Livestock Science 181 (2015) 16

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Livestock Science
journal homepage: www.elsevier.com/locate/livsci

Phytoncide, phytogenic feed additive as an alternative to conventional

antibiotics, improved growth performance and decreased excreta gas
emission without adverse effect on meat quality in broiler chickens
H.L. Li, P.Y. Zhao, Y. Lei, M.M. Hossain, I.H. Kim n
Department of Animal Resource & Science, Dankook University, Cheonan, Chungnam 31116, South Korea

art ic l e i nf o

a b s t r a c t

Article history:
Received 26 May 2015
Received in revised form
23 September 2015
Accepted 1 October 2015

A total of 384 one-day-old broiler chickens [initial body weight (BW): 47.47 0.3 g] were used to investigate the effect of dietary phytoncide, phytogenic feed additive extracted from Korean pine, as an
alternative to tylosin on growth performance, excreta microora, excreta gas emission, meat quality, and
relative organ and tissue weight in the 35-d feeding experiment (starter phase, d 014 and nisher
phase, d 1435). Broiler chickens were assigned to 1 of 4 dietary treatments (6 replicate cages/treatment
and 16 boiler chickens/cage) according to their BW. Dietary treatments were: positive control (PC; basal
diet 0.2 g tylosin/kg) and basal diet 0, 0.5, or 1.0 g phytoncide/kg. During d 014 and 035, BW gain
(BWG) of broiler chickens fed the PC diet was greater (P 0.02 and 0.03, respectively) compared with
those fed with basal diet. During d 035, BWG, feed intake, and feed conversion ratio linearly improved
(P 0.01, 0.03, and 0.03, respectively) as dietary phytoncide content increased. Broiler chickens fed the PC
diet had lower (P 0.04) excreta Escherichia coli concentration compared with those fed with basal diet.
Breast muscle linearly increased (P 0.05) and excreta gas emission linearly decreased (Po 0.05) as
phytoncide concentration increased. In conclusion, phytoncide could be considered as an alternative to
replace tylosin to improve growth performance and decrease excreta gas emission in broiler chickens
without any adverse effect on meat quality.
& 2015 Elsevier B.V. All rights reserved.

Keywords:
Broiler chickens
Excreta gas emission
Excreta microora
Growth performance
Phytogenic feed additive

1. Introduction
To increase the protability, a number of antimicrobial compounds, including antibiotics, plant extract, phytogenic feed additive (PFA), and probiotics, have been extensively used in poultry
and pig diets to enhance energy and nutrient digestibility and
growth performance for several decades (Newton et al., 2002; Park
and Kim, 2014; Park and Kim, 2015). Since European Union banned
the use of antibiotics as a growth promoter for animals in 2006,
great effort has been expended to nd alternatives to antibiotics
(Yan et al., 2012a, 2012b). Plant-derived products, which have
proven to be natural, less toxic, and residue free, were thought to
be ideal growth promoters to be included in animal diets (Hashemi et al., 2008). As one of these alternatives, PFA have recently
gained increasing interests, because it was demonstrated to improve growth performance, reduce stress response, and enhance
immune response (Kawakami et al., 2004; Li et al., 2012; Zhang
et al., 2012; Zhou et al., 2013).
n

Corresponding author. Fax: 82 41 565 2949.

E-mail address: inhokim@dankook.ac.kr (I.H. Kim).

http://dx.doi.org/10.1016/j.livsci.2015.10.001
1871-1413/& 2015 Elsevier B.V. All rights reserved.

Phytoncide was extracted from Korean pines and used as a PFA

to suppress activity of microorganisms, including bacteria and
fungi (Peiulyt et al., 2010). The suppressive activity relies on
essential oils, terpenoids, alkaloid, and phenylpropanoids in phytoncide (Durham et al., 1994). Because of these active components,
PFA have a potential capacity to intercalate into the bacterial
membrane, disintegrate the membrane, and cause ion leakage
(Burt, 2004; Hashemi and Davoodi, 2010). In addition, PFA have
also been demonstrated to reduce stress response in stroke-prone,
spontaneously hypertensive rats and enhance immune response
(Kawakami et al., 2004; Li et al., 2012; Zhang et al., 2012). Ammonia emissions of livestock production system could be harmful
to environment, gastrointestinal health, and overall performance
(Apsimon and Kruse-Plass, 1990; Nyachoti et al., 2006). Mcintosh
et al., (2003) observed that growth of Clostridium sticklandii and
Peptostreptococcus anaerobius, which can produce a lot of ammonia, were inhibited by a blend of essential oils.
Based on previous studies, we can make hypothesis that phytoncide may improve growth performance of broiler chickens and
decrease excreta gas emission. However, little has been known on
the effect of including Korean pine extract, phytoncide, in broiler

H.L. Li et al. / Livestock Science 181 (2015) 16

chicken diets. Therefore, we conducted this study to evaluate the

effect of phytoncide on growth performance, excreta microora
shedding, excreta noxious gas emission, meat quality, and relative
organ and tissue weights in broiler chickens.

2. Materials and methods

2.1. Preparation of phytoncide and experimental animals
The experimental protocols were approved by the animal care
and use committee of Dankook University. The phytoncide (PHYLUS Company, Gapyeong, Gyeonggi, Korea), which include 20%
active substances (essential oils, avonoid, phenolic compounds,
alkaloid, tannin, terpene, and saponin) and 80% carrier (dextrin),
used in this study was extracted from Korean pine. Feed grade
tylosin (TYLOSIN 20 W.S.P; Shijiazhuang ZDHF Stock-Raising Co,
Hebei, China) containing 200 g tylosin/kg was used in our present
study. A total of 384 one-day-old broiler chickens [ROSS 308;
Buseong Company, Pyeongtaek, Gyeonggi, Korea; initial BW:
47.47 0.3 g] were housed in battery cages (124 cm width  64 cm
length  40 cm height) in an environmentally regulated house for
this 35-d feeding experiment. Temperature of the animal facility
on the rst day of experimental period was maintained at 35 C
and lowered by 23 C every week until 22 C in the nal week.
Broiler chickens were individually weighed and assigned to 1 of
4 dietary treatments (6 replicate cages/treatment and 16 broiler
chickens/cage) based on their BW. Dietary treatments were as
follows: PC, basal diet 0.2 g tylosin/kg, and basal diet without
tylosin supplemented with 0, 0.5, or 1.0 g phytoncide/kg. Basal
diets were formulated to meet or little exceed all the nutrient
requirements of broiler chickens for starter (d 014) and nisher
(d 1435) phases based on the recommendations (Ross, 2007;
Table 1). Articial light was provided 24 h/d using uorescent
lights. All diets were fed in mash form, and the experimental diets
and fresh water were provided ad libitum throughout the experimental period.
2.2. Chemical analysis
All feed samples were analyzed in triplicate before initiation of
the experiment. Nitrogen was determined (Kjeltec 2300 Nitrogen
Analyzer; Foss Tecator AB, Hoeganaes, Sweden), and CP was calculated as N  6.25. Lys and Met were measured using an amino
acid analyzer (Beckman 6300; Beckman Coulter Inc., Fullerton, CA)
after acid hydrolysis for 24 h in HCl. The contents of Ca and P were
analyzed according to the methods (984.01 and 965.17, respectively) of AOAC (1995). Chromic oxide (0.2%) was added to the
basal diets as an indicator for determination of ME. Chromium was
analyzed using a UV absorption spectrophotometer (Shimadzu
UV-1201; Shimadzu, Kyoto, Japan) according to the method described by Fenton and Fenton (1979).
2.3. Sampling and measurements
The broilers were weighed by cage and feed intake (FI) was
recorded on d 0, 14, and 35. Then data were used to calculate BWG,
and feed conversion ratio (FCR). A total of 120 broiler chickens, 18
broiler chickens were randomly selected from each treatment (3
broiler chickens per cage), then they were killed via cervical dislocation by trained personnel. The stomach, breast meat, bursa of
Fabricius, liver, spleen, and abdominal fat were then removed by
trained personnel and weighed. Breast muscles were stored at
20 C for the analysis later.
Organ weight and abdominal fat were expressed as a percentage of live BW. The breast muscle Hunter L* (lightness), a*

Table 1
Formula and chemical composition of basal diets (%).
Item

Period
Startera

Finishera

Corn
Soybean meal
Corn gluten meal
Soybean oil
Tricalcium phosphate
Limestone
NaCl
DL-Met
L-Lys  HCl, 78%
Vitamin premixb
Trace mineral premixb

56.67
29.25
6.60
5.40
2.46
0.89
0.20
0.07
0.06
0.20
0.20

62.31
23.51
3.50
4.89
2.29
0.75
0.20
0.07
0.08
0.20
0.20

Calculated analysis (as-fed basis)

ME, MJ/kg
N
Crude protein
Lys
Met
Ca
P

12.97
3.52
22.00
1.10
0.57
1.00
0.80

12.56
3.04
19.00
1.00
0.54
0.90
0.75

Starter phase, d 014; and nisher phase, d 1435.

Provided the following per kilogram of the complete diet: 15,000 IU vitamin
A, 3750 IU vitamin D3, 37.5 mg vitamin E, 2.55 mg vitamin K3, 3 mg vitamin B1,
7.5 mg vitamin B2, 4.5 mg vitamin B6, 24 mg vitamin B12, 51 mg niacin, 1.5 mg folic
acid, 126 mg biotin, 13.5 mg pantothenic acid, 37.5 mg Zn (zinc sulfate), 37.5 mg Mn
(manganese sulfate), 37.5 mg Fe (ferrous sulfate), 3.75 mg Cu (copper sulfate),
0.83 mg I (potassium iodide), and 0.23 mg Se (sodium selenite).
b

(redness), and b* (yellowness) values were determined using a

chromameter (Minolta CR410; Konica Minolta Sensing Inc., Osaka,
Japan). The pH values for each sample were measured in duplicate
using a pH meter (Fisher Scientic, Pittsburgh, PA). The water
holding capacity (WHC) was measured according to the methods
of Kauffman et al. (1986). In brief, a 0.2-g sample was pressed at
20.7 MPa for 3 min on a lter paper (125 mm diameter). Areas of
the pressed sample and expressed moisture were delineated and
then determined with a digitizing area-line sensor (MT-10S; M.T.
Precision Co. Ltd., Tokyo, Japan). Ratio of water to meat area was
calculated, which is the measure of WHC (the smaller ratio indicates the greater WHC). Furthermore, drip loss (%) was measured
by removing 5-cm thick section from broiler chickens breast meat,
weighted, and suspended in a zipper bag for 7 d at 4 C, and each
sample was weighted every other day (d 1, 3, 5, and 7) as described by Honikel (1998).
Total bacteria, including Lactobacillus and Escherichia coli sp., in
fresh excreta collected in the morning were determined at 35 d of
age. Microbial populations were determined by serial dilution
(10  110  7) in anaerobic diluents before inoculation onto petri
dishes of sterile agar as described by Bryant and Burkey (1953),
and Lactobacilli and E. coli species was enumerated. The selective
medium used for Lactobacilli sp. was Rogosa SL agar (Rogosa; Difco
Laboratories, Detroit, MI, US), and the medium used for E. coli was
MacConkey agar (MacConkey; Difco Laboratories). After inoculation, all the dishes were inverted and incubated anaerobically at
37 C for 48 h. The results are presented as log10-transformed
data.
During last 2 d of experimental period, fresh excreta samples
were collected randomly from each cage. Samples were kept in
sealed containers and were immediately stored at  4 C until the
analysis. Subsamples of excreta were taken and stored in 2.6-L
plastic boxes in duplicate, and plastic boxes were sealed carefully.
Each box had a small hole in the middle of one side wall, which
was sealed with adhesive plaster. The samples were permitted to

H.L. Li et al. / Livestock Science 181 (2015) 16

ferment for 5 d at room temperature (25 C). After fermentation

period, a gas sampling pump (Model GV-100; GASTEC Corp. Ayase,
Kanagawa, Japan) was utilized for gas detection. Concentrations of
NH3, H2S, total mercaptans, and acetic acid were measured by
using a detector tube (GASTEC Corp.) within scope of 5.0100.0
(No. 3La, detector tube; GASTEC Corp.), 2.020.0 (No. 4LK, detector
tube; GASTEC Corp.), 0.5120.0 (No. 70, detector tube; GASTEC
Corp.), and 2.050.0 ppm (No.81, detector tube; GASTEC Corp.).
Before measurements, slurry samples were shaken manually for
approximately 30 s to disrupt any crust formation on the surface of
the slurry sample and to homogenize samples. The adhesive
plasters were punctured, and 100 mL of headspace air was sampled approximately 2.0 cm above the slurry surface. Two samples
from each cage were measured and then the average was
calculated.
2.4. Statistical analyses
Data were analyzed by ANOVA using the general linear models
procedure of SAS (2003), with the cage being considered as the
experimental unit. Linear and quadratic effects of dietary treatments (without tylosin; 0, 0.5, and 1.0 g/kg of phytoncide) were
assessed. Also, the different between PC and basal diet without
phytoncide. Results were expressed as the least squares means and
standard error of the mean (SEM). Probability values less than 0.05
were considered signicant.

3. Results
During d 014 and 035, BWG in PC treatment was greater
(P 0.02 and 0.03, respectively) than the treatment containing no
phytoncide (Table 2). There was no difference in FCR among
treatments. The BWG linearly increased (P o0.001, and P 0.04
and 0.01 for d 014, 1435, 035, respectively) as the level of
phytoncide increased throughout the experimental period. During
d 035, FI was linearly increased (P 0.03) with increasing levels
of phytoncide. The FCR linearly improved (P 0.04, 0.04, and 0.03
for d 014, 1435, 035, respectively) with increasing levels of
phytoncide. No difference was observed in meat quality. Most
relative organ and tissues weight among treatments showed no
difference, while breast muscle linearly increased (P 0.05) as

phytoncide supplementation increased (Table 3).

Broiler chickens fed the PC diet had lower excreta E. coli sp.
(P 0.04) and Lactobacillus sp. (P 0.01) concentrations than those
fed the basal diet without phytoncide (Table 4). No linear or
quadratic effect was observed in shedding of microora. The PC
diet decreased (P o0.001) concentration of excreta ammonia
emission compared with the basal diet without phytoncide. Excreta ammonia, total mercaptans, acetic acid, and hydrogen sulde
all linearly decreased (P 0.02, 0.04, 0.05, and 0.02, respectively)
with increasing levels of phytoncide (Table 5).

4. Discussion
Denli et al. (2004) and Hong et al. (2012) reported that supplementation of essential oils, which are the main functional
components of PFA, in a quail or broiler chicken diet increased
BWG and feed efciency. Moreover, Banerjee et al. (2013) found
that phytogenic growth promoter enhanced productive performance of broiler chickens in terms of BWG. In agreement with
those results, our present study indicates that phytoncide have an
obvious capacity to elevate BWG of broiler chickens linearly. The
reason maybe that phytogenic products could increase jejunal
villus height, while decreasing crypt depths of broiler chickens
(Amad et al., 2013), moreover, greater villus width and greater
villus surface area are generally associated with heavier broiler
chickens compared to lighter ones (Incharoen et al., 2010).
However, information concerning the efcacy of PFA on growth
performance in poultry is not consistent. Cross et al., (2007) reported that broiler chickens given a diet with thyme herb (728 d)
had a similar BWG to those given control diet. Khalaji et al. (2011)
reported that using black cumin seed alone or mixed with Artemisia leaves improved broiler chickens health and performance
but Camellia L. plant extract negatively affected broiler chickens
BW and FI. Moreover, numerically different results have also been
reported (Botsoglou et al., 2002; Erdogan et al., 2010; Jang et al.,
2007). According to previous studies, another reason may be microbiota factors because primary mode of action of herbal extract
feed additives is their effect on ecosystem of gastrointestinal microbiota through the control of potential pathogens (Kroismayr
et al., 2007; Roth and Kirchgessner, 1998). Consequently, positive
effects on BWG are most likely due to the antimicrobial properties

Table 2
Effect of dietary phytoncide on growth performance in broiler chickens during d 014, 1435, and 035.a,b
Item

Treatment
PC

SEM
Phytoncide (g/kg)
0

0.5

1.0

P-value
PC vs. basal diet

Linear

Quadratic

d 014
BWG (g)
FI (g)
FCR

554
818
1.479

525
814
1.551

535
821
1.509

555
827
1.495

7
7
0.027

0.02
0.74
0.09

o 0.001
0.08
0.04

0.47
0.39
0.77

d 1435
BWG (g)
FI (g)
FCR

1,107
1,842
1.664

1,096
1,859
1.696

1,109
1,871
1.687

1,122
1,879
1.675

10
35
0.052

0.24
0.53
0.10

0.04
0.32
0.04

0.84
0.29
0.07

d 035
BWG (g)
FI (g)
FCR

1,661
2,639
1.589

1,621
2,654
1.637

1,644
2,670
1.624

1,677
2,683
1.600

11
32
0.028

0.03
0.33
0.06

0.01
0.03
0.03

0.55
0.38
0.76

a
b

BWG body weight gain, FIfeed intake, FCR feed conversion ratio, and SEM standard error of the mean.
Basal diet without tylosin or phytoncide, and PC basal diet 0.2 g tylosin/kg0 g phytoncide/kg.

H.L. Li et al. / Livestock Science 181 (2015) 16

Table 3
Effect of dietary phytoncide on meat quality and relative organ weight in broiler chickens at the end of experiment (d 35).a,b
Item

Treatment

SEM

PC

Phytoncide (g/kg)
0

pH value

0.5

P-value
PC vs. basal diet

Linear

Quadratic

1.0

5.73

5.78

5.83

6.10

0.13

0.77

0.06

0.47

Breast muscle color

Lightness (L*)
Redness (a*)
Yellowness (b*)
WHC (%)

55.17
14.66
13.81
51.71

55.04
15.31
12.17
51.19

54.87
15.21
13.04
50.80

55.31
15.01
12.24
51.87

0.95
0.42
0.99
2.22

0.62
0.13
0.08
0.20

0.84
0.09
0.51
0.91

0.36
0.27
0.70
0.66

Drip loss (%)

d1
d3
d5
d7

2.52
4.13
7.59
10.10

2.54
4.49
7.98
10.21

2.52
4.46
7.41
10.20

2.48
4.31
7.38
10.07

0.33
0.34
0.39
0.40

0.84
0.22
0.41
0.36

0.10
0.12
0.06
0.34

0.52
0.69
0.82
0.18

3.21
0.18
0.15
15.81
1.24
1.28

3.21
0.17
0.16
15.99
1.15
1.27

3.33
0.21
0.17
16.27
1.17
1.23

0.21
0.02
0.01
0.67
0.06
0.10

0.31
0.47
0.66
0.11
0.70
0.52

0.20
0.64
0.07
0.05
0.27
0.09

0.59
0.76
0.39
0.88
0.71
0.46

Relative organ and tissue weight (%)c

Liver
3.28
Spleen
0.20
Bursa of Fabricius
0.16
Breast muscle
16.67
Abdominal fat
1.23
Gizzard
1.22
a
b
c

SEM standard error of the mean, and WHC water holding capacity.
Basal diet without tylosin or phytoncide, and PC basal diet 0.2 g tylosin/kg 0 g phytoncide/kg.
Values are expressed as g/100 g body weight.

Table 4
Effect of dietary phytoncide on excreta microbial population in broiler chickens (log10 cfu/g) at the end of experiment (d 35).a,b
Item

Treatment
PC

E. coli sp.
Lactobacillus sp.
a
b

6.15
7.08

SEM
Phytoncide (g/kg)
0

0.5

1.0

6.38
7.43

6.04
7.36

6.00
7.43

0.02
0.09

P-value
PC vs. basal diet

Linear

Quadratic

0.04
0.01

0.13
0.74

0.51
0.07

PC vs. basal diet

Linear

Quadratic

o 0.001
0.06
0.08
0.32

0.02
0.04
0.05
0.02

0.84
0.41
0.69
0.72

SEM standard error of the mean.

Basal diet without tylosin or phytoncide, and PC basal diet 0.2 g tylosin/kg 0 g phytoncide/kg.

Table 5
Effect of dietary phytoncide on excreta gas emission in broiler chickens at the end of experiment (d 35).a,b
Item (ppm)

Treatment
PC

NH3
Total mercaptans
H2S
Acetic acid
a
b

20.08
1.68
1.40
1.18

SEM
Phytoncide (g/kg)
0

0.5

1.0

25.45
1.98
1.75
1.28

22.06
1.80
1.68
1.08

19.98
1.63
1.48
1.03

1.29
0.25
0.20
0.13

P-value

SEM standard error of the mean.

Basal diet without tylosin or phytoncide, and PC basal diet 0.2 g tylosin/kg 0 g phytoncide/kg.

of PFA. Similar to antibiotics, dietary PFA being used as a growth

promoter do not provide benecial results when broiler chickens
are kept at optimal conditions such as provided with highly digestible diets and clean environment (Botsoglou et al., 2002; Jang
et al., 2004). Additionally, different part of plant, harvesting season, application methods, geographical origin, phytogenic production process, age of poultry, environment condition, and many
other factors can all contribute to inconsistent results (Jin et al.,

1998; Patterson and Burkholder, 2003). Indeed, take all previous

studies and our present study into consideration, further studies
are necessary to evaluate the various effects of phytogenic products and to clarify their specic modes of action in broiler
chickens.
The microora of gut inuences host nutrition, health, and
growth performance (Barrow, 1992) by inuencing the nutrient
utilization and the development of the gastrointestinal system of

H.L. Li et al. / Livestock Science 181 (2015) 16

the host. Even though, unlike ruminants and nonruminant herbivores, chickens derive little nutritional benet from the microora,
microora still plays a crucial role in broiler chickens performance
(Cowan, 1999). Consequently, it is important to consider the fact
that feed additives originating from plant products may have a
profound direct and indirect impact on gut microora (Cowan,
1999).
In our present study, dietary tylosin reduced the population of
E. coli sp., as well as Lactobacillus sp., in excreta. Antibiotics are
natural metabolites of fungi that inhibit the growth and colonization of numerous non-pathogenic species of bacteria in the
gastrointestinal tract, including Lactobacilli, Bidobacteria, Bacteroides, and Enterococci species (Tannock, 1997). In the present
study, phytoncide supplementation reduced E. coli sp. in excreta,
the result was consistent with previous studies that herbs and
their extracted essential oils possess antimicrobial actions (Burt,
2004; Si et al., 2006). Chao et al., (2000) investigated the effects of
45 essential oils on a broad range of microorganisms, and it seems
that antimicrobial effects of phytoncide are potentially more valuable than antibiotics for the gut ecosystem in broiler chickens.
According to Sommer and Husted (1995), pH is very important
for the ammonia emission from slurry. Reduction of Gram-negative bacteria in intestinal tract enhanced the benecial bacteria
activities in the hindgut of broiler chickens and in the slurry
during storage, and increased VFA formation in feces and slurry
can lead to lower pH of slurry and reduction of ammonia emission
(Cambra-Lpez et al., 2010). In line with previous studies, we observed reduction of population of Gram-negative bacteria, E. coli
sp., and concentration of ammonia in excreta of those on dietary
antibiotics and phytoncide treatments. In contrast, Cross et al.
(2007) and Peri et al. (2010) observed no differences between
control and PFA treatments in gut microbiota. Nevertheless, more
study is needed to evaluate the effect of PFA on intestinal microbiota. Furthermore, PFA, especially essential oils, could decrease
gas emission from excreta through increasing nutrient digestion.
Amad et al., (2013) reported that PFA improved nutrient digestibility in the small intestine of broiler chickens. Lee et al., (2003)
demonstrated that essential oils stimulate the secretion of pancreatic digestive enzymes, such as amylase, lipase, and trypsin in
broiler chickens. The improved utilization of dietary nutrients results in reduction in N, P, and other nutrients excreted into the
environment (Cromwell, 2002). In another word, PFA may enable
broiler chickens to better utilize nutrients in feed, which decreases
the fermented substrate, resulting in the reduction of gas emission
from excreta.

5. Conclusion
In general, our present results indicated that phytoncide, a
novel phytogenic feed additive extracted from Korean pine, has the
potential to replace tylosin as a growth promoter to improve
growth performance and health status, decrease excreta gas
emission, and increase breast muscle without any adverse effect
on meat quality in broiler chickens. Hence, further study need to
be done to determine the optimal dosage of phytoncide in broiler
chicken diet.

Conict of interest statement

There was no conict of interest. Papers are accepted for publication in the Journal on the understanding that the content has
not been published or submitted for publication elsewhere.

Acknowledgments
The authors gratefully acknowledge the support by Department
of Animal Resource & Science, Dankook University, we wish to
express our appreciation to our lab members for their help and
support. All authors have contributed signicantly, and all authors
are in agreement with the content of the manuscript.

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