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Department of Animal Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada; Department of
Animal Resources Science, Kongju National University, Yesan, Chungnam 32439, Republic of Korea; and
Department of Poultry Science, University of Georgia, Athens, Georgia 30602-2772, United States
Key words: fructooligosaccharide, Salmonella Enteritidis LPS, immune response, intestinal morphology,
broiler chicken
2015 Poultry Science 00:111
http://dx.doi.org/10.3382/ps/pev275
INTRODUCTION
press the competition between the host and its intestinal microbes (Dibner and Richards, 2005). However,
indiscriminate use of antibiotics may lead to the emergence of antibiotic resistant mutants, and these genes
may be further transferred to humans, causing foodborne diseases (Van Immerseel et al., 2009; Gagg`a
et al., 2010). Developing feeding strategies as alternatives to AGP has paramount social, economic, and environmental importance in poultry production.
Fructooligosaccharides (FOS) are one of the common
types of prebiotics, which consist of short-chain and
non-digestible carbohydrates (Gibson and Roberfroid,
1995). Previous studies on broiler chickens have shown
that dietary supplementation of FOS has the ability to
improve growth performance (Ammerman et al., 1988;
Bailey et al., 1991; Yusrizal and Chen, 2003), enhance
innate and acquired immune response (Khodambashi
Emami et al., 2012), improve intestinal mucosa structures (Xu et al., 2003), and beneficially change the
SHANG ET AL.
chicks were housed in electrically heated battery brooders (Petersime Incubator Company, Gettysburg, OH)
for 21 d. The temperature was monitored daily and
was gradually reduced from 32 to 24o C from d one
to 21. Light was provided for 24 h throughout the experimental period. Upon arrival, birds were individually weighed and sorted into 5 weight classes. Groups
of 5 birds, one from each weight class, were then randomly assigned to 36 battery pens so that the average
initial BW was similar across pens. The experimental
protocol was approved by the University of Manitoba
Animal Care Protocol Management and Review Committee, and birds were handled in accordance with the
guidelines established by the Canadian Council on Animal Care (CCAC, 1993).
Experimental Design
Six treatment groups were randomly assigned to 6
replicate pens of 5 birds each. The experiment was designed as a 3 2 factorial arrangement based on 2 main
factors, as shown in Table 1. The main factors were: 1) 3
dietary treatments: positive control (PC), wheat-cornsoybean meal based diet contained antibiotics: 5.5 mg
virginiamycin (Stafac-44) and 99 mg monensin sodium
(Coban) per kilogram of diet; negative control (NC),
as PC without antibiotics; and NC + FOS, as NC supplemented with 0.5% FOS (Nutraflora P-95; Ingredion,
Etobicoke, ON, Canada) and 2) 2 intraperitoneal injections: 2 mL/kg sterile 1 phosphate buffered saline
(PBS) (AVL82762, HyClone Laboratories, Inc., Logan, UT) as control, or 2 mg/kg Salmonella Enteritidis LPS (ATCC 13076; Sigma-Aldrich, St. Louis,
MO) were executed on d 21. Water and feed were allowed ad libitum. The basal diet was formulated to
meet or exceed the National Research Council nutrient requirements for broiler chickens (NRC, 1994).
Compositions of the experimental diets are shown in
Table 2.
Salmonella Enteritidis
lipopolysaccharides (LPS)
(2). PC + LPS (n = 6)
(4). NC + LPS (n = 6)
(6). NC + FOS + LPS (n = 6)
NC
NC + FOS
35.80
29.80
20.54
4.45
4.50
1.38
1.76
0.10
0.12
0.05
0.50
1.00
35.80
29.80
20.54
4.45
4.50
1.38
1.76
0.10
0.12
0.05
0.50
1.00
0.5
35.72
29.75
20.42
4.20
4.50
1.38
1.76
0.10
0.12
0.05
0.50
1.00
3,118
21.4
1.00
0.45
0.98
0.51
1.08
0.81
3,118
21.4
1.00
0.45
0.98
0.51
1.08
0.81
3,106
21.2
1.00
0.45
0.97
0.50
1.08
0.80
20.7
90.3
20.4
89.9
20.2
89.9
1
Positive control (PC), wheat-corn-soybean meal basal diet
supplemented with 5.5 mg virginiamycin (Stafac-44) and 99 mg
monensin sodium (Coban) in the vitamin premix. Negative control
(NC), wheat-corn-soybean meal basal diet omitted with Stafac-44
and Coban. NC + FOS, NC diet supplemented with 0.5% fructooligosaccharides (FOS).
2
Nutraflora P-95, Short-Chain Fructooligosaccharides (scFOS), contains 4.5% sugar (fructose + glucose + sucrose), 34.2%
GF2 (glucose + 2 molecules fructose), 48.9% GF3 (glucose + 3
molecules fructose), and 12.4% GF4 (glucose + 4 molecules fructose) on DM basis. Ingredion, Etobicoke, ON, Canada.
3
Supplied per kilogram of diet: Mn (manganese oxide), 70 mg;
Zn (zinc oxide), 80 mg; Fe (ferrous sulfate), 80 mg; Cu (copper
sulfate), 10 mg; Se (sodium selenite), 0.3 mg; I (calcium iodate),
0.5 mg; and NaCl (non-iodized white salt), 4.3g.
4
Supplied per kilogram of diet: vitamin A, 8,250 IU; vitamin
D3 , 3,000 IU; vitamin E, 30 IU; vitamin B12 , 0.013 mg; vitamin K,
2 mg; riboflavin, 6 mg; pantothenic acid, 11 mg; niacin, 41.6 mg;
choline, 1,300.8 mg; folic acid, 4 mg; biotin, 0.25 mg; pyridoxine,
4 mg; thiamine, 4 mg; endox (anti-ox), 125 mg; and dl-methionine,
500 mg.
5
Concentrations were calculated based on NRC (1994) guidelines.
number, and their individual BW was recorded for immunological challenge. Four hours after the immunological challenge, approximately 6 mL of blood were collected from the wing vein of each bird and were divided
into 2 aliquots (3 mL each) in a 4-mL Vacutainer containing 7.2 mg K2 EDTA and a 4 mL Serum tube (BD,
Franklin Lakes, NJ) for determination of white blood
cell (WBC) composition and serum immunoglobulin
Y (IgY) concentrations, respectively. Birds were then
euthanized by cervical dislocation for the collection of
tissue samples. Two lymphoid organs (spleen and bursa
of the Fabricius) were excised, and the relative weight
was expressed as a percentage to the individual BW.
Spleen and terminal ileum tissues were aseptically ex-
Ingredient (% of diet)
FOS2
Wheat
Corn
Soybean meal
Canola meal
Canola oil
Calcium carbonate
Dicalcium phosphate
DL-Metionine
L-Lysine HCl
Threoninie
Mineral premix3
Vitamin premix4
Calculated composition5
ME (kcal/kg)
CP (%)
Ca (%)
Available P (%)
Met + Cys (%)
Met (%)
Lys (%)
Thr (%)
Analyzed composition
CP (%)
DM (%)
PC
SHANG ET AL.
Gene2
GenBank
Accession No.
IL-1
IL-2
IL-6
IL-10
IL-18
IFNTLR-4
-actin
CACAGAGATGGCGTTCGTTC
CGTAAGTGGATGGTTTTCCTCT
TTCGACGAGGCAAGGAACC
GCTCTCCTTCCACCGAAACC
ACTGCCAGAAGAGACATGGTG
GCATCTCCTCTGAGACTGGC
TCCGTGCCTGGAGGTAAGT
CAACACAGTGCTGTCTGGTGGTA
GCAGATTGTGAGCATTGGGC
GGCTAAAGCTCACCTGGGTC
AGGTCTGAAAGGCGAACAGG
GGAGCAAAGCCATCAAGCAG
CTCTGAGGGGTGTTCTGGTG
GCTCTCGGTGTGACCTTTGT
TGCCTTGGTAACAGCCTTGA
ATCGTACTCCTGCTTGCTGATCC
NM204524
NM204153
NM204628
AJ621614
NM204608
NM205149
NM001030693
X00182
1
2
Product length
(base pair)
Annealing
temperature
(o C)
118
161
175
198
143
159
155
560
58
55
59
58
56
58
57
58
The listed oligonucleotides were used to analyze intestinal gene expression via quantitative real-time PCR.
IL = interleukin; IFN = interferon; TLR = Toll-like receptor.
Statistical Analysis
The main effects of diet, immunological challenge,
and their interaction were subjected to 2-way ANOVA
by using the GLM procedure of SAS 9.2 (SAS Inst.,
2001). Differences among groups were considered significant at P < 0.05.
RESULTS
Growth Performance and Lymphoid Organ
Weight
Dietary effects related to growth performance such as
BWG, FI, feed conversion ratio, and mortality were not
significantly different (P > 0.05) among dietary treatments (data not shown). Similarly, the relative weight
of spleen (0.086% of BW, 0.093% of BW, and 0.100% of
BW, respectively) and bursa of the Fabricius (0.234% of
BW, 0.259% of BW, and 0.260% of BW, respectively)
did not show significant differences (P > 0.05) among
PC, NC, and NC + FOS groups (data not shown).
Intestinal Morphology
Villus height was significantly higher in the ileum of
broiler chickens fed NC + FOS diet (P = 0.007), when
compared with those fed PC or NC (Table 4). Significantly higher crypt depth and total mucosa thickness
also were observed in the ileum of NC + FOS fed birds
(P = 0.046 and P = 0.010, respectively), when compared with those fed PC. However, no difference was
observed on the VH:CD ratio in the ileum segments.
The duodenum and jejunum did not exhibit any difference (P > 0.05) on villus height, crypt depth, VH:CD
ratio, or total mucosa thickness among all dietary treatment groups.
PC
1,972.81
1454.77
734.12b
214.25
202.66
152.00b
1903.11
1375.37
787.14b
212.95
189.39
175.10a,b
9.37
7.33
4.92
9.43
7.45
4.61
2388.18
1876.87
1110.53b
2322.82
1785.38
1223.62a,b
NC + FOS
SEM
P-value3
2029.20
1363.31
910.03a
51.066
40.639
23.480
0.603
0.628
0.007
226.06
181.85
192.27a
7.074
5.589
6.628
0.708
0.362
0.046
0.394
0.251
0.137
0.992
0.912
0.628
50.277
47.437
34.193
0.452
0.663
0.010
9.30
7.60
4.83
2476.80
1779.97
1362.70a
1
Means of one cross-section from each of the three intestinal segments per bird, 12 birds
per diet, and 6 measurements of each villus height, crypt depth, and mucosa thickness per
cross-section for a total of 216 measurements for each of the intestinal segments per dietary
treatment.
2
Positive control (PC), wheat-corn-soybean meal basal diet supplemented with 5.5 mg
virginiamycin (Stafac-44) and 99 mg monensin sodium (Coban) in the vitamin premix. Negative control (NC), wheat-corn-soybean meal basal diet omitted with Stafac-44 and Coban.
NC + FOS, NC diet supplemented with 0.5% fructooligosaccharides (FOS).
3
Only dietary effects were presented (n = 12/ treatment) in the table; immunological
challenge on d 21 exhibited no effect on the small intestine morphology.
4
Total thickness of villus, crypt, and muscularis mucosa.
a,b
Means with different superscripts within a row differ significantly (P < 0.05).
Table 5. Main effects of diet and challenge on relative percentage of leucocytes composition
of broiler chickens at 21 days of age.1
Diet2
Relative %
Heterophils
Lymphocytes
Monocytes
Basophil
Eosinophil
H:L ratio
PC
a,b
59.13
29.50
6.75a,b
3.75
0.88
2.42
NC
Challenge3
NC + 0.5% FOS
a
63.00
27.00
5.33b
3.67
1.00
2.84
55.56
29.22
9.78a
5.00
0.44
3.14
P-value
0.011
0.769
0.049
0.478
0.343
0.672
Saline
b
47.09
39.64a
7.73
4.82
0.73
1.23b
LPS
a
68.13
20.40b
7.00
3.67
0.80
3.98a
P-value
SEM
< 0.001
< 0.001
0.753
0.282
0.893
< 0.001
2.297
2.353
0.754
0.495
0.160
0.404
1
Values are the means of 6 birds per treatment, in a total of 12 birds per diet and 18 birds per
challenge.
2
Positive control (PC), wheat-corn-soybean meal based diet supplemented with 5.5 mg virginiamycin
(Stafac-44) and 99 mg monensin sodium (Coban) in the vitamin premix. Negative control (NC), wheatcorn-soybean meal based diet omitted with Stafac-44 and Coban. NC + FOS, NC diet supplemented
with 0.5% fructooligosaccharides (FOS).
3
Chickens were intraperitoneally injected with either 2 mL/kg of BW Salmonella Enteritidis
Lipopolysaccaride (LPS) or sterile phosphate buffered saline (PBS).
a,b
Means with different superscripts within a row differ significantly (P < 0.05).
Villus height ( m)
Duodenum
Jejunum
Ileum
Crypt depth ( m)
Duodenum
Jejunum
Ileum
Villus height: crypt depth
Duodenum
Jejunum
Ileum
Total mucosa thickness4 ( m)
Duodenum
Jejunum
Ileum
NC
SHANG ET AL.
Figure 1. a) Natural serum immunoglobulin (Ig)G concentration ( g/mL) in broiler chickens fed positive control (PC), basal diet supplemented with antibiotics virginiamycin and monensin sodium; negative control (NC), PC diet that omitted antibiotics; and NC + FOS, NC diet
supplemented with 0.5% fructooligosaccharides (FOS) under phosphate buffered saline (PBS) injection as control or Salmonella lipopolysaccharide (LPS) challenge conditions. b) Specific serum IgY level (expressed as optical density) in response to salmonella LPS of broiler chickens fed
PC, NC, and NC + FOS diet under PBS injection or Salmonella LPS challenge conditions (n = 6/treatment). Error bars represent standard
error of the mean. Different letters (a to c) represent treatments that differed significantly (P < 0.05), # and represent significant main effects
of immunological challenge (P < 0.05), and asterisks represent significant diet challenge interactions (P < 0.0001).
Figure 3. Relative splenic gene expressions of toll-like receptor (TLR) -4, interleukin (IL)-1, IL-2, IL-6, IL-10, IL-18, and interferon (IFN)
of chickens fed positive control (PC), basal diet supplemented with antibiotics virginiamycin and monensin sodium; negative control (NC),
PC diet that omitted antibiotics; and NC + FOS, NC diet supplemented with 0.5% fructooligosaccharides (FOS) under phosphate buffered saline
(PBS) injection as control or Salmonella lipopolysaccharide (LPS) challenge conditions (n = 6/treatment). Gene expressions were calculated
relative to the housekeeping gene -actin. Error bars represent means standard errors of the mean. Symbols # and represent significant main
effects of immunological challenge (P < 0.05). Error bars represent means standard errors of the mean. Symbols # and represent significant
main effects of immunological challenge (P < 0.05), as analyzed by 2-way ANOVA for 3 2 factorial arrangement. Diet challenge interactions
and dietary effect were not significant.
broiler chickens, as expected, the Salmonella LPS challenge also significantly increased the expression of IL1, -2, -6, and -18 (P = 0.0002, P = 0.0477, P =
0.0190, and P = 0.0006, respectively), but not TLR-4,
IL-10, or IFN- , when compared to the PBS injection
groups (Figure 3). No dietary or diet challenge interaction effects were observed in spleen samples.
Figure 2. Relative ileal expressions of toll-like receptor (TLR) -4, interleukin (IL)-1, IL-2, IL-6, IL-10, IL-18, and interferon gamma (IFN )
mRNA in chickens fed basal diet supplemented with antibiotics (PC); basal diet without antibiotics (NC); and antibiotic free diet supplemented
with 0.5% fructooligosaccharides (NC + FOS) under phosphate buffered saline (PBS) or Salmonella lipopolysaccharide (LPS) challenges (n =
6/treatment). Error bars represent means standard errors of the mean. Symbols # and represent significant main effects of immunological
challenge (P < 0.05). Bars with asterisks represent significant main dietary effect (P < 0.05) within each mRNA expression, as analyzed by 2-way
ANOVA for 3 2 factorial arrangement. Diet challenge interactions were not significant.
SHANG ET AL.
DISCUSSION
Dietary Effects on Growth Performance
and Lymphoid Organ Weight
10
SHANG ET AL.
ACKNOWLEDGMENT
This project was financially supported by the National Sciences and Engineering Research Council
(NSERC)-Engage grant, the Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ009422), Rural Development Administration, Republic of Korea, and University of
Georgia startup grant. The authors would like to thank
Drs. Bogdan A. Slominski and Anna Rogiewicz (University of Manitoba, Winnipeg, MB, Canada) for their
insight and guidance throughout the poultry trial. Special thanks to Mohammed Alizadeh for his helpful discussions and technical assistance on immune response
related analyses. The authors also appreciate the generous support from Ingredion Inc. (Etobicoke, ON,
Canada) for providing FOS for this research.
REFERENCES
Ammerman, E., C. Quarles, and P. V. Twining. 1988. Broiler response to the addition of dietary fructooligosaccharides. Poult.
Sci. 67:46. (Abstr.)
Bailey, J. S., L. C. Blankenship, and N. A. Cox. 1991. Effect of
fructo-oligosaccharide on Salmonella colonization of the chicken
intestine. Poult. Sci. 70:24332438.
Beal, R. K., C. Powers, T. F. Davison, and A. L. Smith. 2006.
Immunological development of the avian gut. Pages 85103 In:
Avian Gut Function in Health and Disease. G. C. Perry, ed. CABI
Publishing, Oxford.
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