3), suggesting a reduction in Salmonella
Enteritidis shedding into the environment (Table 6)
that is in agreement with our previous reports (Tellez
et al., 2001; Farnell et al., 2006; Higgins et al., 2007,
2008, 2011; Vicente et al., 2007, 2008; Wolfenden et al.,
2007a,b). The results of this study confirmed that early
feeding had a significant impact in villus height, vil-
lus width, villus:crypt ratio, and villus surface area in-
dex (Tables 4 and 5), as has been previously described
by several investigators (Cook and Bird, 1973; Uni et
al., 1999b, 2003a,b; Bigot et al., 2003; Yi et al., 2005).
In experiment 3, 24 h of fasting (water and feed) was
enough time to affect the morphology of the intestine,
particularly in the control (nontreated) chickens. Even
though the groups that received the perinatal supple-
ment showed a significant increase in villus height, vil-
lus width, villus:crypt ratio, and villus surface area in-
dex compared with the control or probiotic groups at
24 h, 48 h, 7 d, and 14 d, it was interesting to observe
that the group that received the probiotic in spray had
an improvement in the morphology variables compared
with the control nontreated group at the same days
of evaluation. These significant morphometric changes
were maintained and observed throughout the study
with duodenum and distal ileum development (Tables
4 and 5), which were also associated with a significant
increase in the performance of the chickens (Table 3).
The use of EB during chick transportation significantly
reduced BWL during the simulated shipping period of
48 h and resulted in greater BW and BWG at 7 and 14
d (Table 3). Again we did not consider a repetition for
this trial, the answer for which is 2-fold. First, experi-
ment 3 evaluated and conclusively proved the hypoth-
esis on a much larger scale, involving a timeline study
(24 h, 48 h, 7 d, and 14 d) clearly supported by the
histological studies. Second, results from experiment 1,
even though an incidence study, would complement the
results of experiment 3 and act as a secondary proof for
the efficacy of EB + FM in reducing Salmonella Enter-
itidis recovery in ceca (Tables 2 and 6).
We can further contend these experiments are sup-
ported by numerous previously published results, eval-
uating each individual component, be it effects of EB
on BW and BWL of broiler chickens (Henderson et
al., 2008) or FM on Salmonella colonization individu-
ally. Experiment 3 has confirmed these findings, but
in combination, indicating the compounded effects of
 EFFECTS OF EARLY FEEDING IN COMBINATION WITH PROBIOTICS
early feeding along with early probiotic exposure, with
a 3-fold benefit involving BW, gut health, and preven-
tion of Salmonella colonization.
In conclusion, holding of neonatal chicks or poults is
often inevitable. Our data indicate that early provision
of an exogenous feed and water supplement resulted
in greater BW and BWG. Providing EB as an early
feeding supplement during simulated shipping result-
ed in increased performance. Although early feeding
supplementation does not completely compensate for
the delayed exposure to feed and water, providing an
early feeding supplement may help alleviate the nega-
tive effects of delayed feeding. The results of this study
demonstrated that the combination of EB and FM im-
proved gut morphology (villus height, width, surface
area, and villus:crypt ratio), reduced the amount of
Salmonella that could be recovered, as well as improved
BW compared with controls and each product individ-
ually. These data are relevant to the poultry industry
and address both animal welfare and food safety con-
cerns faced by the poultry industry.
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