Effect of Using Organic Acids to Substitute Antibiotic Growth Promoters on Performance and Intestinal Microflora of Broilers

Abstract

A grower broiler experiment (from 14 to 35 days of age) was conducted to study the effect of using two commercial mixtures of organic acids (Galliacid and Biacid) to substitute antibiotic growth promoter (Eneramycin) on performance, carcass characteristics and intestinal microflora. 400 (Ross 308) broiler chicks were used. A basal corn-soybean meal diet were formulated and served as a control treatment. The control diet was supplemented with either 0.06% Galliacid, 0.1% Biacid or 0.02% Eneramycin. Birds fed the Galliacid-supplemented diet had 16% (p

Full text

1348
INTRODUCTION
Poultry are vulnerable to potentially pathogenic
microorganisms such as Escherichia coli, Salmonella ssp.,
and Clostridium. Pathogenic microflora in the small
intestine compete with the host for nutrients and also reduce
the digestion of fat and fat-soluble vitamins due to
deconjugating effects of bile acids (Engberg et al., 2000).
This depresses growth performance and increases incidence
of disease.
Antibiotics have been given at sub therapeutic dosage
(as feed additive) to stabilize the intestinal microflora and
improve the general performances and prevent some
specific intestinal pathology promote growth for about 50
years (Dibner and Richards, 2005). The long term and
extensive use of antibiotics has resulted in selection of
resistant bacterial strains. Resistance among Gram-negative
bacteria, like E. coli and Salmonella spp., has generated the
strongest objection to antibiotic use (Gustafson and Bowen,
1997). Nayak and Kenney (2002) showed that 25% of the
Salmonella isolates from turkey flocks in West Virginia
were resistant to one or more antibiotics, including
gentamycin, streptomycin, tetracycline, tobramycin, and
trimethoprim. Therefore, using antibiotic growth promoters
have been under scrutiny for many years (Ratcliff, 2000).
Recently, poultry industry has paid more attention towards
addressing public concern for environmental and food
safety. Thus, the non-prescription use of antibiotics in
poultry feeds has been eliminated or severely limited. The
European Union banned the use of sub-therapeutic levels of
antibiotics to prevent disease or promote growth, starting
with a ban on avoparcin in 1997 and a ban on virginiamycin,
bacitracin, spiromycin, and tylosin in 1999. Antimicrobials
banned by January 2006 included avilamycin, bambermycin,
salinomycin and monensin.
So, alternatives to antibiotics are of great interest to the
poultry industry (Waldroup et al., 2003). These alternatives
Asian-Aust. J. Anim. Sci.
Vol. 23, No. 10 : 1348 - 1353
October 2010
www.ajas.info
Effect of Using Organic Acids to Substitute Antibiotic Growth
Promoters on Performance and Intestinal Microflora of Broilers
H. M. A. Hassan*, M. A. Mohamed, Amani W. Youssef and Eman R. Hassan
1
Department of Animal Production, National Research Center, 12311, Dokki, Egypt
ABSTRACT :
A grower broiler experiment (from 14 to 35 days of age) was conducted to study the effect of using two commercial
mixtures of organic acids (Galliacid
®
and Biacid
®
) to substitute antibiotic growth promoter (Eneramycin
®
) on performance, carcass
characteristics and intestinal microflora. 400 (Ross 308) broiler chicks were used. A basal corn-soybean meal diet were formulated and
served as a control treatment. The control diet was supplemented with either 0.06% Galliacid, 0.1% Biacid or 0.02% Eneramycin. Birds
fed the Galliacid-supplemented diet had 16% (p<0.001) more gain than the control, while those fed the Biacid- or Enramycin-
supplemented diets recorded 3 and 5.5% more gain, respectively. Organic acids mixtures and Enramycin supplementation significantly
(p<0.001) improved feed conversion ratio. These results indicated that birds fed either organic acid mixtures or Enramycin-
supplemented diets utilized feed more efficiently than those fed the control diet. Galliacid significantly (p<0.01) increased dressing
percentage and bursa weight (% body weight). No significant differences were detected on liver, spleen and thymus (% body weight)
among treatments. Galliacid or Biacid significantly (p<0.001) decreased intestinal Escherichia coli and Salmonella compared to the
control and Enramycin-supplemented diets. Dietary Enramycin significantly (p<0.001) decreased Escherichia coli, but had no effect on
Salmonella counts. In conclusion, organic acid mixtures are more efficient than antibiotic growth promoter (Enramycin) in improving
broiler performance and decreasing intestinal Escherichia coli and Salmonella spp., and could be successfully used to substitute
antibiotic growth promoters in broiler diets. However, not all of the organic acid mixtures gave the same effect either on performance or
intestinal bacterial counts. (Key Words : Organic Acids, Antibiotic, Broiler, Performance, Carcass, Intestinal Bacteria)
* Corresponding Author: H. M. A. Hassan.
E-mail: husseinhma@yahoo.com
1
Department of Poultry Diseases, National Research Center,
12311 Dokki, Egypt.
Received March 9, 2010; Accepted April 23, 2010

Hassan et al. (2010) Asian-Aust. J. Anim. Sci. 23(10):1348-1353
1349
include acidifiers (organic acids), prebiotics, probiotics,
enzymes, herbal products, microflora enhancers, and
immuno-modulators.
Most of these alternatives have effects on microflora,
directly or indirectly (Richards et al., 2005). Organic acids
have strong bacteriostatic effects and have been used as
Salmonella-control agents in feed and water supplies for
poultry (Ricke, 2003). Acidification with various weak
organic acids to diets such as formic, fumaric, propionic,
lactic and sorbic acid have been reported to decrease
colonization of pathogen and production of toxic
metabolites, improve digestibility of protein and of Ca, P,
Mg and Zn and serve as substrates in the intermediary
metabolism (Kirchgessner and Roth, 1988). Several studies
demonstrated that supplementation of organic acids to
broiler diets increased growth performance, reduced
diseases and management problems (Vlademirova and
Sourdjiyska, 1996; Runho et al., 1997; Jin et al., 1998;
Gunal et al., 2006; Islam et al., 2008; Ao et al., 2009). Most
previous studies have used a single organic acid as a dietary
supplement. Few studies have been conducted concerning
the effect of mixtures of different organic acids and their
capability of such mixtures to substitute antibiotic as
growth promoters in broiler diets. Therefore, the objective
of this study was to determine the effect of using two
different commercial mixtures of organic acids to substitute
dietary antibiotic growth promoters on performance, carcass
characteristics, and intestinal microflora of broiler chickens.
MATERIALS AND METHODS
Birds, diets and housing
A total number of 500 unsexed one day old (Ross 308)
broiler chicks were used in this study. Chicks were brooded
in a warmed fumigated brooder house and fed on a starter
diet to 14 days of age. Birds were then individually weighed
and 400 chicks with almost the same body weight were
divided into four groups (5 replicates of 20 chicks, each).
The average initial live body weight of all replicates was
similar. Replicates were randomly allocated in batteries of
three-tier system that has 20 compartments (5 replicates×4
dietary treatments).
Two commercial mixtures of organic acids (Galliacid
®
and Biacid
®
) as acidifiers and antibiotic (Enramycin
®
) as
growth promoters were used in this study. Galliacid
®
consisted of a mixture of fumaric acid, calcium format,
calcium propionate, potassium sorbate and hydrogenated
vegetable oil. These organic acids are coated and protected
(microencapsulated) by a matrix of fatty acids. Biacid
®
consisted of a mixture of citric acid, calcium formate,
calcium butyrate, calcium lactate, essential oils and
flavoring compounds. Enramycin
®
is a polypeptide
antibiotic produced by Streptomyces fungicidus.
A basal grower diet was formulated to cover all the
nutrient requirements of (Ross 308) broilers. The basal diet
included phytase and xylnase enzyme. The formulation and
nutrient composition of the starter and the basal grower diet
is shown in Table 1. Phytase addition allows reduction of
dietary available phosphorus by 0.1% (Sohail and Roland,
1999) while xylanase allows to reduction of the apparent
metabolizble energy by 3 to 4% in feed formulas (Cowan et
al., 1996; Zhou et al., 2009).
Four experimental grower diets were used. The basal
diet served as a control treatment. The control diet was
supplemented with Galliacid
®
(0.06%), Biacid
®
(0.10%) or
Enramycin
®
(0.02%). Levels of supplementation were
recommended by the producers. Birds were fed the
experimental diets for ad libtum consumption from 14 to 35
days of age.
Gas heaters were used to keep the required temperature
and light was provided 23 h daily during the experiment.
Birds were vaccinated against AI, ND, IB and IBD
throughout the experimental period. After such medical
treatments, a dose of vitamins (AD
3
E) was offered in the
drinking water for the successive 3 days.
Table 1. Formulation and nutrient composition of starter and
grower basal diets
Ingredients % Starter diet Grower diet
Yellow corn 54.31 58.50
Soybean meal (48%) 36.50 32.20
Corn gluten meal (60%) 2.00 2.00
Vegetable oil 2.50 3.30
Limestone 1.37 1.40
Dicalcium phosphate 1.80 1.30
Vitamin and mineral mix
1
0.35 0.35
Salt 0.35 0.35
L-lysine HCl 0.37 0.20
DL-methionine 0.30 0.25
Phytase 0.10 0.10
Xylase 0.05 0.05
Total 100 100
Calculated composition
2
(%)
Crude protein 24.00 22.00
ME (kcal/kg) 2,950 3,050
Lysine 1.42 1.20
Methionine 0.72 0.64
Methionine+cystine 1.00 0.93
Calcium 1.02 0.90
Nonphytate P 0.45 0.37
1
Vitamin-mineral mixture supplied per kg of diet: Vit. A, 12,000 IU; Vit.
D
3
, 2,200 IU; Vit. E, 10 mg; Vit. K
3
, 2 mg; Vit. B
1
, 1 mg; Vit. B
2
, 4 mg;
Vit. B
6
, 1.5 mg; Vit. B
12,
10 μg; Niacin, 20 mg; Pantothenic acid, 10 mg;
Folic acid, 1 mg; Biotin, 50 μg; Choline chloride, 500 mg; Copper, 10
mg; Iodine, 1 mg; Iron, 30 mg; Manganese, 55 mg; Zinc, 50 mg and
Selenium, 0.1 mg.
2
Calculated values based on feed composition Tables of NRC (1994).

Hassan et al. (2010) Asian-Aust. J. Anim. Sci. 23(10):1348-1353
1350
After fasting overnight, birds were individually weighed
and feed consumption was recorded per replicate at 21, 28
and 35 days of age. Body weight gain and feed conversion
ratio were calculated weekly and for the entire period (from
14 to 35 days of age).
Carcass measurements
At day 35, ten birds per treatment were randomly taken
to study carcass characteristics. Chicks were fasted for
approximately 12 h; and then individually weighed,
slaughtered, feathered and eviscerated. Weights of carcass,
liver, spleen and bursa were recorded. The percentage of
carcass and organs (% of live body weight) was calculated.
The preparation of competitive exclusion native gut
microflora
Caecal contents were immediately collected from the
slaughtered birds and tested for the absence of Salmonella
ssp. or E. coli. Half a gram of such material was inoculated
into 10 ml Trypticase Soya broth (Weinack et al., 1979) and
incubated at 37°C for 48 h; anaerobically. Using 0.2 ml of
the broth culture was then transferred to anther 10 ml tube
of Trypticase Soya broth and incubated for 48 h; an
aerobically at 37°C. Salmonella and E. coli colonies
counting, (Collins and Lyne, 1984) caecal content
specimens were taken a septically one gram of each caecal
content was mixed with 9 ml saline for preparation of a ten
fold dilution. One ml from each dilution was spread on
brilliant green agar plate and incubated at 37°C for 24 h.
The total colony count for salmonella and E. coli was then
calculated as the number of colonies by reciprocal of the
dilution. The microbial counts were determined as colony
forming units (cfu) per gram of sample.
Statistical analysis
Data were statistically analyzed for analysis of variance
using the General Liner Model of SAS Institute (1990).
Significant differences among treatment means were
separated by Duncan’s new multiple rang test (Duncan,
1955) with a 5% level of probability.
RESULTS AND DISCUSSION
Table 2 shows the effect of dietary treatments on the
productive performance (body weight gain (BWG), feed
intake (FI) and feed conversion ratio (FCR)) recorded
weekly and for the entire period (from 14 to 35 days of age).
The results of BWG showed that birds fed Galliacid
supplemented diet exhibited significant (p<0.05 for the first
and second periods; and p<0.001 for the third and entire
periods) more gain than the other groups. At 35 day of age,
this group gave 16% more gain compared to the control
group. Birds fed the Biacid or Enramycin supplemented
diets recorded 3 and 5.5% more gain, respectively, than the
control birds.
Feed intake did not differ significantly among
treatments during the periods from 14 to 21 and from 22 to
28 days of age. However, during the period from 29 to 35
days of age and the entire period (14-35) birds fed Galliacid
supplemented diet consumed significantly (p<0.05) more
feed than the other groups. No significant differences were
detected on either BWG or FI between the control group
and those fed Biacid or Enramycin supplemented diets
during the different intervals or the entire period. The
results of FCR showed that addition of organic acids
mixture or Enramycin did significantly (p<0.01) improve
FCR. Birds fed Biacid or Enramycin supplemented diets
gave almost the same values of FCR during the different
intervals and the entered period. Birds fed Galliacid
supplemented diets showed significant (p<0.01) posterior
value of FCR compared to the control and the other
supplemented diets. Although, birds fed Galliacid
supplemented diets consumed significantly (p<0.05) more
feed than the other group it recorded the best value of FCR.
An improvement in value of FCR being 7, 3 or 2% was
obtained when the control diet was supplemented with
Galliacid, Biacid or Enramycin, respectively. These results
indicated the superiority of Galliacid compared to Biacid or
Enramycin. Addition of either organic acid mixtures or
Enramycin improved the performance of growing broilers
expressed as BWG or FCR. Birds fed such supplemented
Table 2. Effect of dietary treatments on growth performance of broiler chicks
Diets
Dietary
treatments
14-21 days 22-28 days 29-35 days 14-35 days
WG FI FCR WG FI FCR WG FI FCR WG FI FCR
Control 347
b
566 1.63
a
407
b
680 1.67
a
458
b
833
b
1.82
a
1,212
b
2,079
b
1.72
a
Diet 1 0.06%
galliacid
374
a
592 1.58
d
457
a
728 1.59
c
574
a
925
a
1.61
c
1,405
a
2,245
a
1.60
c
Diet 2 0.1% biacid 348
b
559 1.60
c
426
ab
702 1.65
b
479
b
836
b
1.75
b
1,253
b
2,097
b
1.67
b
Diet 3 0.02%
enramycin
349
b
562 1.61
b
440
ab
727 1.65
b
490
b
856
b
1.75
b
1,279
b
2,145
b
1.68
b
SE of means
±4.3 ±5.7 ±0.01
±
6.9
±
10.6
±
0.01
±
12.9
±
12.8
±
0.02 ±21 ±22.2
±
0.02
Significances * NS ** * NS ** *** * *** *** * ***
a-d
Mean within each column with no common superscript differ significantly (p<0.05).
* p<0.05, ** p<0.01, *** p<0.001. NS = Not significant (p>0.05).

Hassan et al. (2010) Asian-Aust. J. Anim. Sci. 23(10):1348-1353
1351
diets utilized feed more efficient than the control diets.
An improvement on values of BWG by 16% and FI by
8% was observed when the control diet was supplemented
with Galliacid.
The effects of dietary treatments on carcass
characteristics of 35 days old broilers are shown in Table 3.
Using Galliacid significantly (p<0.05) increased dressing
percentage and bursa weights relative to live body weight.
No significant differences were detected on liver, spleen
and thymus (% body weight) among treatments. However,
there were minor increases in the relative weight of liver,
spleen and thymus of birds fed Galliacid or Biacid
supplemented diets compared to those fed the control or
Enramycin supplemented diets.
The effects of dietary treatments on intestinal microflora
(Escherichia coli and Salmonella) of chicks fed the
different dietary treatments are shown in Table 4. The
results showed that the addition of Galliacid or Biacid
significantly (p<0.001) decreased Escherichia coli and
Salmonella compared to the control and Enramycin
supplemented diet. Dietary Enramycin significantly
(p<0.001) decreased Escherichia coli, but had no effect on
Salmonella. The pronounced decreased of Escherichia coli
and Salmonella ssp. bacteria counts were observed with
galliacid treatment. From the economic point of view, using
organic acid mixture costed about 5.25 dollars/ton feed
compare to 8.50 dollars for eneramycin antibiotic.
The results of the present study confirmed those
obtained by Naidu (2000), Fushimi et al. (2001), Gunes et
al. (2001), Gornowicz and Dziadek (2002), Wolfenden et al.
(2007) and Abd El-Hakim et al. (2009) who concluded that
organic acids could be used in poultry, not only as a growth
promoter but also as a meaningful tool of controlling
intrinsic pathogenic bacteria (E. coli and Salmonella). They
found that organic acids feeding improved feed conversion
ratio, growth performance, enhanced mineral absorption
and speeding recovery from fatigue. In addition, Gauthier
(2002) reported that, contrary to antibiotics, organic acids
have other properties like; lowering of the chyme pH
consequently, enhancing of protein digestion. On the other
hand, Denli et al. (2003) found that dietary organic acids
had no effect on the carcass yield and liver weight of broiler
chickens at 42 d old.
Abdel-Fattah et al. (2008) found that broiler chicks fed
dietary organic acids had superior improvement in live body
weight, body weight gain and feed conversion ratio
compared to those of unsupplemented diet. Owens et al.
(2008) reported that total live weight gain (12%) and gain:
feed (9%) of broiler chicks were significantly improved for
diets containing organic acids additives, compared to the
control diets. Ao et al. (2009) found that the basal diet
supplemented
with 2% citric acid of broilers significantly
(p<0.05) increased feed intake, weight gain,
AME
n
of the
diets, and retention of CP and neutral detergent
fiber (NDF).
Islam et al. (2008) concluded that fumaric acid (FA)
may promote growth of broilers, 1.25% FA group showed
significantly (p<0.05) better weight gain and better feed
efficiency than the groups with 5.0 and 7.5% FA. Higher
gain was associated with higher feed intake. Similar to the
present results, the relative weight of heart, liver and spleen
was not affected by the treatment.
The obtained results proved those of Abdel-Fattah et al.
(2008) who found that organic acids significantly increased
the relative weight of bursa. The resulted improvement in
Table 3. Effect of dietary treatments on dressing percent, and organ weights as percent of live body weight of broiler chicks at 35 days o
f
age
Diets
Dietary
treatments
Live body
weight
Carcass
weight
Dressing
%
Liver
%
Spleen
%
Thymus
%
Bursa
%
Control 1,564
b
1,130
b
72.21
b
2.27 0.12 0.37 0.105
c
Diet 1 0.06% galliacid 1,760
a
1,302
a
73.98
a
2.45 0.14 0.39 0.138
a
Diet 2 0.1% biacid 1,604
b
1,159
b
72.26
b
2.34 0.14 0.38 0.110
b
Diet 3 0.02% enramycin 1,628
b
1,179
b
72.43
b
2.19 0.12 0.36 0.107
c
SE of means
±21.24
±
19.46
±
0.26
±
0.065
±
0.003 ±0.011
±
0.004
Significances *** *** * NS NS NS **
a-d
Mean within each column with no common superscript differ significantly (p<0.05).
* p<0.05, ** p<0.01, *** p<0.001. NS = Not significant (p>0.05).
Table 4. Effect of dietary treatments on intestinal bacteria o
f
broiler chicks at 35 days of age
Diets
Dietary
treatments
E. coli ssp.
log
10
cfu/g
Salmonela ssp.
log
10
cfu/g
Control 6.392
a
5.491
a
Diet 1 0.06%
galliacid
4.415
c
3.690
c
Diet 2 0.1% biacid 4.459
c
4.194
b
Diet 3 0.02%
enramycin
5.151
b
5.513
a
SE of mean
±
0.21
±
0.21
Significances *** ***
a-c
Mean within each column with no common superscript differ
significantly (p<0.05).
*** p<0.001.

Hassan et al. (2010) Asian-Aust. J. Anim. Sci. 23(10):1348-1353
1352
growth performance associated with significant increase in
bursa weight proved that addition of Galliacid did
positively affect the immune system and resistance of
broilers againest desises. In this respect, Katanabdef et al.
(1989) reported that the increase in the relative immune
organs weight is considered as an indication of the
immunological advances.
Other feed additives like B-mannanase (Zou et al.,
2006), mashroom extract, probiotics (Willis et al., 2007),
ascorbic acid (Amakye-amin et al., 2000) could increase
bursa weight and improve growth performance and
immunity of broilers. Also, Mos (mannan oligosacchride)
exerts a significant growth-promoting effect by enhancing
the bird’s resistance to enteric pathogens (Ferket, 2004,
Mohamed et al., 2008).
Brul and Coote (1999) explained that the key basic
principle on the mode of action of organic acids on bacteria
is that non-dissociated organic acids can penetrate the
bacteria cell wall and disrupt the normal physiology of
certain types of bacteria that we call “pH sensitive”
meaning that they cannot tolerate a wide internal and
external pH gradient. Lee (2005) added that, more likely,
the organic acids in poultry might play a direct role on the
GIT bacteria population, reducing the level of some
pathogenic bacteria and mainly controlling the population
of certain types of bacteria that compete with the birds for
nutrients.
Dietary acidification inhibits of intestinal bacteria
competing with the host for available nutrients, and a
reduction of possibly toxic bacterial metabolites, thus
improving weight gain of the host animal. Furthermore, the
growth inhibition of potential pathogen bacteria and
zoonotic bacteria, e.g. E. coli and Salmonella, in the feed
and in the GI- tract are of benefit with respect to animal
health. In poultry production organic acids have mainly
been used in order to sanities the feed considering problems
with Salmonella infections (Iba and Berchieri, 1995;
Berchieri and Barrow, 1996; Thompson and Hinton, 1997).
The superiority of Galliacid over the Biacid may be
because of the microencapsulation the organic acids of
Galliacid are coated and protected by a matrix of fatty acids.
Thus, the organic acids can reach the intestine without
modification, where they are released slowly under the
action of lipase secretions. Non dissociated organic acids
can be active on bacteria and modulate the intestinal flora.
CONCLUSIONS
It could be concluded that, under the condition of the
present study, organic acids mixtures are more efficient than
antibiotic growth promoter (Enramycin) on improving
broiler performance and decreasing intestinal Escherichia
coli and Salmonella ssp. Not all of the organic acids
mixtures gave the same effect either on performance or
intestinal bacterial counts. From the obtained results and the
forgoing discussion, it could be reported that if organic
acids mixtures were used correctly along with nutritional,
managerial and biosecurity measures, they can be a
powerful tool in maintaining the health of the
gastrointestinal tract of poultry, thus improving their
performances and successfully used as growth promoters. It
is well known that, antibiotics at sub-dosage could prevent
necrotic enteritis. Organic acids mixtures proved to have the
same effect on stabilizing the intestinal microflora.
However, in case of necrotic enteritis challenge organic
acids mixtures are not comparable with antibiotics at
therapeutic dosage. Further, studies are needed to compare
using organic acids mixtures with antibiotics in different
experimental conditions.
REFERENCES
Abd El-Hakim, A. S., G. Cherian and M. N. Ali. 2009. Use of
organic acids, herbs and their combination to improve the
utilization of commercial low protein broiler diets. Int. J. Poult.
Sci. 8(1):14-20.
Abdel-Fattah, S. A., M. H. El-Sanhoury, N. M. El-Mednay and F.
Abdel-Azeem. 2008. Thyroid activity, some blood constituents,
organs morphology and performance of broiler chicks fed
supplemental organic acids. Int. J. Poult. Sci. 7(3):215-222.
Amakye-amin, J., T. L. Lin, P. Y. Hester, D. Thiagarajan, P. A.
Watkins and C. C. Wu. 2000. Ascorbic acid supplementation
improved antibody response to infection bursal disease
vaccination in chicks. Poult. Sci. 79:680-688.
Ao, T., A. H. Cantor, A. J. Pescatore, M. J. Ford, J. L. Pierce and K.
A. Dawson. 2009. Effect of enzyme supplementation and
acidification of diets on nutrient digestibility and growth
performance of broiler chicks. Poult. Sci. 88:111-117.
Berchieri, A., Jr. and P. A. Barrow. 1996. Reduction in incidence
of experimental fowl typhoid by incorporation of a commercial
formic acid preparation (Bio-Add
TM
) into poultry feed. Poult.
Sci. 75:339-341.
Brul, S. and P. Coote. 1999. Preservative agents in foods, mode of
action and microbial resistance mechanisms. Int. J. Food
Microbiol. 50:1-17.
Collins, C. H. and P. M. Lyne. 1984. Microbiological methods.
Fifth Edition, Butterworth's, London, 331- 345.
Cowan, W. D., A. Korsbak, T. Hastrup and P. B. Rasmussen. 1996.
Influence of added microbial enzymes on energy and protein
availability of selected feed ingredients. Anim. Feed Sci.
Technol. 60:311-319.
Denli, M., F. Okan and K. Celik. 2003. Effect of dietary probiotic,
organic acid and antibiotic supplementation to diets on broiler
performance and carcass yield. Pak. J. Nutr. 2:89-91.
Dibner, J. J. and J. D. Richards. 2005. Antibiotic growth promoters
in agriculture: history and mode of action. Poult. Sci. 84:634-
643.
Duncan, D. B. 1955. Multiple range test and multiple F tests.
Biometrics 11:1-42.
Engberg, R. M., M. S. Hedemann, T. D. Leser and B. B. Jensen.

Hassan et al. (2010) Asian-Aust. J. Anim. Sci. 23(10):1348-1353
1353
2000. Effect of zinc bacitracin and salinomycin on intestinal
microflora and performance of broilers. Poult. Sci. 79:1311-
1319.
Ferket, P. R. 2004. Alternatives to antibiotics in poultry
production: response, practical experience and
recommendations. In: Biotechnology in the Feed and Food
Industry (Ed. T. P. Lyons and K. A. Jacques), Proceedings of
Alltech’s 20th Annual Symposium, pp: 57-66. Nottingham
University Press, UK.
Fushimi, T., K. Tayama, M. Fukaya, K. Kitakoshi, N. Nakai, Y.
Tsukamoto and Y. Sato. 2001. Acetic acid feeding enhances
glycogen repletion in liver and skeletal muscle of rats. J. Nutr.
131:1973-1977.
Gauthier, R. 2002. Intestinal health, the key to productivity (The
case of organic acids) XXVII Convention ANECA-WPDSA
Puerto Vallarta, Jal. Mexico. 30 April 2002.
Gornowicz, E. and K. Dziadek. 2002. The effects of acidifying
preparations added to compound feeds on management
conditions of broiler chickens. Ann. Anim. Sci. (Suppl. 1):93-
96.
Gunal, M., G. Yayli, O. Kaya, N. Karahan and O. Sulak. 2006. The
effects of antibiotic growth promoter, probiotic or organic acid
supplementation on performance, intestinal microflora and
tissue of broilers. Int. J. Poult. Sci. 5(2):149-155.
Gunes, H., H. Cerit and A. Altinel. 2001. Etlik piliçlerin verim
özellikleri üzerine pre-probiotigin (Fermacto-500) etkisi. Ist.
Univ. Vet. Fak. Derg. 27:217-229.
Gustafson, R. H. and R. E. Bowen. 1997. Antibiotic use in animal
agriculture. J. Appl. Micro. 83:531-541.
Iba, A. M. and A. JR. Berchieri. 1995. Studies on the use of a
formic acid propionic acid mixture (Bio-add
TM
) to control
experimental Salmonella infection in broiler chickens. Avian
Pathol. 24:303-311.
Islam, K. M. S., A. Schuhmacher, H. Aupperle and J. M. Gropp.
2008. Fumaric acid in broiler nutrition: a dose titration study
and safety aspects. Int. J. Poult. Sci. 7(9):903-907.
Jin, L. Z., Y. W. Ho, N. Abdullah, M. A. Ali and S. Jalaluddin.
1998. Effects of adherent Lactobacillus cultures on growth,
weight of organs and intestinal microflora and volatile fatty
acids in broilers. Anim. Feed Sci. Technol. 70:197-209.
Katanbaf, M. N., E. A Dunington and P. B. Siegel. 1989.
Restricted feeding in early and late feathering chickens,
growth and physiological responses. Poult. Sci. 68:344-351.
Kirchgessner, V. M. and F. X. Roth. 1988. Ergotrope effekte durch
organische sauren in der ferkelaufzucht und schweinemast.
Ubers. Tierernaehr, 16:93-108.
Lee, M. D. 2005. Molecular basis for AGP effects in animals, p.
37-38. Antimicrobial Growth Promoters: Worldwide Ban on
the Horizon. Noordwijk and Zee, the Netherlands, Jan 31-Feb
1.
Mohamed, M. A., H. M. A.
Hassan and E. M. A.
El-Barkouky.
2008. Effect of mannan oligosaccharide on performance and
carcass characteristics of broiler chicks. J. Agric. Soc. Sci.
4:13-17.
Naidu, A. S. 2000. Natural food antimicrobial systems. CRC Press
USA. pp. 431-462.
National Research Council (NRC). 1994. Nutrient requirements of
poultry. 9th rev. ed. National Academy press, Washington, DC.
Nayak, R. and P. B. Kenney. 2002. Screening of Salmonella
isolates from a turkey production facility for antibiotic
resistance. Poult. Sci. 81:1496-1500.
Owens, B., L. Tucker, M. A. Collins and K. J. McCracken. 2008.
Effects of different feed additives alone or in combination on
broiler performance, gut microflora and ileal histology. Br.
Poult. Sci. 49(2):202-212.
Ratcliff, J. 2000. Antibiotic bans-a European perspective. Pages:
135-152 in Proceeding of the 47 Maryland Nutrition
Conferences for Food the Manufacturers, March 22-24.
Richards, J. D., J. Gong and C. F. M. de Lange. 2005. The
gastrointestinal microbiota and its role in monogastric nutrition
and health with an emphasis on pigs: Current understanding,
possible modulations and new technologies for ecological
studies. Can. J. Anim. Sci. 85:421-435.
Ricke, S. C. 2003. Perspectives on the use of organic acids and
short chain fatty acids as antimicrobials. Poult. Sci. 82:632-
639.
Runho, R. C., N. K. Sakomura, S. Kuana, D. Banzatto, O. M.
Junoqueria and J. H. Stringhini. 1997. Uso do acido organico
(acido fumarico) nas racoes de frangos de corte. Revista
Brasileira de Zootecnia, 26:1183-1191.
SAS Institute. 1990. SAS
®
/STAT User’s Guide: Statistics, Version
6, 4
th
edition. SAS Institute Inc, Cary, NC.
Sohail, S. S. and D. A. Roland. 1999. Influence of supplemental
phytase on performance of broilers four to six weeks of age.
Poult. Sci. 78:550-555.
Thompson, J. L. and M. Hinton. 1997. Antibacterial activity of
formic and propionic acids in the diet of hens on salmonellas
in the crop. Br. Poult. Sci. 38:59-65.
Vlademirova, L. and S. Sourdjiyska. 1996. Test on the effect from
adding probiotics to the combined feeds for chicks. J. Anim.
Sci. 3:36-39.
Waldroup, P. W., E. O. Oviedo-Rondon and C. A. Fritts. 2003.
Comparison of Mos and antibiotic feeding programs in broiler
diets containing copper sulfate. Int. J. Poult. Sci. 2:28-31.
Weinack, O. M., C. F. Smyser and G. H. Soneyenbos. 1979.
Evaluation of several methods of detecting salmonella in
groups of chickens. Avian Dis. 23:179-193.
Willis, W. L., O. S. Isikuemhen and S. A.Ibrahim. 2007.
Performance assessment of broiler chickens given mashroom
extract alone or in combination with probiotics. Poult. Sci.
86:1856-1860.
Wolfenden, A. D., J. L. Vicente, J. P. Higgins, R. L. Andreatti
Filho, S. E. Higgins, B. M. Hargis and G. Tellez. 2007. Effect
of organic acids and probiotics on salmonella enteritidis
infection in broiler chickens. Int. J. Poult. Sci. 6:403-405.
Zhou, Y., Z. Jiang, D. Lv and T. Wang. 2009. Improved energy-
utilizing efficiency by enzyme preparation supplement in
broiler diets with different metabolizable energy levels. Poult.
Sci. 88:316-322.
Zou, X. T., X. J. Oiao and Z. R. Xu. 2006. Effect of β-mannanase
(hemicell) on growth performance and immunity of broilers.
Poult. Sci. 85:2176-2179.