PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

Influence of dietary inclusion of Bacillus licheniformis on laying performance,

egg quality, antioxidant enzyme activities, and intestinal barrier
function of laying hens

K. Lei,1 Y. L. Li,1 D. Y. Yu,1 I. R. Rajput,1 and W. F. Li1,2

Key Laboratory of Molecular Feed Sciences, Institute of Animal Nutrition and Feed Sciences,
College of Animal Science, Zhejiang University, Zhejiang, China 310058

ABSTRACT This experiment was conducted to evalu-
ate the effects of dietary inclusion of Bacillus licheni-
formis on laying performance, egg quality, antioxidant
enzyme activities, and intestinal barrier function of lay-
ing hens. Hy-Line Variety W-36 hens (n = 540; 28 wk
of age) were randomized into 6 groups, each group with
6 replications (n = 15). The control group received the
basal diet formulated with maize and soybean meal.
The treatment groups received the same basal diets
supplemented with 0.01, 0.02, 0.03, 0.06, and 0.09%
Bacillus licheniformis powder (2 × 1010 cfu/g) for an
8-wk trial. The results showed that dietary supplemen-
tation with 0.01 and 0.03% B. licheniformis significant-
ly increased egg production and egg mass. However,
no significant differences were observed in egg weight,
feed consumption, and feed conversion efficiency among
Key words: Bacillus licheniformis, laying performance, egg quality, hormone
the 6 groups. Supplementation with different levels of
B. licheniformis was found to be effective in improve-
ment of egg quality by increasing egg shell thickness
and strength. Compared with control, d-lactate con-
tent, diamine oxidase activity, and adrenocorticotropic
hormone level in serum decreased significantly, and the
level of estradiol and follicle-stimulating hormone in-
creased significantly in plasma of all the experimental
groups. Dietary supplementation with B. licheniformis
increased the intestinal villus height and reduced the
crypt depth. In conclusion, dietary inclusion of B. li-
cheniformis could improve laying performance and egg
quality significantly in a dose-dependent manner by de-
creasing the stress response, upregulating the growth
hormone, and improving intestinal health.
2013 Poultry Science 92:2389–2395
http://dx.doi.org/10.3382/ps.2012-02686

INTRODUCTION Probiotics are nonpathogenic bacteria that exert a ben-

Antibiotics can improve health and productive per-
formance of animals, but also result in the development
of drug-resistant microorganisms, which can then pass
the resistance on to infectious microorganisms in hu-
mans (Li et al., 2006). The European Union has banned
the use of antibiotics as growth-promoting agents in
the poultry industry, and many countries are restrict-
ing the use of antibiotic as growth-promoting agents.
Therefore, it is essential to find possible alternatives to
antibiotics for growth promotion and improvement in
poultry production. Since Tortuero first applied a pro-
biotic (Lactobacillus acidophilus) as an alternative to
antibiotics in poultry (Tortuero, 1973), application of
probiotics as feed additives has been persistently tested
and used in a variety of poultry production settings.
©2013 Poultry Science Association Inc.
Received August 15, 2012.
Accepted May 10, 2013.
1 All the authors of this research contributed equally.
2 Corresponding author: wfli@zju.edu.cn
eficial influence on health, physiology, or both of the
host (Rajput et al., 2013), which can improve intestinal
structure, aid in development of immunity to defend
against pathogens, and subsequently improve growth
performance. It also has been shown that application
of probiotics could improve weight gain and feed con-
version rate and reduce mortality in broiler chickens
(Tortuero, 1973; Jin et al., 1998; Kurtoglu et al., 2004;
Lutful Kabir, 2009). Supplementation of probiotics in a
basal diet has been shown to be useful for ameliorating
the adverse influence of stress (Deng et al., 2012), pro-
moting the activities of antioxidant enzymes (Rajput et
al., 2012), and improving the health of the host beyond
their inherent basic nutrition (Fuller, 1989). Recently,
studies have shown that supplementation of probiot-
ics has the ability to inhibit the adhesion of patho-
genic bacteria to the intestinal wall and to enhance
immune potency (Balevi et al., 2001), which suggests
that probiotics have a significant role in normalization
of colonic physiologic function and barrier integrity of
conjunctions of the cells with a reduction in mucosal

2389
2390 Lei et al.

pro-inflammatory cytokine levels (Mack et al., 1999; Table 1. Composition and nutrition of the basal experimental
Madsen et al., 2001). diet (%) to which Bacillus was added1
Several selected probiotics have been applied in poul- Item Value
try production including Lactobacillus, Streptococcus,
Ingredient
Saccharomyces, Aspergillus, and Bacillus species (Tan-  Maize 60.20
nock, 2001). Bacillus species are ideally suited as feed   Soybean meal 24.00
additives because of their stability as spore-forming   Wheat bran 3.11
  Fish meal 2.24
bacteria and ability to produce a variety of enzymes  CaHPO4 1.00
such as protease, amylase, and lipase (Hagedorn et al.,  Limestone 7.00
1985). Diets supplemented with Bacillus subtilis, Ba-  Salt 0.33
  dl-Methionine 0.11
cillus subtilis fermented product, or probiotic powder   l-Lysine 0.01
can improve weight gain and feed efficiency (Santoso et   Soybean oil 1.00
al., 1995; Li et al., 2006). Bacillus licheniformis has a  Premix2 1.00
Calculated value
strong ability to produce protease, lipase, and amylase,   ME (MJ/kg) 11.50
which facilitates the degradation of feed for nutrient,   CP (g/kg) 187
absorption, and utilization of feed (Rozs et al., 2001).   Calcium (%) 3.34
  Total phosphorus (%) 0.59
It is reported that B. licheniformis can produce antimi-   Available phosphorus (%) 0.38
crobial active substances and has a unique mechanism   Lysine (%) 1.04
about reacting with oxygen that inhibits the growth   Methionine + cysteine (%) 0.75
and reproduction of pathogens (Kim et al., 2004), and 1Bacillus licheniformis cells were suspended in skim milk powder (2 ×

promotes the growth and homeostasis of the intestine
to adjust the intestinal flora for the recovery of bowel
functions. However, little research has been conducted
on the use of B. licheniformis in laying hens; therefore,
1010 cfu/g); B. licheniformis powders (2 × 1010 cfu/g) were added to the
basal diet at 0 (control), 0.01% (2 × 106 cfu/g of diet), 0.02% (4 × 106
cfu/g of diet), 0.03% (6 × 106 cfu/g of diet), 0.06% (1.2 × 107 cfu/g of
diet), and 0.09% (1.8 × 107 cfu/g of diet) level.
2Premix provided per kilogram of diet: retinyl palmitate, 3.96 mg;

a dearth of information about the effects of probiotics
in vivo on laying hens inspired us to focus on the issue.
Thus, the objectives of this study were to investigate
the effect of dietary supplemental B. licheniformis on
laying performance and egg quality, serum hormone,
antioxidant enzyme activities, and intestinal barrier
functions.
MATERIALS AND METHODS
Birds and Management
A total of 540 Hy-Line Variety W-36 hens, 28 wk of
age, were randomly divided into 6 groups, each of which
had 6 replicates of 15 hens. Three hens were housed per
cage under the same management conditions in a win-
dowed poultry house. This trial lasted from 28 to 38
wk of age, including a 2-wk acclimatization period and
8-wk experimental period. During the experimental pe-
riod, birds were fed the diets ad libitum twice daily at
0800 and 1600 h and allowed free access to water with a
photoperiod of 16L:8D. The average ambient RH inside
the barn was 55 ± 5% and the mean daily temperature
was 23 ± 2°C. The experiment was carried out in ac-
cordance with the Chinese guidelines for animal welfare
and approved by the animal welfare committee of Ani-
mal Science College, Zhejiang University.
Diets and Bacterial Strains
All hens were fed the same basal diet to which Ba-
cillus cultures were added to derive treatments (Table
1). Bacillus licheniformis cells were suspended in skim
milk powder (2 × 1010 cfu/g) by the Laboratory of
cholecalciferol, 0.07 mg; dl-α-tocopheryl acetate, 20 mg; menadione so-
dium bisulfite, 4 mg; thiamine mononitrate, 1.63 mg; riboflavin, 5 mg;
niacin, 30 mg; pantothenic acid, 10 mg; folic acid, 0.5 mg; biotin, 0.22
mg; choline chloride, 250 mg; cyanocobalamin, 0.012 mg; Cu, 8 mg; Fe,
30 mg; I, 0.6 mg; Mn, 50 mg; Se, 0.12 mg; Zn, 40 mg.
Microbiology, Institute of Feed Sciences, Zhejiang Uni-
versity, PR China. Bacillus licheniformis powders (2 ×
1010 cfu/g) were added to the basal diet at levels of 0
(control), 0.01% (2 × 106 cfu/g of diet), 0.02% (4 ×
106 cfu/g of diet), 0.03% (6 × 106 cfu/g of diet), 0.06%
(1.2 × 107 cfu/g of diet), and 0.09% (1.8 × 107 cfu/g
of diet). The experimental diet was stored in a dry and
well-ventilated storeroom.
Laying Performance and Egg Quality
Hen-day egg production, feed consumption, egg
weight, and hen mortality were recorded daily. Feed
conversion ratio was calculated as grams of feed in-
take per gram of egg mass. At 38 wk of age, 12 eggs
from each replicate were randomly collected to assess
egg quality parameters. Albumen height, Haugh units,
yolk color, eggshell thickness, and eggshell strength
were measured with a digital egg tester after eggs were
weighed and cracked open.
Blood Sampling
At the end of the experiment, after 12 h of feed with-
drawal, blood samples of 12 hens (2 birds per replicate)
were drawn from the axillary vein into vacuum tubes
(5 mL) containing coagulant and centrifuged for 10 min
(3,000 × g) at 4°C. Pure serum samples were aspirated
 DIETARY INCLUSION OF BACILLUS LICHENIFORMIS
by pipette, stored in sterilized 1.5-mL Eppendorf tubes
at −80°C, and thawed at 4°C before analysis.
Antioxidant Enzyme Estimation
Assay kits for superoxide dismutases (SOD), gluta-
thione (GSH), catalse (CAT), total antioxidant capac-
ity (T-AOC), malondialdehyde (MDA), glutathione
reductase (GR), glutathione S-transferase (GST), and
thioredoxin reductase protein (TrxR) were obtained
from the Nanjing Jiancheng Bioengineering Institute
(Nanjing, China). The GR, GST, TrxR, d-lactate, and
diamine oxidase levels were measured by ELISA with
commercial kits provided by Hangzhou Nuoyang Bio-
technology Co. Ltd. (Hangzhou, China), whereas SOD
(Winterbourn et al., 1975), GSH (Zakowski and Tap-
pel, 1978), CAT (Beers and Sizer, 1952), T-AOC (Erel,
2004), and MDA (Gomez et al., 1998) levels were mea-
sured by spectrophotometric methods using a spectro-
photometer.
Serum Hormone Determination
Concentrations of serum luteinizing hormone
(LH), progesterone (P), follicle-stimulating hormone
(FSH), estradiol (E2), adrenocorticotrophic hormone
(ACTH), and corticosterone (CORT) were measured
by ELISA with commercial kits provided by Hangzhou
Nuoyang Biotechnology Co. Ltd. (Hangzhou, China).
Histological Structure and Assessment
of Intestinal Function
One-centimeter lengths from the medial portions of
jejunum were washed in physiological saline solution
and fixed in 10% buffered formalin. Tissue samples
were later embedded in paraffin, and a 20-μm section
of each sample was placed on a glass slide and stained
with hematoxylin and eosin (Thompson and Richter,
1960). The villus was observed under a Nikon micro-
scope (Nikon Corp., Tokyo, Japan). Villus height was
measured from the top of the villus to the villus crypt
junction and crypt depth measured as the depth of the
invagination between adjacent villus (total of 5 samples
for intestinal segments and 30 measurements of each
intestinal segment per group).
2391
Statistical Analysis
Data were statistically analyzed by one-way ANOVA
procedure of SPSS 16.0 for Windows (SPSS Inc., Chi-
cago, IL). When significant differences were found (P <
0.05), Tukey’s test was further performed. Statements
of significance were based on P < 0.05. The data were
expressed as the means ± SEM.
RESULTS AND DISCUSSION
Laying Performance and Egg Quality
Dietary supplementation with different levels of B.
licheniformis did not show any adverse effects on laying
performance of hens. The results clearly showed that
all treatment groups had higher egg production and egg
mass output compared with the control through a 56-d
trial period (Table 2). Hens receiving B. licheniformis
at 0.01 and 0.06% had improved egg production over
hens fed the basal diet devoid of Bacillus. The highest
egg production (98.4%) was from the 0.01% B. licheni-
formis group, and the lowest (94.0%) from the con-
trol group. Although diets containing 0.02, 0.03, and
0.09% B. licheniformis resulted in intermediate hen egg
production, it has been shown that hens fed 250, 500,
and 750 mg of probiotic (3.2 × 109 cfu/g)/kg of diet
had improved egg production (Kurtoglu et al., 2004).
However, the impact of probiotics on hen egg produc-
tion varies as Li et al. (2006) and Yalçin et al. (2010)
showed no effect of probiotics on hen egg production.
Similar to egg production, hens fed 0.01 and 0.06% B.
licheniformis had higher egg mass than hens fed the
control diet, and hens fed diets containing 0.02, 0.03,
and 0.09% B. licheniformis had intermediate hen egg
mass. However, differences among treatments for egg
weight did not occur. There were also no significant
differences for feed consumption and feed-conversion ef-
ficiency among all 6 groups, which were in agreement
with previous findings (Goodling et al., 1987; Nahashon
et al., 1994; Mohan et al., 1995). In contrast, Nahashon
et al. (1994) showed that addition of probiotics could
improve feed consumption and decrease feed conversion
efficiency, whereas Thanh et al. (2009) showed that
feeding metabolite combinations produced by Lactoba-
cillus plantarum decreased feed conversion ratio.

Table 2. Laying performance of the hens fed diets without or with Bacillus licheniformis
B. licheniformis Egg production Egg weight Egg mass Feed consumption Feed conversion
(%) (%) (g) (g/hen per d) (g/hen per d) (g/g)

0 94.0 ± 0.4b 57.8 ± 0.8 54.3 ± 0.6b 112.8 ± 0.7 2.06 ± 0.03
0.01 98.4 ± 0.6a 57.9 ± 0.5 57.0 ± 0.8a 113.1 ± 4.1 1.99 ± 0.10
0.02 96.6 ± 0.6ab 57.9 ± 0.6 55.7 ± 0.5ab 116.6 ± 2.0 2.09 ± 0.03
0.03 96.3 ± 0.3ab 58.3 ± 0.3 56.1 ± 0.1ab 116.0 ± 2.0 2.06 ± 0.04
0.06 97.9 ± 0.2a 58.2 ± 0.4 57.0 ± 0.5a 112.8 ± 1.9 1.97 ± 0.03
0.09 96.1 ± 0.7ab 58.1 ± 0.1 55.8 ± 0.5ab 113.9 ± 0.1 2.04 ± 0.02
a,bDifferent superscripts in the same column indicate values significantly different (P < 0.05) among the groups.
2392 Lei et al.
Table 3. Internal and external quality of the eggs from hens fed diets without or with Bacillus licheniformis at different levels
B. licheniformis Albumen height Eggshell thickness Eggshell strength
(%) (mm) Yolk color Haugh units (mm) (N)

0 6.50 ± 0.15b 6.86 ± 0.09b 79.6 ± 1.4b 0.303 ± 0.004d 33.91 ± 0.08c
0.01 6.78 ± 0.16ab 7.00 ± 0.11ab 82.0 ± 1.4b 0.332 ± 0.004ab 38.12 ± 0.08ab
0.02 6.64 ± 0.12ab 6.95 ± 0.10ab 82.1 ± 0.7b 0.324 ± 0.003bc 37.44 ± 0.08ab
0.03 6.95 ± 0.12a 7.20 ± 0.10a 82.7 ± 1.0b 0.342 ± 0.005a 38.51 ± 0.09a
0.06 6.93 ± 0.13a 6.93 ± 0.11ab 86.5 ± 1.5a 0.327 ± 0.004bc 38.51 ± 0.10a
0.09 6.67 ± 0.11ab 6.51 ± 0.08c 81.6 ± 0.7b 0.319 ± 0.003c 36.85 ± 0.06ab
a–dDifferent superscripts in the same column indicate values significantly different (P < 0.05) among the groups.

Some dietary levels of B. licheniformis improved egg
quality, but responses were not dose dependent (Table
3). Compared with the control, albumen height was
significantly increased in 0.03 and 0.06% B. lichenifor-
mis group, whereas Haugh units increased only in the
0.06% group. This effect may be related to increased
protein synthesis and the transfer of water from yolk.
Our results differ from those of Aghaii et al. (2010),
who demonstrated that the addition of probiotics had
no effects on albumen height and Haugh units. The
thickness and strength of egg shell were significantly in-
creased in all B. licheniformis treated groups compared
with the control. The 0.09% B. licheniformis group,
among all the 5 treatments, showed the least improve-
ment in thickness and strength of eggshell. There were
no significant differences in the Haugh units and egg-
shell strength among all 5 treatment groups. That may
be associated with the ability of probiotics to decrease
pH and improve intestinal barrier function (Resta-Len-
ert and Barrett, 2003). Lower pH and intestinal epi-
thelium functions both are required for the dissolution
of calcium and phosphorus to promote absorption and
utilization (Li et al., 2006). Another study reported
that calcium and phosphorus retention were improved
in layers when the diet was supplemented with Lac-
tobacillus (Nahashon et al., 1994). The darkest yolk
color score was from the 0.03% B. licheniformis group,
whereas the lightest was from the control. The findings
are in agreement with those of Nahashon et al. (1994)
and Mohan et al. (1995), who reported that a slight im-
provement in yolk color was observed when hens were
supplemented with probiotics during the peak period
of laying. Because our treatments varied greatly, we
conclude that the impact of probiotics on egg yolk color
is unclear. The difference in laying performance and
egg quality among the various studies might be due to
the different probiotic species, trial length, and envi-
ronmental conditions.
Intestinal Barrier Function
d-Lactate is a byproduct of bacterial metabolism;
it is neither produced nor metabolized by mammalian
cells. The resident microflora in an ischemic segment of
bowel rapidly proliferate and overpopulate; consequent-
ly, the mucosal barrier of the gut begins to break down
(Murray et al., 1993). Plasma d-lactate levels may be a
useful marker to evaluate the degree of intestinal injury
and gut barrier dysfunction. Diamine oxidase (DAO)
is an enzyme found in high concentration in the intes-
tinal mucosa of humans and other mammalian species,
and its activity serves as a marker of mucosal matura-
tion and integrity (Luk et al., 1980). The DAO activity
in serum is a marker of the total mass of functional to
enterocytes; a decreased level during gastroenteritis is
reflected in a decrease of serum DAO activity (Forget
et al., 1985). This study showed that B. licheniformis
supplementation in the diet significantly decreased the
d-lactate level and diamine oxidase activity (Table 4).
The lowest values for d-lactate and diamine oxidase
were from the 0.01 and 0.03% B. licheniformis groups,
respectively. These changes are due to the ability of
probiotics to modulate the intestinal microbial environ-
ment in favor of the host.
The structure of the intestinal mucosa can reveal
some information on gut health. Increased villus height
indicates increased surface area for nutrient absorp-
tion, and deeper crypt indicates fast tissue turnover
and a high demand for new tissue. In the present study,
compared with the control, the B. licheniformis treated
groups showed better intestinal integrity (Figure 1).
Hence, the addition of different levels of B. lichenifor-

Table 4. Intestinal barrier function of laying hens fed diets without or with Bacillus licheniformis at
different levels
B. licheniformis d-Lactate Diamine oxidase Villus height Crypt depth
(%) (μmol/L) (ng/L) (μm) (μm)

0 5.3 ± 0.7b 761.6 ± 24.5c 211.2 ± 1.9c 45.7 ± 1.1b

0.01 2.2 ± 0.3a 337.1 ± 25.6a 228.8 ± 1.2b 31.1 ± 0.6d
0.03 2.6 ± 0.6a 320.3 ± 49.7a 232.5 ± 2.4b 41.7 ± 0.9c
0.06 2.7 ± 0.3a 483.6 ± 32.1b 286.0 ± 2.0a 49.6 ± 1.0a
a–dDifferent superscripts in the same column indicate values significantly different (P < 0.05) among the groups.
 DIETARY INCLUSION OF BACILLUS LICHENIFORMIS 2393

Figure 1. Structure of the intestinal mucosa of hens fed control versus hens fed various Bacillus licheniformis levels. Color version available
in the online PDF.

mis significantly increased villus height and decreased
the crypt depth (Table 4). Similarly, feeding of the me-
tabolite combination produced by Lactobacillus plan-
tarum increased small intestinal villus height (Thanh
et al., 2009). The improvement of the structure of the
intestinal mucosa in B. licheniformis-treatment groups
might lead to prolific nutrient absorption, and increase
disease resistance and decrease diarrhea-producing fac-
tors (Xu et al., 2003).
Serum Hormone
No differences were observed among the 6 groups for
CORT and LH (Table 5). The levels of E2 and FSH
in the probiotic-treated groups were increased signifi-
cantly, whereas ACTH level was significantly decreased
compared with the control. The level of P was signif-
icantly decreased in 0.01 and 0.06% B. licheniformis
supplemented groups compared with the control, but
the level of P was numerically improved in 0.03% B.
licheniformis group to the control level. The ACTH is
often produced in response to biological stress (Raikh-
instein and Hanukoglu, 1993). Follicle-stimulating hor-
mone is synthesized and secreted by gonadotrophs of
the anterior pituitary gland, which regulates the devel-
opment, growth, pubertal maturation, and reproductive
processes and stimulates the growth and recruitment of
immature ovarian follicles in the ovary (Radu et al.,

Table 5. Serum hormone levels of laying hens fed diets without or with Bacillus licheniformis at different levels1
B. licheniformis ACTH CORT E2 FSH LH P
(%) (ng/L) (ng/L) (ng/L) (mIU/mL) (mIU/mL) (ng/mL)

0 136.9 ± 10.5b 363.3 ± 12.2 21.0 ± 7.2b 2.7 ± 0.1b 5.6 ± 2.0 218.3 ± 40.0ab
0.01 87.2 ± 10.0a 218.8 ± 13.6 78.1 ± 8.8a 5.3 ± 0.6a 5.4 ± 0.4 106.4 ± 25.1c
0.03 75.9 ± 5.9a 327.0 ± 17.7 94.1 ± 20.7a 5.1 ± 0.6a 6.4 ± 1.9 235.4 ± 46.3a
0.06 95.4 ± 6.0a 205.5 ± 10.9 81.0 ± 17.0a 5.2 ± 0.2a 5.3 ± 1.2 119.5 ± 28.2bc
a–cDifferentsuperscripts in the same column indicate values significantly different (P < 0.05) among the groups.
1Concentrations of serum luteinizing hormone (LH), progesterone (P), follicle-stimulating hormone (FSH), estradiol (E2), adrenal cortical hormone
(ACTH), and corticosterone (CORT) were measured by ELISA with commercial kits provided by Hangzhou Nuoyang Biotechnollgy Co. Ltd. (Hang-
zhou, China).
2394 Lei et al.
Table 6. Antioxidant enzyme activities of laying hens fed diets without or with Bacillus licheniformis at different levels1
B. licheniformis CAT GR GST TrxR T-AOC SOD GSH MDA
(%) (U/mL) (ng/mL) (ng/mL) (U/L) (U/mL) (U/mL) (μmol/L) (nmol/mL)

0 19.8 ± 3.3 2.3 ± 0.5 2.8 ± 0.7c 42.8 ± 7.9 4.3 ± 0.5 462.9 ± 19.0 375.0 ± 49.6ab 10.3 ± 1.3
0.01 15.7 ± 0.7 1.8 ± 0.1 3.3 ± 0.2bc 45.9 ± 5.7 4.3 ± 0.6 457.9 ± 32.4 394.1 ± 5.1ab 8.1 ± 1.3
0.03 19.2 ± 2.2 2.6 ± 0.4 5.5 ± 0.7a 57.4 ± 9.4 3.0 ± 0.4 366.6 ± 21.7 535.0 ± 70.1a 11.4 ± 0.4
0.06 15.7 ± 2.1 2.1 ± 0.2 4.5 ± 0.3ab 48.8 ± 5.2 4.2 ± 0.7 392.3 ± 22.6 365.5 ± 40.0b 9.9 ± 0.4
a–cDifferent
superscripts in the same column indicate values significantly different (P < 0.05) among the groups.
1Catalase(CAT), glutathione reductase (GR), glutathione S-transferase (GST), thioredoxin reductase protein (TrxR), total antioxidant capacity
(T-AOC), superoxide dismutases (SOD), glutathione (GSH), and malondialdehyde (MDA).

2010). The lower level of ACTH implied that addition
of B. licheniformis could alleviate biological stress. The
increased levels of E2 and FSH resulting from addi-
tion of B. licheniformis may be related to lower serum
ACTH, which reduces inhibition of the reproductive
hormone secretion (Matteri et al., 1984). It was also
reported that increased serum ACTH inhibits gonado-
tropin secretion (Dobson and Smith, 1995).
Activity of Antioxidant Enzymes
The antioxidant defenses include antioxidants (vi-
tamin C, vitamin E, and uric acid) and antioxidant
enzymes present in the biological system (Sies, 1991).
Supplementation with 0.03 and 0.06% B. licheniformis
improved GST activity (Table 6) over the control, and
there were no differences for other antioxidant enzyme
activities among all groups. The GST is a soluble pro-
tein that catalyzes the conjugation of GSH with many
xenobiotics, and their reactive metabolites could form
more water-soluble compounds (Cai and Harrison,
2000). The finding of Matés (2000) showed that expres-
sion of GST was regulated by a common antioxidant re-
sponse element, which suggested that their gene prod-
ucts play a protective role against oxidative damage in
various tissues by neutralizing reactive oxygen species.
Consistent with the antioxidant enzyme activity of se-
rum, there was no significant effect of treatment on
T-AOC and MDA among groups, but a slight numeri-
cal decrease was observed in MDA contents. The MDA
endogenously reflects the lipid peroxidation (Yang
et al., 2008), which is the consequence of diminished
antioxidant protection when levels of reactive oxygen
species increase (Yang et al., 2008). The findings of
Capcarova et al. (2010) revealed that T-AOC in broiler
chickens was significantly increased in both Lactobacil-
lus fermentum and Enterococcus faecium supplemented
groups, but the activities of antioxidant enzymes were
not determined. The discrepancy among these studies is
likely due to the different animals, physiological status,
diet compositions, the probiotic source, and their ap-
plication levels. All together, our results demonstrated
that supplementation of B. licheniformis has no impact
on improving the antioxidant enzymes activities in lay-
ing hens except for GST.
In conclusion, inclusion of B. licheniformis in the hen
diet was effective in increasing egg production perfor-
mance of hens by improving villus structure, sustain-
ing a balanced intestinal barrier function, decreasing
the stress response, and regulating hormone secretion.
Therefore, the probiotic B. licheniformis may be useful
for ameliorating certain adverse influences on produc-
tion and gut health of laying hens.
ACKNOWLEDGMENTS
This study was supported by the Key Science and
Technology Program of Zhejiang Province, China (no.
2006C12086). The research was designed by W. F. Li;
Kai Lei, Y. L. Li, and I. R. Rajput conducted the re-
search; D. Y. Yu analyzed the data; and Kai Lei com-
pleted writing the paper.
REFERENCES
Aghaii, A., M. Chaji, T. Mohammadabadi, and M. Sari. 2010. The
effect of probiotic supplementation on production performance,
egg quality, and serum and egg chemical composition of lying
hens. J. Anim. Vet. Adv. 9:2774–2777.
Balevi, T., U. S. Ucan, B. Coskun, V. Kurtoglu, and I. S. Cetingul.
2001. Effect of dietary probiotic on performance and humoral im-
mune response in layer hens. Br. Poult. Sci. 42:456–461.
Beers, R. F., Jr., and I. W. Sizer. 1952. A spectrophotometric meth-
od for measuring the breakdown of hydrogen peroxide by cata-
lase. J. Biol. Chem. 195:133–140.
Cai, H., and D. G. Harrison. 2000. Endothelial dysfunction in cardio-
vascular diseases: The role of oxidant stress. Circ. Res. 87:840–
844.
Capcarova, M., J. Weiss, C. Hrncar, A. Kolesarova, and G. Pal.
2010. Effect of Lactobacillus fermentum and Enterococcus faecium
strains on internal milieu, antioxidant status and body weight of
broiler chickens. J. Anim. Physiol. Anim. Nutr. (Berl.) 94:e215–
e224.
Deng, W., X. F. Dong, J. M. Tong, and Q. Zhang. 2012. The pro-
biotic Bacillus licheniformis ameliorates heat stress-induced im-
pairment of egg production, gut morphology, and intestinal mu-
cosal immunity in laying hens. Poult. Sci. 91:575–582.
Dobson, H., and R. F. Smith. 1995. Stress and reproduction in farm
animals. J. Reprod. Fertil. Suppl. 49:451–461.
Erel, O. 2004. A novel automated direct measurement method for
total antioxidant capacity using a new generation, more stable
ABTS radical cation. Clin. Biochem. 37:277–285.
Forget, P., Z. Saye, J. L. Van Cutsem, and G. Dandrifosse. 1985. Se-
rum diamine oxidase activity in acute gastroenteritis in children.
Pediatr. Res. 19:26–28.
Fuller, R. 1989. Probiotics in man and animals. J. Appl. Bacteriol.
66:365–378.
Gomez, E., D. S. Irvine, and R. J. Aitken. 1998. Evaluation of a
spectrophotometric assay for the measurement of malondialde-
hyde and 4-hydroxyalkenals in human spermatozoa: Relation-
ships with semen quality and sperm function. Int. J. Androl.
21:81–94.
 DIETARY INCLUSION OF BACILLUS LICHENIFORMIS
Goodling, A. C., G. J. Cerniglia, and J. A. Hebert. 1987. Production
performance of White Leghorn layers fed lactobacillus fermenta-
tion products. Poult. Sci. 66:480–486.
Hagedorn, S. R., G. Bradley, and P. J. Chapman. 1985. Glutathione-
independent isomerization of maleylpyruvate by Bacillus mega-
terium and other gram-positive bacteria. J. Bacteriol. 163:640–
647.
Jin, L. Z., Y. W. Ho, N. Abdullah, and S. Jalaludin. 1998. Growth
performance, intestinal microbial populations, and serum choles-
terol of broilers fed diets containing Lactobacillus cultures. Poult.
Sci. 77:1259–1265.
Kim, Y., J. Y. Cho, J. H. Kuk, J. H. Moon, J. I. Cho, Y. C. Kim,
and K. H. Park. 2004. Identification and antimicrobial activity
of phenylacetic acid produced by Bacillus licheniformis isolated
from fermented soybean, Chungkook-Jang. Curr. Microbiol.
48:312–317.
Kurtoglu, V., F. Kurtoglu, E. Seker, B. Coskun, T. Balevi, and E.
S. Polat. 2004. Effect of probiotic supplementation on laying hen
diets on yield performance and serum and egg yolk cholesterol.
Food Addit. Contam. 21:817–823.
Li, L., C. L. Xu, C. Ji, Q. Ma, K. Hao, Z. Y. Jin, and K. Li. 2006.
Effects of a dried Bacillus subtilis culture on egg quality. Poult.
Sci. 85:364–368.
Luk, G. D., T. M. Bayless, and S. B. Baylin. 1980. Diamine oxidase
(histaminase). A circulating marker for rat intestinal mucosal
maturation and integrity. J. Clin. Invest. 66:66–70.
Lutful Kabir, S. M. 2009. The role of probiotics in the poultry indus-
try. Int. J. Mol. Sci. 10:3531–3546.
Mack, D. R., S. Michail, S. Wei, L. McDougall, and M. A. Holling-
sworth. 1999. Probiotics inhibit enteropathogenic E. coli adher-
ence in vitro by inducing intestinal mucin gene expression. Am.
J. Physiol. 276:G941–G950.
Madsen, K., A. Cornish, P. Soper, C. McKaigney, H. Jijon, C.
Yachimec, J. Doyle, L. Jewell, and C. De Simone. 2001. Probiotic
bacteria enhance murine and human intestinal epithelial barrier
function. Gastroenterology 121:580–591.
Matés, J. M. 2000. Effects of antioxidant enzymes in the molec-
ular control of reactive oxygen species toxicology. Toxicology
153:83–104.
Matteri, R. L., J. G. Watson, and G. P. Moberg. 1984. Stress or
acute adrenocorticotrophin treatment suppresses LHRH-induced
LH release in the ram. J. Reprod. Fertil. 72:385–393.
Mohan, B., R. Kadirvel, M. Bhaskaran, and A. Natarajan. 1995. Ef-
fect of probiotic supplementation on serum/yolk cholesterol and
on egg shell thickness in layers. Br. Poult. Sci. 36:799–803.
Murray, M. J., J. J. Barbose, and C. F. Cobb. 1993. Serum d(-)-
lactate levels as a predictor of acute intestinal ischemia in a rat
model. J. Surg. Res. 54:507–509.
Nahashon, S. N., H. S. Nakaue, and L. W. Mirosh. 1994. Production
variables and nutrient retention in single comb White Leghorn
laying pullets fed diets supplemented with direct-fed microbials.
Poult. Sci. 73:1699–1711.
Radu, A., C. Pichon, P. Camparo, M. Antoine, Y. Allory, A. Couve-
lard, G. Fromont, M. T. Hai, and N. Ghinea. 2010. Expression of
2395
follicle-stimulating hormone receptor in tumor blood vessels. N.
Engl. J. Med. 363:1621–1630.
Raikhinstein, M., and I. Hanukoglu. 1993. Mitochondrial-genome-
encoded RNAs: Differential regulation by corticotropin in bovine
adrenocortical cells. Proc. Natl. Acad. Sci. USA 90:10509–10513.
Rajput, I. R., L. Y. Li, X. Xin, B. B. Wu, Z. L. Juan, Z. W. Cui, D.
Y. Yu, and W. F. Li. 2013. Effect of Saccharomyces boulardii and
Bacillus subtilis B10 on intestinal ultrastructure modulation and
mucosal immunity development mechanism in broiler chickens.
Poult. Sci. 92:956–965.
Resta-Lenert, S., and K. E. Barrett. 2003. Live probiotics protect
intestinal epithelial cells from the effects of infection with entero-
invasive Escherichia coli (EIEC). Gut 52:988–997.
Rozs, M., L. Manczinger, C. Vagvolgyi, and F. Kevei. 2001. Secre-
tion of a trypsin-like thiol protease by a new keratinolytic strain
of Bacillus licheniformis. FEMS Microbiol. Lett. 205:221–224.
Santoso, U., K. Tanaka, and S. Ohtani. 1995. Effect of dried Ba-
cillus subtilis culture on growth, body composition and hepatic
lipogenic enzyme activity in female broiler chicks. Br. J. Nutr.
74:523–529.
Sies, H. 1991. Oxidative stress: From basic research to clinical ap-
plication. Am. J. Med. 91:31S–38S.
Tannock, G. W. 2001. Molecular assessment of intestinal microflora.
Am. J. Clin. Nutr. 73:410S–414S.
Thanh, N. T., T. C. Loh, H. L. Foo, M. Hair-Bejo, and B. K. Azhar.
2009. Effects of feeding metabolite combinations produced by
Lactobacillus plantarum on growth performance, faecal microbial
population, small intestine villus height and faecal volatile fatty
acids in broilers. Br. Poult. Sci. 50:298–306.
Thompson, J. H., and W. R. Richter. 1960. Hemotoxylin-eosin
staining adapted to automatic tissue processing. Stain Technol.
35:145–148.
Tortuero, F. 1973. Influence of the implantation of Lactobacillus aci-
dophilus in chicks on the growth, feed conversion, malabsorption
of fats syndrome and intestinal flora. Poult. Sci. 52:197–203.
Winterbourn, C. C., R. E. Hawkins, M. Brian, and R. W. Carrell.
1975. The estimation of red cell superoxide dismutase activity. J.
Lab. Clin. Med. 85:337–341.
Xu, Z. R., C. H. Hu, M. S. Xia, X. A. Zhan, and M. Q. Wang. 2003.
Effects of dietary fructooligosaccharide on digestive enzyme ac-
tivities, intestinal microflora and morphology of male broilers.
Poult. Sci. 82:1030–1036.
Yalçin, S., K. Cakin, O. Eltan, and L. Dagasan. 2010. Effects of
dietary yeast autolysate (Saccharomyces cerevisiae) on perfor-
mance, egg traits, egg cholesterol content, egg yolk fatty acid
composition and humoral immune response of laying hens. J.
Sci. Food Agric. 90:1695–1701.
Yang, R. L., W. Li, Y. H. Shi, and G. W. Le. 2008. Lipoic acid pre-
vents high-fat diet-induced dyslipidemia and oxidative stress: A
microarray analysis. Nutrition 24:582–588.
Zakowski, J. J., and A. L. Tappel. 1978. A semiautomated system
for measurement of glutathione in the assay of glutathione per-
oxidase. Anal. Biochem. 89:430–436.