Individual and combined effects of genistein and hesperidin on immunity

and intestinal morphometry in lipopolysacharide-challenged broiler chickens

A. A. Kamboh*† and W-y. Zhu*1

*Laboratory of Gastrointestinal Microbiology, College of Animal Science and Technology, Nanjing Agricultural
University, Nanjing 210095, China; and †Department of Veterinary Microbiology, Faculty of Animal Husbandry
and Veterinary Sciences, Sindh Agriculture University, Tandojam 70060, Pakistan

ABSTRACT Genistein and hesperidin have been shown
to have beneficial effects in several animal models in-
cluding mice, rats, pigs, and humans. This study inves-
tigated the individual and combined effects of genistein
(an isoflavone) and hesperidin (a flavanone) on immu-
nity and intestinal morphometry in lipopolysaccharide
(LPS)-challenged broiler chickens. Seven hundred twen-
ty 1-d-old commercial Arbor Acres broiler chicks were
randomly divided into 6 treatments, with 6 replicates
of 20 birds each. Chicks were fed a basal diet without
any additive (control), supplemented with 5 mg of ge-
nistein/kg of feed (G5) and 20 mg of hesperidin/kg of
feed (H20), or a mixture of genistein and hesperidin
(1:4) with doses of 5 (GH5), 10 (GH10), and 20 (GH20)
mg/kg of feed for 42 d. On d 16, 18, and 20, one-half
the birds from each group were separated and injected
intraperitoneally with Escherichia coli LPS (250 μg/
kg of BW) to induce the immunological stimulation.
Samples were collected on 21 and 42 d. The results
showed that LPS treatment exerts immunomodulatory
effects (P < 0.05) in phagocytic activity at 21 d, where-
Key words: genistein, hesperidin, immunity, lipopolysaccharide, morphometry
as a few negative effects including reduced villus length
and increased crypt depth were observed in some seg-
ments of the small intestine. Both genistein and hes-
peridin seemed to modify the immunity positively by
altering the phagocytic activity (P < 0.01). Parameters
of intestinal morphometry such as villus length, crypt
depth, villus width, and villus length/crypt depth ra-
tios were also improved (P < 0.01 or P < 0.05) by the
supplemental genistein and hesperidin in both LPS-un-
challenged and -challenged groups. However, no effect
(P > 0.05) was observed for BWG, FI, and FCR of
broilers. Overall, genistein and hesperidin improved the
immunity and the morphometry of small intestine in a
dose-dependent manner. These findings provided the
first account on the in vivo effects of genistein and hes-
peridin for immunostimulation and morphometric gut
development in LPS-challenged chickens. Thus, both
compounds may be used as alternative feed additives
in the poultry industry to promote gut health and im-
prove immunity against infections.
2014 Poultry Science 93:2175–2183
http://dx.doi.org/10.3382/ps.2014-03971

INTRODUCTION In China, some bacterial diseases such as fowl cholera,

In recent years, growing interest in discontinuing in-
feed antibiotics has greatly enhanced the search for al-
ternative feed additives including prebiotic, probiotic,
and phytogenic additives to improve the immune sys-
tem, gut function, and productivity of farm animals
(Giannenas et al., 2010). In contrast to antibiotics,
these products do not have any known side effects and
are thus considered safe in the food chain. Flavonoids,
a group of polyphenolic compounds found mainly in
fruits and vegetables, are some of the widely researched
alternatives to in-feed antibiotics in animal nutrition.
©2014 Poultry Science Association Inc.
Received February 13, 2014.
Accepted May 8, 2014.
1 Corresponding author: zhuweiyun@njau.edu.cn
infectious coryzea, avian bordetellosis, and mycoplas-
mosis; and viral diseases such as avian influenza, viral
enteritis, infectious bursal disease, infectious bronchitis,
and Newcastle disease have been common in poultry
birds recently (Wang et al., 2013). In addition to these
obvious diseases, subclinical forms of these diseases and
several others such as colibacillosis, necrotic enteritis,
and fowl typhoid are very devastating because these
cause several billion dollars of loss per annum (Frolich
et al., 2013). These subclinical diseases cause low-dose
endotoxemia. In contrast to high-dose endotoxemia
that induces a robust momentary inflammatory re-
sponse, subclinical low-dose endotoxin causes low-grade
yet persistent inflammatory responses that result in the
initiation and propagation of chronic pathological dis-
eases (Maitra et al., 2012). Lipopolysaccharide (LPS),
an endotoxin, is an essential component of the cell wall

2175
2176 Kamboh and Zhu

of gram-negative bacteria. Many studies have been re- Table 1. Feed ingredients and analytical composition of basal
ported on LPS challenge, but all those demonstrated diet for broiler chickens (%, unless otherwise indicated)
a severe challenge (high-dose endotoxemia, 500–1,000 Starter Grower
µg/kg of BW; Xie et al., 2000; Bowen et al., 2009). Item (1 to 21 d) (22 to 42 d)
Little information is available on subclinical low-dose Ingredient
exposure of endotoxin LPS in broilers.  Corn 60.2 65.65
Genistein and hesperidin are 2 naturally occurring   Soybean meal 30.0 24.0
  Corn gluten meal 2.5 3.0
flavonoids, abundant in soybeans and citrus fruits, re-   Soybean oil 2.8 3.0
spectively. Flavonoids are well known for improving  Limestone 1.2 1.2
chicken health and egg production (Ting et al., 2011),   Dicalcium phosphate 1.7 1.6
  Vitamin-mineral premix1 1.0 1.0
gut protective efficiency, attenuation of LPS effects,  Salt 0.3 0.3
and upregulation of both innate and acquired immune   l-Lysine 0.15 0.15
responses (Calixto et al., 2004; Kawaguchi et al., 2004).   dl-Methionine 0.15 0.1
Analyzed chemical composition
In some studies, flavonoid-rich alfalfa extract and soy   ME (kcal/kg) 3,122 3,188
isoflavones have been reported for significant effects on  CP 20.72 18.93
growth performance, T and B lymphocyte prolifera-   Ether extract 4.14 5.79
 DM 89.1 88.4
tion, and stimulation of immune cells in broiler chickens  Lysine 1.17 1.0
(Dong et al., 2007; Jiang et al., 2007). In some recent  Methionine 0.5 0.41
studies, unpurified flavonoids in their native forms (fla-   Methionine + cysteine 0.65 0.6
 Threonine 0.7 0.6
vonoid-rich botanicals) or their extracts (or both) were  Tryptophan 0.17 0.16
reported for immunomodulatory and gut-promoting ef-  Calcium 0.9 0.84
fects, including increased villus length and surface area  Phosphorus 0.6 0.5
of small intestine in growing chickens (Hanieh et al., 1Mineral premix provided per kilogram of diet: transretinyl acetate,

2010; Hassanpour et al., 2010; Wallace et al., 2010). To
our knowledge, however, little research has been con-
ducted to investigate the supplementation of purified
flavonoids in immunologically challenged broiler chick-
ens. Therefore, the present study aimed to evaluate
the growth performance, immunomodulatory, and gut
morphometric effects of 2 purified flavonoids genistein
and hesperidin individually and in combination, in low
levels in immunologically challenged broiler chickens.
MATERIALS AND METHODS
Experimental Design, Diets, and Housing
All the experimental procedures were reviewed and
approved by the Animal Care and Use Committee of
the Nanjing Agricultural University, China. A total of
720 one-day-old, mixed sex commercial broiler chicks
(Arbor Acres) were obtained from a local hatchery (He-
wei Agriculture Development Co., Anhui, China). The
birds (average BW 44 g) were randomly divided into 6
treatment groups as follows: 1) basal diet without any
additive (control); 2) basal diet with 5 mg of genistein/
kg (G5); 3) basal diet with 20 mg of hesperidin/kg
(H20); 4) basal diet with 5 mg of genistein + hesperi-
din/kg (1:4, GH5); 5) basal diet with 10 mg of genis-
tein + hesperidin/kg (1:4, GH10); and 6) basal diet
with 20 mg of genistein + hesperidin/kg (1:4, GH20).
The dose and ratios of genistein and hesperidin were
based on previous work done in our laboratory (unpub-
lished data). Each group had 6 replicates of 20 birds
each that were assigned to wire cages (size: 102 × 60
× 32 cm) and housed in a full automatically controlled
room maintained at 34°C during the first day, 31 to
33°C during the first week, and gradually reduced by
3°C per week to reach a minimum of 26°C, after which
25 mg; cholecalciferol, 6 mg; menadione, 1.2 mg; thiamine, 2.3 mg; ri-
boflavin, 8 mg; nicotinamide, 42 mg; choline chloride, 400 mg; calcium
pantothenate, 10 mg; pyridoxine HCl, 4 mg; biotin, 0.04 mg; folic acid,
1 mg; cobalamin, 0.012 mg; Fe (from ferrous sulfate), 82 mg; Cu (from
copper sulfate), 7.5 mg; Mn (from manganese sulfate), 110 mg; Zn (from
zinc oxide), 64 mg; I (from calcium iodate), 1.1 mg; Se (from sodium
selenite), 0.28 mg.
it was maintained at room temperature till the end of
the experiment (at 42 d). Relative humidity was main-
tained in the house between 65 to 75% during the entire
experimental period. Twenty-four hour light availabil-
ity was ensured in the house. Corn-soybean basal diets
in the form of mash (starter mash for 1 to 21 d and
grower mash for 22 to 42 d) and water were offered ad
libitum to all birds. The birds were not supplemented
with any antibacterial or anticoccidial agent and did
not receive any therapeutic intervention during the en-
tire study period. The composition of experimental di-
ets is shown in Table 1.
A 2 × 6 factorial design was used as genistein or
hesperidin (or both) treatment as one factor and LPS/
saline injection as another factor. Genistein and hes-
peridin were purchased from a commercial company
(Sigma Chemical Co. USA, St. Louis, MO) with 98%
purity. Their purity was further confirmed before they
were mixed with feed (Mahmoud et al., 2013). On d 16,
one-half the birds from each replicate were given 0.9%
saline solution and the other half were injected intra-
peritoneally with highly purified Escherichia coli LPS
(serotype 055:B5, Sigma) at 250 µg/kg of BW. This
procedure was repeated on d 18 and 20; however, chick-
ens are very tolerant of the effect of LPS, so repeated
exposure is needed to induce immunological stimula-
tion (Zhang et al., 2010). Body weight, feed refusal, and
mortalities were recorded for both starter and grower
periods.
 GENISTEIN AND HESPERIDIN EFFECTS IN BROILERS
Sample Collection
At 21 and 42 d of age, broilers were deprived of feed
for 12 h, weighed to calculate performance, and one
broiler per replicate was randomly selected and killed
by decapitation. The whole GIT was removed and 2-cm
segments were taken from the middle of the duodenum
(from the gizzard outlet to pancreatic and bile ducts),
jejunum (from the point of entry of the bile ducts to
Meckel’s diverticulum), and ileum (from Meckel’s di-
verticulum to ileocecal junction). These samples were
washed with cold PBS and kept in 10% neutral buff-
ered formalin at 4°C for fixation until morphometric
analysis.
In Vivo Mononuclear Phagocytosis
Assessment
The individual and combined effects of genistein and
hesperidin on the phagocytic activity of the reticuloen-
dothelial system were assessed by measuring the car-
bon clearance rate as described previously (Damre et
al., 2003). On d 21 and 42, 6 birds from each group
were given colloidal carbon (black indian ink, Shang-
hai Xinde Oriental Co., Shanghai, China) intravenously
(0.1 mL/300 g of BW). At 2 and 20 min intervals, 30-
µL blood samples were collected from the wing vein
after the carbon injection. Each blood sample was he-
molyzed immediately by the addition of 2 mL of 0.1%
Na2CO3 solution. The concentration of carbon particles
was analyzed in plasma samples by measuring optical
density (OD) at wavelength of 600 nm. Phagocytic ac-
tivity was calculated in terms of the phagocytic index
by using the following equations (Shukla et al., 2009):
rate of carbon clearance (K) = log OD2
− log OD10/T2 − T1;
phagocytic index = K1/3
× BW of animal/liver weight + spleen weight,
where OD2 is the log absorbance of blood at 2 min,
OD10 is log absorbance of blood at 10 min, T2 is the
last time point of blood collection, and T1 is the first
time point of blood collection.
Morphometric Analysis
Formalin-fixed intestinal tissues were embedded with
optimal cutting temperature compound (OCT, Sigma)
and cut into transverse sections (7 µm) at −20°C. These
were stained by the hematoxylin and eosin (H&E, Sig-
ma) method and examined with a Nikon phase con-
trast microscope coupled with a Microcomp integrated
digital imaging analysis system (Nikon Eclipse 80i,
Nikon Co., Tokyo, Japan). Images were viewed (4×) to
measure morphometric parameters of intestinal archi-
2177
tecture. Villus length was measured as the vertical dis-
tance from the villus tip to villus-crypt junction. Villus
width was recorded at its mid-height. Crypt depth was
measured as the vertical distance from the villus-crypt
junction to the lower limit of the crypt (Giannenas et
al., 2010). All the measurements were recorded from 10
well-oriented villi × 3 sections/chicken.
Statistics
Data were analyzed using the GLM procedure of
SPSS 16.0 for Windows (SPSS, 1999) as 6 × 2 factorial
arrangement with dietary treatments of genistein/hes-
peridin and LPS challenge as main effects. Tukey’s mul-
tiple range test was used to evaluate the significant dif-
ferences among treatment means. Level of significance
was determined at P < 0.05. All results were presented
as mean and pooled SEM.
RESULTS
Growth Performance
As shown in Table 2, dietary treatments of genis-
tein and hesperidin and LPS challenge did not affect
BW gain (BWG), feed intake (FI), and feed conver-
sion ratio (FCR, intake:BWG) of broilers during the
starter (1–21 d) and grower period (22–42 d). A slight
reduction was observed in FI and BWG during 1 to
21 d in LPS-challenged groups; however, it was found
to be nonsignificant (P > 0.05). No effect of dietary
treatments or LPS were found in the mortality ratio
throughout the experimental period (data not shown).
Phagocytic Response
Effect of Dietary Treatments. Dietary treatments
of genistein and hesperidin elevated (P < 0.01) the
phagocytic index of broiler chickens at 21 and 42 d
in a dose-dependent fashion (Table 3). In LPS-unchal-
lenged groups, all dietary treatments except G5 showed
a higher level (P < 0.01) of phagocytic activity com-
pared with the control on d 21 (i.e., H20, +15.7%;
GH5, +8.4%; GH10, +45.9%; and GH20, +92.9%).
At 42 d, all dietary treatments except low dose groups
(G5 and GH5) had improved (P < 0.01) phagocytic
activity (i.e., H20, +16.0%; GH10, +17.0%; and GH20,
+51.5%) compared with the unsupplemented control
group.
Effect of LPS. A marked increase (P = 0.001) in
phagocytic activity was observed for the LPS challenge
(control: +27.3%) on 21 d. In LPS-challenged groups,
phagocytic activity was improved in all groups except
GH20 (i.e., control, +27.3%; G5, +50.5%; H20, +33.8%;
GH5, +29.1%; and GH10, +20.9%). The interaction
between challenge and diet was also statistically signifi-
cant (P < 0.01) on 21 d, whereas LPS challenge did not
affect phagocytic activity on 42 d.
 2178

Table 2. Effect of genistein and hesperidin supplementation on growth performance in lipopolysaccharide (LPS)-induced broiler chickens from 1 to 42 d of age
Dietary treatment2

LPS (−) LPS (+) P-value3

Parameter1 CON G5 H20 GH5 GH10 GH20 CON G5 H20 GH5 GH10 GH20 SEM Diet Challenge Interaction

1–21 d                                
  BWG (g) 794 792 779 791 799 783 734 782 785 780 789 788 8.320 0.064 0.056 0.033
  FI (g) 1,033 1,042 1,048 1,049 1,014 1,025 960 1,019 1,020 1,027 998 1,013 9.431 0.109 0.042 0.333
 FCR 1.301 1.315 1.345 1.326 1.269 1.309 1.307 1.303 1.299 1.316 1.264 1.285 0.011 0.505 0.061 0.669
22–42 d                                
  BWG (g) 1,125 1,135 1,202 1,137 1,118 1,162 1,131 1,156 1,210 1,205 1,202 1,145 11.02 0.074 0.957 0.765
  FI (g) 2,412 2,473 2,475 2,393 2,310 2,492 2,448 2,463 2,426 2,396 2,387 2,454 17.21 0.212 0.856 0.897
 FCR 2.197 2.069 1.990 2.176 2.066 2.144 2.164 2.130 2.004 1.988 1.985 2.143 0.019 0.406 0.775 0.985
1BWG = BW gain; FI = feed intake; FCR = feed conversion ratio.
2CON = control; G5 = 5 mg of genistein/kg; H20 = 20 mg of hesperidin/kg; GH5 = 5 mg of genistein + hesperidin/kg (1:4); GH10 = 10 mg of genistein + hesperidin/kg (1:4); GH20 = 20 mg of
genistein + hesperidin/kg (1:4).
3P-values represent the main effect of the diet, the main effect of LPS challenge, and the interaction between dietary treatments and LPS challenge.
Kamboh and Zhu

Table 3. Effect of genistein and hesperidin supplementation on phagocytic index in lipopolysaccharide (LPS)-induced broiler chickens at 21 and 42 d of age
Dietary treatment2

LPS (−) LPS (+) P-value3

Phagocytic
index1 CON G5 H20 GH5 GH10 GH20 CON G5 H20 GH5 GH10 GH20 SEM Diet Challenge Interaction

21 d 0.561g 0.487h 0.649f 0.608f 0.819d 1.082a 0.714e 0.733e 0.868c 0.785d 0.990b 1.031b 0.062 0.001 0.001 0.001
42 d 0.619c 0.607c 0.718b 0.584c 0.724b 0.938a 0.621c 0.613c 0.693b 0.600c 0.709b 0.955a 0.055 0.001 0.979 0.622
a–hMean values (n = 6) in a row not sharing a common superscript are significantly different (P < 0.05).
1Relative rate of carbon clearance.
2CON = control; G5 = 5 mg of genistein/kg; H20 = 20 mg of hesperidin/kg; GH5 = 5 mg of genistein + hesperidin/kg (1:4); GH10 = 10 mg of genistein + hesperidin/kg (1:4); GH20 = 20 mg of
genistein + hesperidin/kg (1:4).
3P-values represents the main effect of the diet, the main effect of LPS challenge, and the interaction between dietary treatments and LPS challenge.
 GENISTEIN AND HESPERIDIN EFFECTS IN BROILERS
Intestinal Morphometrics
Effect of Dietary Treatments. In LPS-unchallenged
groups, the duodenal villus length was significantly lon-
ger (P < 0.01) at both 21 and 42 d for GH10 (+5.9%
at 21 d, +12.5% at 42 d) and GH20 (+11.2% at 21
d, +12.0% at 42 d) groups compared with the control
group (Tables 4 and 5). However, there was no effect
of dietary treatments on duodenal villus width at 21
d, whereas at 42 d high-dose groups (H20 and GH20)
were significantly improved (P < 0.01) regardless of
LPS challenge. Among LPS-unchallenged groups, duo-
denal crypt depth was reduced significantly (P < 0.05)
in GH10 group (−11.2%) at 21 d. Villus length/crypt
depth ratio of duodenum was elevated (P < 0.01) in
GH5 (at 21 d), GH10, and GH20 (at 21 and 42 d)
groups, regardless of LPS challenge.
The means of villus length, villus width, crypt depth,
and villus length/crypt depth ratios in jejunum are
presented in Tables 4 and 5, respectively. It was ob-
served that, in LPS-unchallenged groups villus length
altered significantly (P < 0.01) in H20 (+14.1%), GH5
(+9.7%), GH10 (+19.6%) at 21 d, and GH20 groups
(+18.3% at 21 d and +10.4% at 42 d) in comparison
with the control group. A similar trend was observed
for villus width, but the differences were significant (P
< 0.05) only on 42 d for the H20 group (+13.3%). Like-
wise, villus length/crypt depth ratio was also increased
(P < 0.01) for GH10 (+38.0%) and GH20 (+39.1%)
groups at 21 d, compared with the control group. How-
ever, crypt depth was not affected by supplemented
genistein and hesperidin (P > 0.05).
As shown in Tables 4 and 5, among LPS-unchallenged
groups, villus length of the ileum in the G5 (+17.4%)
group, crypt depth in the H20 (−11.7%) and GH10
(–10.8%) groups, and villus length/crypt depth in the
G5 (+22.5%), H20 (+20.8%), GH10 (+17.4%), and
GH20 (+15.3%) groups were significantly (P < 0.05 or
P < 0.01) altered in comparison with control group at
21 d. At 42 d, no effect of supplemental genistein and
hesperidin was observed in morphometric structures
of the ileum (Table 5). In LPS treated groups, villus
length and villus width was improved at 21 d, and the
interaction between diet and LPS challenge was statis-
tically significant (P < 0.05) for the crypt depth and
villus length/crypt depth ratio (21 d).
Effect of LPS. Tables 4 and 5 indicate that LPS
treatment reduced the villus length (control: −3.6%)
and increased the crypt depth (control: +4.5%) at 21
d, whereas no effect was recorded in the morphometry
of the duodenum at 42 d. However, in genistein- and
hesperidin-treated groups, these deteriorations were
not observed. In LPS-challenged groups, duodenal vil-
lus length were significantly raised (P < 0.05) in GH10
(+12.4% on 42 d) and GH20 (+14.0% on 21 and +12.5%
on 42 d) groups. However, duodenal crypt depth was
promoted by LPS challenge with significant differences
(P < 0.01) in all treated groups compared with the
control group (at 21 d). The interaction between diet
2179
and challenge was nonsignificant for all parameters of
duodenum except for villus length at 21 d (P = 0.01,
Table 4). The LPS injection reduced the villus length
(control: −8.7%) and villus length/crypt depth ratio
(control: −9.8%) at 21 d, whereas no effect was record-
ed in the morphometry of jejunum at 42 d. Significant
interaction (P = 0.04) was recorded between the diet
and LPS challenge for the villus length at 21 d (Table
4). In LPS-challenged groups, reduction in crypt depth
and elevation in villus length/crypt depth ratio of il-
eum was recorded by the LPS injection with significant
differences (P < 0.05) in H20, GH10, and GH20 groups
(21 d).
DISCUSSION
Circulating subclinical LPS, a glycoprotein from
gram-negative bacteria, occurs in animals in disease
conditions and sometimes in very low levels in healthy
individuals. It activates innate immunity through Toll-
like receptor 4 (TLR4); however, persistent elevation
of circulating LPS is known to cause chronic subclinical
diseases. It may induce apoptosis by activating the car-
diac renin-angiotensin system, and thus decrease sur-
vival (Lew et al., 2013). The LPS extracted from E. coli
is used at subclinical levels to induce immunological
stress in experimental animals because it is a valuable
indicator to evaluate the efficacy of immunomodulatory
agents, health status, and capability to acclimatize or
recover from subclinical infections (Cheng et al., 2004).
The present study revealed that both individual and
combined dietary treatments of genistein and hesperi-
din did not influence the growth performance (BWG,
FI, and FCR) of broiler chickens challenged with E.
coli LPS. Previous studies have also found that in-
feed flavonoids did not affect BW, BWG, and FI in
broilers (Peña et al., 2008; Kamboh and Zhu, 2013a).
However, it was reported that LPS challenge (500 µg/
kg of BW) retarded the FI and daily gain of growing
turkeys (Hu et al., 2011), whereas, in the present study,
similar effects could not be detected in genistein and
hesperidin-supplemented chickens. This might be due
to the low dose of LPS used in our study, anti-stress
effects of these compounds, or both, like other phyto-
chemicals (Young et al., 2003). Our previous studies
have also documented the anti-stress and antioxidative
effects of these flavonoidal compounds (Kamboh and
Zhu, 2013b,c).
The current study has demonstrated the improved
phagocytic activity of mononuclear cells by the supple-
mentation of genistein and hesperidin in a dose-depen-
dent manner, which is consistent with a study of rats fed
200 to 500 mg of flavonoid-rich Caesalpinia bonducella
extract/kg (Shukla et al., 2009). Moreover, the results
suggest the possibility that supplemental genistein and
hesperidin could potentiate the phagocytic activity not
only of peritoneal leukocytes and monocytes but also
of tissue macrophages, liver Kupffer cells, and spleenic
macrophages. Thus, important visceral organs such
 2180

Table 4. Effect of genistein and hesperidin supplementation on intestinal morphometric analysis in lipopolysaccharide (LPS)-induced broiler chickens at 21 d of age1
Dietary treatment

LPS (−) LPS (+) P-value2

Parameter CON G5 H20 GH5 GH10 GH20 CON G5 H20 GH5 GH10 GH20 SEM Diet Challenge Interaction

Duodenum                                
  Villus length (µm) 1,027def 985f 1,063cd 1,076bcd 1,087bc 1,142a 990ef 984f 1,043cde 1,027def 1,034cdef 1,129ab 16.742 0.001 0.001 0.015
  Villus width (µm) 148ab 143ab 149ab 152ab 159ab 157ab 140b 141b 149ab 141b 154ab 164a 3.028 0.001 0.207 0.315
  Crypt depth (µm) 134ab 122bc 130abc 121bc 119c 124bc 140a 124bc 121bc 125bc 124bc 132abc 2.477 0.016 0.175 0.432
  Villus length/ 7.70cd 8.06bcd 8.17abcd 8.89ab 9.06ab 9.19a 7.08d 8.00bcd 8.64abc 8.20abc 8.34abc 8.56abc 1.091 0.001 0.008 0.261
  crypt depth
Jejunum                                
  Villus length (µm) 838de 845cd 956ab 919bc 1,002a 991ab 765e 841de 931ab 934ab 993ab 998a 19.937 0.001 0.173 0.049
  Villus width (µm) 104 107 115 107 111 110 97 107 112 108 110 111 4.276 0.025 0.554 0.354
  Crypt depth (µm) 91 89 82 87 83 78 93 90 84 83 82 80 3.206 0.002 0.954 0.806
  Villus length/ 9.2cd 9.4bcd 11.8abc 10.6abcd 12.8a 12.7a 8.3d 9.3cd 11.0abcd 11.2abcd 12.3abc 12.5ab 1.251 0.001 0.397 0.665
  crypt depth
Kamboh and Zhu

Ileum                                
  Villus length (µm) 591b 694a 633ab 618b 626ab 647ab 601b 660ab 647ab 632ab 625ab 651ab 15.040 0.363 0.932 0.770
  Villus width (µm) 117 117 118 113 120 122 118 119 120 120 123 121 7.003 0.166 0.492 0.903
  Crypt depth (µm) 111a 106abc 98cd 108ab 99bcd 105abc 111a 107ab 92de 108ab 86e 100bcd 3.191 0.001 0.038 0.036
  Villus length/ 5.34f 6.54abc 6.45bcd 5.71def 6.27bcd 6.16cde 5.38ef 6.17cde 7.01ab 5.84cdef 7.25a 6.47abcd 1.080 0.001 0.062 0.048
  crypt depth
a–fMean values (n = 6) in a row not sharing a common superscript are significantly different (P < 0.05).
1CON = control; G5 = 5 mg of genistein/kg; H20 = 20 mg of hesperidin/kg; GH5 = 5 mg of genistein + hesperidin/kg (1:4); GH10 = 10 mg of genistein + hesperidin/kg (1:4); GH20 = 20 mg of
genistein + hesperidin/kg (1:4).
2P-values represent the main effect of the diet, the main effect of LPS challenge, and the interaction between dietary treatments and LPS challenge.
Table 5. Effect of genistein and hesperidin supplementation on intestinal morphometric analysis in lipopolysaccharide (LPS)-induced broiler chickens at 42 d of age
Dietary treatment1

LPS (−) LPS (+) P-value2

Parameter CON G5 H20 GH5 GH10 GH20 CON G5 H20 GH5 GH10 GH20 SEM Diet Challenge Interaction

Duodenum                                
  Villus length (µm) 1,497b 1,493b 1,514b 1,522b 1,684a 1,676a 1,499b 1,474b 1,527b 1,520b 1,685a 1,686a 31.778 0.001 0.939 0.782
  Villus width (µm) 179c 189abc 192ab 182bc 185abc 194a 178c 188abc 193ab 182bc 186abc 195a 6.167 0.001 0.975 0.763
  Crypt depth (µm) 164a 160a 162a 158ab 142c 154abc 161a 163a 163a 160a 144bc 155abc 2.939 0.001 0.588 0.635
  Villus length/ 9.1c 9.3c 9.3c 9.7bc 11.8a 10.9ab 9.3c 9.0c 9.3c 9.5c 11.7a 10.9ab 0.738 0.001 0.760 0.849
  crypt depth
Jejunum                                
  Villus length (µm) 1,134bc 1,194ab 996d 1,045cd 1,208ab 1,252a 1,129bc 1,189ab 1,123bc 1,048cd 1,206ab 1,247a 19.919 0.004 0.365 0.789
  Villus width (µm) 143b 140b 162a 149ab 156ab 153ab 142b 140b 163a 154ab 154ab 156ab 5.033 0.001 0.760 0.769
  Crypt depth (µm) 112 115 106 108 107 108 112 113 106 107 107 109 5.408 0.074 0.935 0.818
  Villus length/ 10.2abc 10.4abc 9.4c 9.7bc 11.3ab 11.7a 10.1abc 10.5abc 10.6abc 9.7bc 11.3ab 11.6a 1.134 0.001 0.410 0.753
  crypt depth
Ileum                                
  Villus length (µm) 870 953 861 988 946 869 869 903 859 989 942 870 32.673 0.433 0.670 0.734
  Villus width (µm) 139 143 142 135 143 146 139 144 143 136 144 145 6.666 0.445 0.893 0.972
  Crypt depth (µm) 117 117 105 112 111 106 118 116 105 111 111 106 5.745 0.052 0.912 0.946
  Villus length/ 7.46 8.28 8.20 8.91 8.56 8.35 7.41 8.37 8.20 8.99 8.56 8.38 2.144 0.019 0.836 0.782
  crypt depth
a–dMean values (n = 6) in a row not sharing a common superscript are significantly different (P < 0.05).
1CON = control; G5 = 5 mg of genistein/kg; H20 = 20 mg of hesperidin/kg; GH5 = 5 mg of genistein + hesperidin/kg (1:4); GH10 = 10 mg of genistein + hesperidin/kg (1:4); GH20 = 20 mg of
GENISTEIN AND HESPERIDIN EFFECTS IN BROILERS

genistein + hesperidin/kg (1:4).

2P-values represent the main effect of the diet, the main effect of LPS challenge, and the interaction between dietary treatments and LPS challenge.
2181
2182 Kamboh and Zhu
as the liver and spleen could obtain more protection
from pathogenic bacteria and viruses. It is well known
that Kupffer cells and macrophages are critical compo-
nents of the mononuclear phagocytic system of the liver
and spleen, respectively. Both Kupffer cells and mac-
rophages are derived from the circulating monocytes;
hence, a substance that can modulate the activity of
monocytes will also affect the monocytes fixed in the
tissues (Kupffer cells and spleen macrophages). It is
also well recognized that the enhancement of phagocyte
function is applicable for therapy of cancer because el-
evated phagocyte number or activity results in the re-
lease of H2O2, which causes the destruction of cancer-
ous cells (Gu et al., 2010). Furthermore, we observed
the immunostimulatory effect of LPS on phagocytic
cells (21 d) for the elimination of carbon particles. That
is consistent with a recent in vitro study that revealed
that subclinical low-dose endotoxins could activate
macrophages (Maitra et al., 2012). Muller et al. (2005)
also documented that constituents of bacterial cell wall,
including LPS are the potent activators of monocytes,
neutrophils, and macrophages. Phagocytosis by mono-
nuclear cells in response to LPS is accompanied by a
dramatic increase in oxygen consumption by an abun-
dant production of reactive oxygen intermediates such
as superoxide anion that ultimately damage the visceral
organs and decrease the function of immune cells. Bio-
flavonoids could neutralize reactive oxygen species and
also inhibit the phosphorylation of neutrophil proteins,
which is suggested as an important intracellular event
for neutrophil activation (Pilkhwal et al., 2010). It has
been hypothesized that TLR4 is the critical receptor
for the LPS signaling in host defense, and the innate
immune system may be involved in the regulation of
adaptive immunity, which is mediated by lymphocytes
(Ogikubo et al., 2004). Our results demonstrating a
significant effect of a genistein and hesperidin combi-
nation possibly indicate its synergism. From available
literature, it is manifested that flavonoids have a strong
potential for making synergism for platelet aggregation
(Pignatelli et al., 2000) and antimicrobial activity (Al-
varez et al., 2008). Nevertheless, whether or not there is
synergism between these compounds needs to be evalu-
ated, possibly by special-purpose experiment design.
This study is the first to evaluate the impact of pu-
rified genistein and hesperidin on gut development in
broiler chickens. Previously, some attempts have been
made on commercial chickens to explore the gut-pro-
moting effects of herbal plants containing adequate
amounts of flavonoids, and the incredible differences
in terms of increased villus length and decreased crypt
depth were recorded in the small intestine (Hassanpour
et al., 2010; Awad et al., 2011). In the present study,
a remarkable increase in gut villus length and villus
width (21 and 42 d) and reduction in crypt depth (in
duodenum and ileum on 21 d and duodenum on 42 d)
in supplemented chickens also indicating the novel po-
tential of these compounds to improve gut morphology.
Differences measured in villus length and width indi-
cated that these compounds could stimulate the epi-
thelial cell mitosis because longer or thicker (or both)
villi are associated with activated cell mitosis, with
reduced crypt depth reflecting the prolonged survival
of villi without the need for renewal. This is because
crypt is regarded as the villus factory and large crypt
indicates rapid tissue turnover due to inflammation
from pathogens, their toxins, or both (Giannenas et al.,
2010; Awad et al., 2011). Moreover, the increased vil-
lus length/crypt depth ratios by supplemental genistein
and hesperidin could be considered as a better ratio for
digestive tract maintenance (Hu and Guo, 2007).
Hu et al. (2011) reported that subclinical level injec-
tion of LPS could cause deleterious effects on the small
intestine structure and functions by decreasing the vil-
lus length and villus width in broilers. In the present
study, the LPS-challenged control group showed similar
responses of gut morphometry by decreasing the villus
length and increasing crypt depth, whereas genistein- or
hesperidin-treated groups did not show any deleterious
effect on intestinal structures. These intestinal morpho-
metric caring effects of supplemental compounds might
be a protective effect of these compounds from pro-
apoptotic oxidant stress to gut epithelial cells (Miller
et al., 2001) or modulating the intestinal microflora
that play an instructive role in the regulation of villus
morphology (Hooper et al., 2001). In some recent stud-
ies, poor morphometry (shorter villi and deeper crypts)
of the intestinal tract have been associated with gut
stressors such as pathogens and inflammation produced
by them and their toxins (Giannenas et al., 2010).
Compounds used in the present study (genistein and
hesperidin) are well known for their anti-inflammatory
and prebiotic-like effects on gut microbiota, as reviewed
by Laparra and Sanz (2010).
In conclusion, the current study has demonstrated
the effects of genistein and hesperidin individually and
in combination (1:4) for immunostimulation and mor-
phometric gut development in low level LPS-challenged
broiler chickens. It was established that genistein and
hesperidin have the potential to improve immunity, and
those also develop the morphometric structures of the
small intestine in a dose-dependent manner. Moreover,
both compounds were found to be effective in attenu-
ating the LPS-induced deterioration of intestinal ar-
chitecture. Further experiments should be established
using the live pathogen challenge, positive control, or
both (of antibiotics) to validate the present findings, so
these compounds can be used as alternative feed addi-
tives in the poultry industry.
ACKNOWLEDGMENTS
This work was supported by National public benefit
research program (200903003). Asghar Ali Kamboh is
highly thankful to Sindh Agriculture University, Tan-
dojam/Higher Education Commission of Pakistan, for
granting monetary support for his study at Nanjing
Agricultural University.
 GENISTEIN AND HESPERIDIN EFFECTS IN BROILERS
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