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A 57-day study was performed to determine the effects of two aqueous extracts (AEs) on broiler performance, immune function and intestinal microflora. Four groups of 75 one-day-old female broilers (Ross308) received one of the following treatments: (1) a standard commercial feed (C); (2) C supplemented with 2 g/kg rosemary AE (R); (3) C supplemented with 2 g/kg oregano AE (O); (4) C supplemented with 1 g/kg oregano AE + 1 g/kg rosemary AE (OR). Individual body weight, average daily gain, average daily feed intake and feed conversion efficiency were determined at 1, 11, 22, 36 and 57 days. Sample collections for IgG titration and intestinal microflora examination were performed at 22 and 57 days. The addition of oregano AE alone or in combination with rosemary AEs improved body weight up to 36 days of age (P < .01). A time effect was recorded for total serum IgG in all groups (P < .001) and the percentage increase of the value was positively (P < .05) influenced by the AE supplementation. Lactobacilli raised (P < .001) in ileum and cecum of all groups supplemented with AEs. Staphylococcus spp. population was constantly lower in both intestinal tracts of the AE supplemented groups. On the basis of our results, AEs could improve broiler performance and immune function and contribute to a balanced gut microflora, essential for the digestion process and protection against enteropathogenic organisms.
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Journal of Applied Animal Research
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Effects of oregano (Origanum vulgare L.) and
rosemary (Rosmarinus officinalis L.) aqueous
extracts on broiler performance, immune function
and intestinal microbial population
Maria Pia Franciosini, Patrizia Casagrande-Proietti, Claudio Forte, Daniela
Beghelli, Gabriele Acuti, Dario Zanichelli, Alessandro dal Bosco, Cesare
Castellini & Massimo Trabalza-Marinucci
To cite this article: Maria Pia Franciosini, Patrizia Casagrande-Proietti, Claudio Forte,
Daniela Beghelli, Gabriele Acuti, Dario Zanichelli, Alessandro dal Bosco, Cesare Castellini &
Massimo Trabalza-Marinucci (2016) Effects of oregano (Origanum vulgare L.) and rosemary
(Rosmarinus officinalis L.) aqueous extracts on broiler performance, immune function and
intestinal microbial population, Journal of Applied Animal Research, 44:1, 474-479, DOI:
10.1080/09712119.2015.1091322
To link to this article: http://dx.doi.org/10.1080/09712119.2015.1091322
Published online: 18 Oct 2015.
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Effects of oregano (Origanum vulgare L.) and rosemary (Rosmarinus ofcinalis L.)
aqueous extracts on broiler performance, immune function and intestinal microbial
population
Maria Pia Franciosini
a
, Patrizia Casagrande-Proietti
a
, Claudio Forte
a
, Daniela Beghelli
b
, Gabriele Acuti
a
,
Dario Zanichelli
c
, Alessandro dal Bosco
d
, Cesare Castellini
d
and Massimo Trabalza-Marinucci
a
a
Department of Veterinary Medicine, University of Perugia, Perugia, Italy;
b
School of Biosciences and Veterinary Medicine, University of Camerino,
Macerata, Italy;
c
Phenbiox srl, Bologna, Italy;
d
Department of Agricultural, Food and Environmental Sciences, University of Perugia, Perugia, Italy
ABSTRACT
A 57-day study was performed to determine the effects of two aqueous extracts (AEs) on broiler performance,
immune function and intestinal microora. Four groups of 75 one-day-old female broilers (Ross308) received
one of the following treatments: (1) a standard commercial feed (C); (2) C supplemented with 2 g/kg
rosemary AE (R); (3) C supplemented with 2 g/kg oregano AE (O); (4) C supplemented with 1 g/kg
oregano AE + 1 g/kg rosemary AE (OR). Individual body weight, average daily gain, average daily feed
intake and feed conversion efciency were determined at 1, 11, 22, 36 and 57 days. Sample collections for
IgG titration and intestinal microora examination were performed at 22 and 57 days. The addition of
oregano AE alone or in combination with rosemary AEs improved body weight up to 36 days of age (P
< .01). A time effect was recorded for total serum IgG in all groups (P < .001) and the percentage increase
of the value was positively (P < .05) inuenced by the AE supplementation. Lactobacilli raised (P < .001) in
ileum and cecum of all groups supplemented with AEs. Staphylococcus spp. population was constantly
lower in both intestinal tracts of the AE supplemented groups. On the basis of our results, AEs could
improve broiler performance and immune function and contribute to a balanced gut microora, essential
for the digestion process and protection against enteropathogenic organisms.
ARTICLE HISTORY
Received 25 March 2014
Accepted 28 April 2015
KEYWORDS
Aqueous extracts; broiler
performance; immune
function; intestinal
microora; oregano;
rosemary
1. Introduction
Since 2006 the banning of the use of antibiotics by the European
Commission has focused the attention of the scientic commu-
nity on the development of alternative products, able to
replace antibiotics while achieving the same productivity. In
this view, aromatic plants and extracts obtained from these
plants have become increasingly important due to their several
positive effects on animals. Currently, the chemical components
of most plant extracts are generally recognized to be safe and
are commonly used in the food industry (Varel 2002), because
they are well-accepted by consumers who perceive the nal
product to be healthier. Oregano (Origanum vulgare L.) and
rosemary (Rosmarinus ofcinalis L.) have been particularly
studied for their properties. Specically the oregano essential
oils (EOs) possess in vitro antimicrobial (Lambert et al. 2001), anti-
fungal (Kalemba & Kunicka 2003), insecticidal (Karpouhtsis et al.
1998) and antioxidant (Botsoglou et al. 2004) properties, due to
carvacrol and thymol (Adam et al. 1998). Another Labiatae
species with signicant antioxidative properties is rosemary; its
antioxidative activity arises from phenolic terpenes, such as ros-
marinic acid and rosmarol (Cuppett & Hall 1998). Several
studies reported an improvement in the performance of chickens
fed with a combination of plant extracts. The higher average daily
gain (ADG) and greater feed conversion efciency (FCE) achieved
could be attributed to the positive effects of extracts on nutrient
digestibility (Alçiçek et al. 2003; Hernandez et al. 2004; Jamroz
et al. 2006). Wenk (2002) stated that herbs develop their initial
activity by adding avour and, therefore, inuencing the eating
pattern, secretion of digestive uids and total feed intake. The
growing attention, devoted to environmental issues, led to new
techniques capable of extracting plant bioactive compounds
without using non-renewable resources (i.e. solvents) with a con-
sequent reduction in the environmental impact of wastes
because the plant material, remaining after solvent extraction,
is considered hazardous waste (Setti & Zanichelli 2009). The
obtained products are dened as aqueous extracts (AEs) and, in
addition to being solvent-free and more environmentally
friendly, these products contain the complete phytocomplexes
instead of the oily fraction alone. However, there is a lack of
studies concerning the use of AEs in animal nutrition (Javed
et al. 2009; Ogbe & Afku 2012; Onu 2012) if compared to those
concerning EOs. On the basis of these considerations, the aim
of the present study was to give a contribute on the effects of
dietary supplementation with oregano and rosemary AEs, used
alone or in combination, on broiler chicken performance, intesti-
nal microbial population and immune function.
2. Materials and methods
2.1. Experimental design
The study was carried out at the experimental farm of the
Department of Veterinary Medicine Perugia (Italy), according
© 2015 Taylor & Francis
CONTACT Patrizia Casagrande-Proietti patrizia.casagrandeproietti@unipg.it
JOURNAL OF APPLIED ANIMAL RESEARCH, 2016
VOL. 44, NO. 1, 474479
http://dx.doi.org/10.1080/09712119.2015.1091322
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to the European Directive 2010/63/EU on animal welfare for
experimental and other scientic purposes. A tot al of 300, one-
day-old female broilers (Ross 308), were divided into four
groups of 75 birds, each fed a starter diet from 1 to 21 days
and a growernisher diet from 22 to 56 days (Table 1). The
experimental diets were based on a standard commercial feed
used as control (C group) which was supplemented with 2 g/
kg rosemary AE (R group), 2 g/kg oregano AE (O group) and 1
g/kg oregano + 1 g/kg rosemary AEs (OR group). The compo-
sition of the rosemary and oregano AEs is indicated in Table 2.
The diets, formulated according to the nutritional requirements
for broiler chickens (Larbier & Leclerq 1992), were adminis tered
in mash form ad libitum. The trial was performed during the
months of November and December. Birds were submitted to
standard management usually adopted in industrial broiler pro-
duction. All subjects were vaccinated at the hatch against New-
castle disease (ND), Marek disease (MD), infectious bronchitis (IB)
and coccidiosis ND, IB vaccines were again administered in the
farm at 21 days. The individual body weight (BW) was measured
at 1, 11, 22, 36 and 57 days and feed consumed per group was
recorded daily. ADG, average daily feed intake (ADFI) and FCE
of all birds were calculated.
Blood samples from ve chickens/group were collected for
IgG titration at 22 (T1) and 57 days (T2) by wing vein puncture,
and serum samples were stored at 20°C until analysis. At the
same time intervals, eight subjects from each group were
humanely killed for intestinal microora examination.
2.2. Feed analysis
Dry matter was evaluated following AOAC (2000) method 934.01.
Crude protein was determined by measuring the total nitrogen
according to AOAC (1990) Kjeldahl method 954.01 and converting
this value to the protein content by multiplying by 6.25. Crude fat
and ash were determined by the 920.39 and 942.05 methods,
respectively (AOAC 1990). Neutral detergent bre (NDF), acid
detergent ber (ADF) and lignin were analysed according to Van
Soest et al. (1991). The calcium and phosphorous concentrations
were determined according to Julshamn et al. (1998)andAOAC
(1996), respectively. Starch was analysed by the polarimetric
method according to ISO 10520:1997 and Community methods
of analysis for the ofcial control of feeding stuffs (European Direc-
tive 72/1999/CEE).
2.3. Extraction and analysis of AEs
The AEs were obtained by a process of bio-liquefaction based on
enzyme bio-catalysis (Setti & Zanichelli 2009). The plant material
was placed in boiling water, and a specic enzymatic preparation
was then added after cooling. After four hours of hydrolysis, the
plant material was ltered. The AEs obtained were analysed to
quantify antioxidant capacity, total polyphenols and reducing
sugars (Table 2). The antioxidant activity of the oregano and
rosemary AE was measured in terms of radical scavenging
ability using the stable radical DPPH (Donglin & Yasunori 2004),
and the values were expressed in ORAC/L using trolox® as a refer-
ence compound (Davalos et al. 2004). Total polyphenols were
evaluated using the Folin-Ciocalteu reagent (Ainsworth & Gille-
spie 2007). The content of the redu cing sugars was evaluated
using the ADNS method (Bailey et al. 1992).
2.4. IgG titration
The chicken serum IgG value was determined by a commercially
available ELISA kit (Cat. No. E33104. Bethyl Laboratories, Inc.,
USA) and an automated washing and reader instrument
(Mago4S, Diamedix Corporation, Miami, FL, USA). The pro-
cedures for sample assays were performed according to the
manufacturers instructions, and the colorimetric reactions
Table 1. Ingredient and chemical composition of the experimental diets.
Starter GrowerFinisher
Ingredients (kg/100 kg)
Maize 53.99 58.99
Wheat middlings 6.00 6.50
Corn gluten 1.15 1.00
Soybean meal, 46% CP 32.00 25.00
Extruded soybean 3.00
Soybean oil 2.00 1.50
Calcium carbonate 1.00 .50
Dicalcium phosphate 1.50 1.25
Sodium chloride .35 .30
Vitamin and mineral mix
a
.50 .50
Lysine .15 .10
Methionine .20 .20
Fatty acid supplement
b
1.16 1.16
Composition, g/kg
Analysed
Dry matter 90.21 89.82
Crude protein 20.44 19.82
Crude fat 4.59 5.07
NDF 10.20 11.83
ADF 2.19 2.18
Lignin (s.a.) .49 .63
Crude ash 5.99 5.62
Starch (%) 41.85 42.39
Total calcium 1.20 1.20
Total phosphorus .70 .60
Available phosphorus .52 .44
Calculated
Lysine 1.20 1.00
Methionine .50 .52
Methionine + Cystine .88 .83
Threonine .80 .74
Tryptophan .23 .21
Arginine 1.35 1.22
Leucine 1.86 1.73
Isoleucine .88 .79
Valine .98 .90
Histidine .55 .50
ME (Mcal/Kg) 3.03 3.09
a
Supplied per kilogram of diet: vitamin A, 12,500 I.U. (retinol); vitamin D3, 3,000 I.
U.; vitamin E, 50 mg (tocopheryl acetate); vitamin K3, 2 mg; thiamine, 2 mg;
riboavin, 4 mg; pyridoxine, 1 mg; cyanocobalamin, .015 mg; pantothenic acid
15 mg; folic acid, 50 mg; biotin, 10 mg; choline chloride, 60; iodine, 3 mg;
selenium, 20 mg; iron, 3 mg; manganese, 12, mg; copper, 1,5 mg; zinc, 5 mg.
b
Supplied per kilogram of diet: C18:2 9c,11t, 2.5 g; C18:2 10t, 12c, 2.5 g; C14:0, .16
g; C16:0, .45 g; C20:3, .02 g; C20:5, .02 g; C22:6, .63 g; and others, .36 g.
Table 2. Mean and SEM of the composition of the oregano (O) and rosemary (R)
AEs.
AE
Antioxidant capacity
(ORAC/L)
Total polyphenols
(g/L)
Reducing sugars
(g/L)
O 17780 ± 260 2.5 ± .2 3.3 ± .2
R 10460 ± 160 2.3 ± .2 2.9 ± .1
Note: AEs: aqueous extracts.
JOURNAL OF APPLIED ANIMAL RESEARCH 475
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were read at the 450 nm wavelength. All samples were assayed
three times.
2.5. Microbiological analyses
The caecum and ileum (between the Merkels diverticulum and
a point 40 mm proximal to the ileocecal junction) from eight
birds per group were carefully removed and pooled to obtain
four samples for each intestinal region. A sterile stick was
used to place 1 g of intestinal contents into a sterile test tube
containing 2 mL of .9% sterile saline solution. The stool was
pressed and mixed in this solution, and the tube was brought
to volume (10 mL) with .9% sterile saline solution. Each
pooled sample (.1 mL) was 10-fold serially diluted (from 10
1
to 10
10
). Violet red bile agar and KF streptococcus agar were
used for the enumeration of Coliforms and Enterococci, respect-
ively. Mannitol salt agar was used for the enumeration of Sta-
phylococci. All the plates were aerobically incubated at 37°C
for 2448 h, and the number of colonies was counted. For the
enumeration of anaerobic bacteria, reinforced clostridial agar
enriched with 5% sheep blood and 1 mg/mL vitamin K1, was
used as anaerobe blood agar. Anaerobic incubation was per-
formed in anaerobic jars (Oxoid) at 37°C for 48 h. Anaerobic
conditions were obtained using Anaerogen (Oxoid) and
checked using methyl blue strips as oxidation reduction indi-
cators. For the enumeration of Lactobacilli, Rogosa agar
(Oxoid) was used. The plates were incubated for three days at
35°C under microaerophilic conditions. All data were expressed
as CFU × log/g.
2.6. Statistical analysis
Performance data were subjected to statistical analyses with the
GLM (General Linear Model) procedures of SAS (SAS Institute Inc
2010) according to a model for repeated measurements, where
the independent factor was the dietary treatment (four levels).
Microbiological data were analysed using a completely random-
ized design with factorial arrangement 2 × 2 × 4 with factors A
(ileum and cecum), B (sampling time) and C (dietary treatment).
Mean separation and comparison were conducted using Tukeys
test. The microbiological data were adjusted using a log
10
trans-
formation. The chicken serum IgG concentrations were analysed
according to a model where the independent factors were
dietary treatment (four levels), sampling time (two levels) and
their interaction. The estimated marginal means of IgG increases
(T2 vs. T1) were analysed by pairwise comparisons. The IgG per-
centage increases were calculated with the following formula:
IgG increase = [(IgG values at T2IgG values at T1)/IgG values at
T1], and they were expressed as percentage (%). The level of stat-
istical signicance was set at P < .05.
3. Results and discussion
The BW means for all exp erimental groups are summarized in
Table 3. There were no differences in BW means at the begin-
ning of the trial. AE supplementation signicantly affected the
BWs of bir ds at 11, 22 and 36 days of age, while no effects
were observed at the end of the experiment. The general
trend recorded for BW was conrmed by the ADG (Table 4)
and the ADFI (Table 5), with the exception of the data obtained
at the end of the trial. A numerically lower FCE was observed at
36 and 57 days in birds fed diet with the AEs (Table 5).
Table 3. Body weight (g) of broilers fed with the experimental diets up to 57 days.
Diets
Age (days)
111 22 36 57
C 44.16 179.79
B
389.69
B
1001.18
B
2447.20
R 43.76 183.91
B
466.57
AB
1124.01
AB
2437.29
O 43.90 221.40
A
493.92
AB
1157.18
A
2168.64
OR 48.05 173.24
B
504.23
A
1204.47
A
2431.37
SEM 2.05 5.69 30.98 37.13 83.18
P N.S. <.001 <.05 <.01 N.S.
Note: C: control diet; R: diet supplemented with rosemary AE (.2 mg/kg); O: diet
supplemented with oregano AE (.2 mg/kg); OR: diet supplemented with rosem-
ary AE (.1 mg/kg) and oregano AE (.1 mg/kg).
Means within a column lacking a common superscript differ.
N.S.: not signicant.
Table 4. Average daily gain (g) in experimental groups up to 57 days.
Diets
Age intervals (days)
111 1122 2236 3657
C 12.33
B
18.82
B
44.04
B
65.85
A
R 13.52
B
24.88
AB
48.52
AB
54.91
AB
O 16.13
A
24.49
AB
50.18
AB
47.65
B
OR 12.39
B
29.50
A
53.84
A
56.30
AB
SEM .47 2.75 2.38 3.53
P <.001 <.05 <.05 <.01
Note: C: control diet; R: diet supplemented with rosemary AE (.2 mg/kg); O: diet
supplemented with oregano AE (.2 mg/kg); OR: diet supplemented with rosem-
ary AE (.1 mg/kg) and oregano AE (.1 mg/kg).
Means within a column lacking a common superscript differ.
Table 5. Feed conversion efciency (FCE) and average daily feed intake (ADFI, g/die) in experimental groups up to 57 days.
Diets
Age intervals (days)
111 1122 2236 3657
ADFI FCE ADFI FCE ADFI FCE ADFI FCE
C 24.04 1.95 62.12 3.62 76.19
B
1.73 183.72
A
2.79
R 27.31 2.02 66.18 2.66 95.10
AB
1.96 93.90
B
1.71
O 25.08 1.60 59.75 2.44 90.83
AB
1.81 92.44
B
1.94
OR 25.40 2.05 60.77 2.06 104.45
A
1.94 88.39
B
1.57
SEM .84 .05 4.05 .06 6.69 .07 12.32 .06
P N.S. N.S. N.S. N.S. <.05 N.S. <.05 N.S.
Note: C: control diet; R: diet supplemented with rosemary AE (.2 mg/kg); O: diet supplemented with oregano AE (.2 mg/kg); OR: diet supplemented with rosemary AE (.1
mg/kg) and oregano AE (.1 mg/kg).
Means within a column lacking a common superscript differ.
N.S.: not signicant.
476 M. P. FRANCIOSINI ET AL.
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Previous studies have already reported the benecial effects
of some plant extracts (Hernandez et al. 2004; Windisch et al.
2008), indicating reduced feed intake and higher ADG or BW,
responsible for improved FCE. Alçiçek et al. (2004) investigated
the effects of dietary supplementation in the diet of broilers
with EO mixture, alone or in association with organic acid and
probiotic, showing higher yields in the group fed with EO
alone. Hernandez et al. (2004) tested two different types of
plant extract mixtures, including oregano and rosemary, in a
42-day trial. They found no differences in feed intake or FCE
for the entire experimental period, but broilers fed with plant
extracts grew faster than the controls. Hashemipour et al.
(2013) evaluated the effects of supple mentation of different
levels of thymol and carvacrol on performance in broiler chick-
ens. However, Papageorgiou et al. (2003) demonstrated that a
mixture of EOs did not improve the growth performance of
broilers. Phytogenic feed additives were also reported to stimu-
late the intestinal secretion of mucus in broilers, which impairs
the adhesion of pathogens and contributes to the stabilization
of the micro bial ora in the guts of the animal, improving diges-
tive processes (Jamroz et al. 2003).The different effects of plant
extracts on broiler performance may be due to intrinsic and
extrinsic factors, mainly including the experimental approaches
used to test the suitability of these substances as growth-pro-
moting additives for broilers (Windisch et al. 2008). With
respect to studies on AEs, Javed et al. (2009) analysed the
effects of AEs derived from different plants administered in
drinking water in broiler chicks. Their results showed positive
effects of AEs on ADG, FCE, dressing percentage, weight of
some organs and feed intake. Onu (2012) evaluated the effect
of different levels of Telfairia occidentalis AEs on the perform-
ance and haematological indices of starter broilers and
observed a positive inuence on BW, ADG and FCE.
Although the dietary treatments did not appear to signi-
cantly affect mean serum IgG values (Table 6) in terms of absol-
ute numbers, their increases (expressed in percentages) from T1
to T2 revealed a signicant (P < .05) diet effect with the highest
value in the O group.
In this study, the increase in the total IgG in the O and R
groups at T2 may indicate a more intense immune response
to vaccine antigen in the treated subjects. The total IgG
values observed at the end of our trial were greater than
those reported in the literature (Larsson et al. 1993) although
they could be related to the different procedures followed.
However, dietary immunomodulators can amplify or
decrease the magnitude of the reaction to a challenge
through their effects on other immune cells (Dietert et al. 1991).
Hashemipour et al. (2013) found an improved immune
response in broilers fed with a diet supplemented with
thymol and carvacrol, characterized by an enhancement of
hypersensitivity response and an increase of total IgG and IgG
anti-sheep red blood cells with decreased heterophil to lym-
phocyte ratio. Varshney et al. (2013) examined the effects of
Ocimum sanctum and Argemone Mexicana AEs in chickens,
reporting higher antibody titres and improved cell-mediated
immune response compared to control. With respect to the
microbiological investigations, this study was mainly under-
taken to detect possible differences in some bacterial popu-
lations following the administration of diets supplemented
with AEs. We focused on Lactoba cillus spp., Enterococcus spp.,
Coliforms and
Staphylococcus spp. and nally Anaerobics
(including Clostridium spp.).
The results produced by the experimental diets on the intes-
tinal microora composition are shown in Tables 7 and 8. In our
investigation the effects on the intestinal microora population
were encouraging though not conclusive.
At T1, the values for Coliforms were signicantly lower (P
< .001) in the cecum of the O and R groups than the C group.
The Coliform values increased in the ileum of all groups with
age though the O group showed the lowest values. Coliforms
are a large population of bacteria whose effects are not
always qualied as positive, including several potential patho-
genic agents (Amit-Romach et al. 2004). Globally Staphylococcus
spp. population was lower in the cecum and ileum in all groups
fed diets enriched with AE at both sampling times; in particular,
the R and O groups showed the lowest (P < .001) count in the
ileum and caecum at T1, whereas the O group presented the
lowest values in the ileum at T2(P < .001).
Staphylococcus spp. is not considered a benecial species in
the microbial gut population. Most infections in chickens are
Table 6. Effect of the different diets on total serum IgG (mg/mL) at 22 (T1) and 57
days (T2).
Diets T1 T2 % of increase T2T1
C 3.7 11.7 2.2
A
R 4.1 14.9 2.7
A
O 3.3 19.8 5.0
B
OR 3.4 11.9 2.4
A
SEM 2.1 2.1 .5
P N.S. N.S. <.05
Note: C: control diet; R: diet supplemented with rosemary AE (.2 mg/kg); O: diet
supplemented with oregano AE (.2 mg/kg); OR: diet supplemented with rosem-
ary AE (.1 mg/kg) and oregano AE (.1 mg/kg).
Means within a column lacking a common superscript differ.
N.S.: not signicant.
Table 7. Effect of the diets on different bacterial populations in the ileum tract at 22 (T1) and 57 days (T2).
Diets
Coliforms Staphylococcus spp. Enterococcus spp. Total anaerobics Lactic acid bacteria
T1 T2 T1 T2 T1 T2 T1 T2 T1 T2
C 4.47
E
6.80
B
3.78
D
4.80
A
7.64
BC
7.50
CD
11.18
B
9.40
D
10.36
A
6.5
E
R 3.58
G
6.09
C
3.52
E
4.03
C
7.31
D
7.83
B
9.35
D
9.72
C
4.23
F
9.53
B
O 3.26
H
4.20
F
3.36
E
3.86
D
8.28
A
8.38
A
8.69
E
12.52
A
6.64
E
7.22
D
OR 5.41
D
7.34
A
4.52
B
4.12
C
7.31
D
8.50
A
9.73
C
12.57
A
7.94
C
9.40
B
SEM .051 .034 .057 .032 .042
P <.001 <.001 <.001 <.001 <.001
Note: C: control diet; R: diet supplemented with rosemary AE (.2 mg/kg); O: diet supplemented with oregano AE (.2 mg/kg); OR: diet supplemented with rosemary AE (.1
mg/kg) and oregano AE (.1 mg/kg).
Means within each bacterial population lacking a common superscript differ.
JOURNAL OF APPLIED ANIMAL RESEARCH 477
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caused by coagulase-positive staphylococci, particularly
S. aureus, and coagulase-negative staphylococci (Jordan 1996;
McNamee et al. 1998). Staphylococci are frequently found in
poultry products for human consumption (Rosec et al. 1997;
Manie et al. 1998) and can thus spread to humans through
the food chain. With regards to Enterococcus spp., diets sup-
plemented with oregano produced the highest count (P < .001),
particularly in the caecum tract, and its value increased in all
groups at T2 supporting the hypothesis of a time -dependent
positive effect. Antagonistic effect of Enterococcus faecium
against the human and poultry pathogenic Salmonella spp.
was reported by Audisio et al. (1999), though its high level of
bile salt hydrolase activity may be responsible for growth
depression in chickens (Garrido et al. 2004).
Globally, total anaerobes were higher in the cecum than
ileum, with higher values (P < .001) in the O and OR groups in
both intestinal tracts at T2. At T1 their values were signicantly
higher in the C group. Among the AE-treated groups, R showed
the lowest counts in both intestinal tracts at T2. Barnes et al.
(1972) reported that the majority of bacteria in the cecum,
detected by culture-based methods, were strictly anaerobic,
and their number and diversity were inuenced with age. Anae-
robes constitute a large intestinal population of which the clos-
tridia, including C. perfringens, represent a signicant part. An
increase in C. perfringens could determine the occurrence of
clinical and subclin ical necrotic enteritis in birds (Kaldhusdal
et al. 1999; Williams et al. 2003).
In our work the inuence of rosemary, added either alone or
in combination with oregano, on Lactobacillus spp. should be
emphasized. Birds in the C group showed the highest values
for Lactobacilli in the ileum and cecum tracts at T1. Values
increased (P < .001) in all groups fed the AE diets at T2,
especially in the R group, showing, also in this case, a potential
time-dependent effect. The stimulation of benecial bacteria,
such as Lactobacilli, could contribute to balanced gut microora
and provide a favourable condition for digestion processes and
protection against enteropathogenic organisms. The role of this
bacterium in protecting the intestinal environment against
invasions by pathogens, such as C. perfringens, Campylobacter
jejuni subsp. jejuni, Salmonella spp . and patho genic strains of
E. coli (Mead 2000), has been known for a long time. Various
members of the Lactobacillus spp
. are also able to modulate
chicken cytokine and chemokine gene expression (Haghighi
et al. 2008; Brisbin et al. 2010).
In conclusion several limitations are related to culture-based
methods in the identication of the intestinal ecosystem
because up to 99% of bacteria fail to grow under articial
conditions (Amann et al. 1995; Hanson & Henson 1996). In par-
ticular, traditional methods were not always capable of isolating
the anaerobic bacteria and maintaining viability (Mead 1997).
However, Wang et al. (1996) reported a good correlation
between PCR-based and culture-based methods for bacterial
species requiring no particular enrichment media. Although
these results are in agreement with others (Garrido et al.
2004; Cross et al. 2007), further studies are needed to ensure
the actual effectiveness of AE on the performance and
welfare of broiler chickens.
Acknowledgements
The authors would like to thank E. Cassetta and G. Ceccarani for laboratory
analyses and G. Migni, O. Mandoloni and G. Covarelli for assistance and care
of animals. We also acknowledge Phenbiox srl, G.I.Ma. S.p.A. and Clarita
Cavallucci for their technical support in formulating experimental feeds.
Disclosure statement
No potential conict of interest was reported by the authors.
Funding
This work was supported by the project Made in Italy MI01_00148 funded
by the Italian Ministry of Economic Development. It is part of a PhD research
project in Animal Health, Livestock Production and Food Safety (University
of Perugia).
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المستخلص: أجريت الدراسة الحالية في حقل طيور السمان التابع إلى محطة الأبحاث والتجارب الزراعية / كلية الزراعة / جامعة البصرة للفترة من 24/11/2018 ولغاية 16/4/2019 لمعرفة تأثير إضافة مسحوق أوراق (البردقوش، إكليل الجبل) بصورة منفردة او تآزرية وبالإعمار المختلفة في العليقة على الأداء الإنتاجي والفسلجي والمناعي لطائر السمان الياباني وعلى فترتين الاولى (فترة النمو) من عمر يوم واحد ولغاية 42 يوماً، الفترة الثانية (فترة الانتاج) استمرت من عمر 43 يوماً ولمدة 12 اسبوع بعد النضج الجنسي للطيور. استخدم في التجربة 450 فرخاً من أفراخ طائر السمان الياباني البني غير مجنس بعمر يوم واحد. وزعت الأفراخ بشكل عشوائي على عشر معاملات منذ اليوم الأول لبدء التجربة، شملت كل معاملة ثلاث مكررات وبواقع 15 فرخاً لكل مكرر وعلى النحو التالي: T1 : عليقة اساسية بدون أي إضافة، T2 : إضافة 5غم من مسحوق أوراق نبات البردقوش /كغم علف من عمر يوم واحد لغاية 21 يوماً، T3 : إضافة 5غم من مسحوق أوراق نبات البردقوش /كغم علف من عمر 22 لغاية 42 يوماً، T4 : الرابعة إضافة 5غم من مسحوق أوراق نبات إكليل الجبل /كغم علف من عمر يوم واحد لغاية 21 يوماً، T5 : إضافة 5غم من مسحوق أوراق نبات إكليل الجبل /كغم علف من عمر 22 لغاية 42 يوماً، T6 : إضافة 2.5غم من مسحوق أوراق نبات البردقوش و 2.5غم من مسحوق أوراق نبات إكليل الجبل /كغم علف من عمر يوم واحد لغاية 21 يوماً، T7 : إضافة 2.5غم من مسحوق أوراق نبات البردقوش و 2.5غم من مسحوق أوراق نبات إكليل الجبل /كغم علف من عمر 22 لغاية 42 يوماً، T8 : إضافة 5غم من مسحوق أوراق نبات البردقوش /كغم علف من عمر يوم واحد لغاية نهاية التجربة، T9 : إضافة 5غم من مسحوق أوراق نبات إكليل الجبل/كغم علف من عمر يوم واحد لغاية نهاية التجربة، T10 : إضافة 2.5غم من مسحوق أوراق نبات البردقوش و2.5غم من مسحوق أوراق نبات إكليل الجبل /كغم علف من عمر يوم واحد ولغاية نهاية التجربة.
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