Journal of Applied Aquaculture

ISSN: 1045-4438 (Print) 1545-0805 (Online) Journal homepage: http://www.tandfonline.com/loi/wjaa20

Effects of prebiotic mannan oligosaccharide on

the growth, survival, and anxiety-like behaviors of
zebrafish (Danio rerio)

Mohammad Navid Forsatkar, Mohammad Ali Nematollahi, Gholamreza

Rafiee, Hamid Farahmand & Gonzalo Martínez-Rodríguez

To cite this article: Mohammad Navid Forsatkar, Mohammad Ali Nematollahi, Gholamreza
Rafiee, Hamid Farahmand & Gonzalo Martínez-Rodríguez (2017) Effects of prebiotic mannan
oligosaccharide on the growth, survival, and anxiety-like behaviors of zebrafish (Danio rerio),
Journal of Applied Aquaculture, 29:2, 183-196, DOI: 10.1080/10454438.2017.1306732

To link to this article: http://dx.doi.org/10.1080/10454438.2017.1306732

Published online: 25 Apr 2017.

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JOURNAL OF APPLIED AQUACULTURE
2017, VOL. 29, NO. 2, 183–196
http://dx.doi.org/10.1080/10454438.2017.1306732

Effects of prebiotic mannan oligosaccharide on the

growth, survival, and anxiety-like behaviors of zebrafish
(Danio rerio)
Mohammad Navid Forsatkara, Mohammad Ali Nematollahia, Gholamreza Rafieea,
Hamid Farahmanda, and Gonzalo Martínez-Rodríguezb
a
Department of Fisheries, Faculty of Natural Resources, University of Tehran, Karaj, Iran; bInstituto de
Ciencias Marinas de Andalucía, Consejo Superior de Investigaciones Científicas (ICMAN-CSIC), Puerto
Real, Cádiz, Spain

ABSTRACT KEYWORDS
A study was designed to investigate the possible positive Behavior; Danio rerio;
prebiotic effects of mannan oligosaccharides (MOS) on growth growth; starvation tolerance;
and reduction of anxiety behaviors in zebrafish (Danio rerio). stress response; welfare
The experimental diets were supplemented with 0, 2, 4, 6, or
8 g MOS kg−1 and were fed to juvenile fish (0.47 ± 0.017 g) in
triplicates for six weeks. Significant differences were found in
the final body weight, body weight intake, percent body
weight intake, and specific growth rate among treatments.
The regression analyses showed that the optimum MOS level
was 4–4.1 g MOS kg−1 diet. Fish fed 4 g MOS kg−1 feed showed
higher survival than the other treatments. Also, using the novel
tank test, fish fed with 4 g MOS kg−1 diet showed lower anxiety
by swimming in the upper portion of the tank. The other
behavior parameters remained unaffected by the prebiotic.
The results indicated the beneficial effect of the prebiotic
MOS on growth and partly on welfare of zebrafish juveniles.

Introduction
Prebiotics are indigestible carbohydrates that selectively stimulate the growth
and metabolism of health-promoting bacteria already present in the host gut
(Gibson and Roberfroid 1995). Several studies indicate that prebiotics could
significantly increase growth performance, feed utilization, digestibility of
dietary ingredients, disease resistance and stimulate the immune response
of aquatic animals (Akrami et al. 2013; Daniels et al. 2010). Among different
options, prebiotic mannan oligosaccharides (MOS) is one the most applied
prebiotics in aquaculture. MOS is a glucomannoprotein complex derived
from the cell wall of Saccharomyces cerevisiae, containing mannose as the
primary carbohydrate element (Salze et al. 2008). It has been shown that
MOS has advantages for health and growth parameters in several fish species
(Torrecillas et al. 2014). For example, dietary MOS elevated growth, feed

CONTACT Mohammad Ali Nematollahi malahi@ut.ac.ir Department of Fisheries, Faculty of Natural

Resources, University of Tehran, Chamran St., P.O. Box 31585-4314, Karaj, Tehran, Iran.
© 2017 Taylor & Francis
184 M. N. FORSATKAR ET AL.

utilization, enzyme activity, and immunity in red drum (Sciaenops ocellatus;

Zhou et al. 2010), hybrid striped bass (Morone chrysops × M. saxatilis;
Anguiano et al. 2013; Li and Gatlin 2004), and common carp (Cyprinus
carpio; Ebrahimi et al. 2012), and reduced stress response in marron
(Cherax tenuimanus; Sang et al. 2009) and cobia (Rachycentron canadum;
Salze et al. 2008).
Diet can significantly impact physical health and behavior in fish and
therefore can be associated with quality of life (Oliva-Teles 2012). This is
very important given the rapid development of aquaculture and the limited
knowledge on fish physiological stress (Segner et al. 2012), which in turn can
negatively affect the overall performance of cultured species (Santos et al.
2010). To alleviate this problem, many attempts have been made to improve
nutritional status of the animals, and in this regard food additives like probio-
tics, prebiotics, and symbiotics have played a special role (Nakagawa et al.
2007). Considering the beneficial roles of MOS in fish, it is recommended to
understand the best inclusion levels in the diet of different fish species.
Zebrafish (Danio rerio Hamilton-Buchanan) is a model species for devel-
opmental biology, physiology, molecular genetics, toxicology, and behavioral
research due to its extraordinary ability to adapt to laboratory conditions
(Jesuthasan 2012; Lawrence 2007). Moreover, due to the similarity in biolo-
gical and physiological responses to cultured fish species, along with a small
size, rapid development, and generation time, zebrafish has been proposed a
suitable model for finfish aquaculture research (Ribas and Piferrer 2014).
Surprisingly, little information is available regarding effects of dietary pre-
biotics additives to be given to growing zebrafish (Carnevali et al. 2013). This
gap in knowledge is particularly evident on the impacts of diet on zebrafish
behavior and its welfare issues (O’Brine et al. 2015). On the other hand, the
impact of prebiotics as a feed supplement on behavioral traits such as those
involved in stress and anxiety are practically unstudied in fish when com-
pared to physiological endpoints (Anguiano et al. 2013; Torrecillas et al.
2014). This may be important given that alterations in behavior may take
place long before physiological changes occur (Little 2002). Additionally,
animal fitness is strongly dependent on how the individuals interact with
the different stimuli (Lind and Cresswell 2005). Therefore, tracking the
behaviors of individuals can be beneficial to understand their stress condition
in a culture enclosure (Conti et al. 2006; Papadakis et al. 2014).
Anxiety is a major chronic emotional disorder, and its molecular basis
could be evolutionarily conserved between fish and mammals (Jesuthasan
2012). Moreover, the relationship between stress and anxiety is well known in
zebrafish (Chakravarty et al. 2013). Like in open field testing in rodents,
anxiety is operationally defined in fish by how much time the animal spends
at the bottom of a novel tank. The moment at which the fish moves into the
top portion of the tank has been inferred to be the moment at which the fish
 JOURNAL OF APPLIED AQUACULTURE 185

feels safe enough to explore this new environment (Stewart et al. 2012). It is
noteworthy that anxiety is strongly affected by diet (Del Rosario et al. 2012;
Jacka et al. 2013). However, effects of dietary components on zebrafish
behavior remain largely unknown. To address this point, this study aimed
to examine the effects of the prebiotic MOS on growth performance, survival,
and behavioral profile of anxiety in zebrafish.

Materials and methods

Fish and housing
Thirty-day-old zebrafish juveniles reproduced from adult mature albino strain
fish in the laboratory were used in the present experiments. Parent fish were
purchased from a local ornamental fish hatchery, maintained under an artifi-
cial photoperiod of 14 L:10 D (lights on at 08:00) at 27 ± 0.5ºC, and fed twice a
day with commercial fish diet (BioMar, France; contained 35% protein). The
spawning took place in a 15 L rectangular opaque container using four gravid
females and three males, which was equipped with a plastic mesh of 4 mm
mesh size in the bottom to prevent access of parents to the laid embryos.
Breeders were then taken out and embryos were directly incubated in the
spawning tank until hatching, removing dead eggs every 12 h. Larvae were
fed green water (rich in Paramecium spp.) and boiled egg yolk until 5–9 days
after hatching (dph), with Artemia nauplii afterwards until 24 dph, and with
commercial powdered fish food from 20 dph. From 15 dph larva were
transferred to a bigger glass rectangular tank (140 L; water height 20 cm)
until the beginning of feeding experiments. All animal handling and testing
techniques were designed using guidance from the Association for the Study
of Animal Behavior and the Animal Behavior Society (ASAB/ABS 2012).

Prebiotic
The prebiotic used Agrimos® mannan oligosaccharides—MOS—is a specific
combination of mannan oligosaccharides and glucose (B-glucans) extracted
from the yeast cell walls of Saccharomyces cerevisiae (Lallemand Animal
Nutrition, France). Based on the manufacturer guide, MOS is obtained by
the autolysis of yeast cells at high temperature and at a controlled pH. After
yeast autolysis is completed, cell wall and yeast extracts are separated by
centrifugation, and the cell wall is spray dried.

Experimental diet
In order to prepare the experimental diets, the basal diet (BioMar granules;
containing 35% crude protein, 10% lipid) was crushed to form a fine powder,
186 M. N. FORSATKAR ET AL.

mixed with the appropriate level of MOS (0, serving as control; 2, 4, 6, or 8 g

kg−1 diet) diluted in distilled water (Akrami et al. 2013). The resulting paste
was spread on a plastic sheet, air dried, slightly ground, and sieved to
produce a suitable crumble size. We used low-density 1 mm BioMar pellet
in the present study to conform with the feeding practice of the hatchery
where we purchased the parent fish; this hatchery has recorded the best fish
growth using this diet. The prepared diets were stored at 4ºC during the
feeding experiment.

Feeding trials
At 30 dph, 300 zebrafish individuals previously starved for 24 h were sorted
by eye (initial body weight of 30 randomly collected individuals in each
treatment: 0.047 ± 0.017 g; mean ± SD) and randomly distributed into five
treatment groups in triplicate (60 fish in each treatment with three repli-
cates). Experimental feeding units (each replicate) were independent 12 L
circular plastic containers, with a water column of 20 cm and a fish density of
1.6 L−1. The tanks were continuously aerated, and physicochemical proper-
ties of the water used in the experiment were: temperature 27 ± 0.5ºC,
dissolved oxygen 8.1–8.8 mg L−1, pH 7.2–7.8, and total hardness
110–150 mg L−1 as CaCO3. Tank water was changed partly with dechlori-
nated municipal tap water every two days throughout the experiment.
During the six weeks that the experiment lasted, fish were hand-fed with
the prepared diets twice per day at 10:00 h and 16:00 h to apparent satiety
(Yossa et al. 2011).

Growth parameters
At the end of the feeding trial, 30 fish from each treatment were anesthetized
with clove powder (80 mg L−1) and their individual weight and length were
measured. Thereafter, growth performance parameters and survival rate were
calculated according to the following formulae:
Body Weight Increase ðBWIÞ ¼ Wt W0
Percent Body Weight Increase ðPBWIÞ ¼ ððWt W0 Þ=W0 Þ100
Specific Growth Rate ðSGRÞ ¼ ðLn Wt Ln W0 Þ100=t
Condition Factor ðCFÞ ¼ ðWt =Lt 3 Þ100
Survival rate ¼ ðNt =N0 Þ100
where Wt and W0 are final and initial body weights (g), respectively, t is
duration of the experiment in days, Lt is the standard length of fish at the end
of the experiment, N0 is the initial number of fish, and Nt is the final number
of fish.
 JOURNAL OF APPLIED AQUACULTURE 187

Starvation tolerance test

The quality of fish fed different concentrations of MOS was checked by
conducting starvation tolerance test (Furuita et al. 2000; Hedayati Rad et al.
2014) at the end of the feeding trial. The individuals from one replicate in
each treatment were divided into two groups of 10 fish each, left undisturbed,
and starved until they died. Survival activity index (SAI) for each treatment
was then calculated from the following equation:
1X
i¼1
SAI ¼ ðN  hiÞ  i
N k

where N is the total number of larvae, hi is the cumulative mortality by the ith
day, and k is the number of days elapsed until all larvae were dead due to
starvation.

Stress induction
After completion of feeding trial, 24 h-starved fish were subjected to a
simulated live transport as proposed by Pramod et al. (2010) with some
modifications. Fish (20 individuals from each treatment, 6–7 fish per repli-
cate) of the two remaining replicates for each treatment were netted and
placed into separate low-density polyethylene bags (55.5 × 27.5 cm) filled to
15 cm with aquarium treated water. Bags were then filled completely with
oxygen, made airtight, and put in a polystyrene box. The box was then
covered by a lid, sealed, and kept in a warm place. After 24 h in the box,
fish were placed back into their home tanks containing aerated fresh water
and after 1 h were examined for behavioral responses.

Behavioral observation
To assess the possible effects of MOS supplementation on fish behavior, fish
were exposed to a novel environment. The novel tank test is a repeatable and
reliable test to assess behavioral phenotypes related to stress and anxiety in
zebrafish (Stewart et al. 2012). The unfamiliar environment of a novel arena
serves as a potentially dangerous context to fish. Therefore, in this study,
changes in exploratory behavior relative to control treatment would show the
intensity of stress and anxiety of the fish.
The behavioral measures of anxiety in this study were after Stewart
et al. (2015). Fish were placed individually in a 4.5 L glass tank (30 cm
length × 10 cm width × 15 cm height) completely filled with water. The
tank was divided into two equal virtual horizontal sections, demarcated
with a line on the outside walls. The tank was lit from above by a 40 W
lamp, and the back and two sides were covered with pieces of white
188 M. N. FORSATKAR ET AL.

cardboard. A video camera was mounted 70 cm in front of the tanks and

used to record fish behavior during 5 min. The video was visualized and
the following behavioral characteristics were annotated following the
adding of the fish to the tank: time to reach the upper portion of the
tank (s), time spent in the upper portion of the tank (s), number of
entries to the upper section of the tank, number of freezing periods,
and freezing duration (s). Freezing was defined as the total absence of
movement, except for gills and eyes, for 1 s or longer. A decrease in
exploration (longer times to reach the top, fewer entries to the top, longer
freezing periods) represent behaviors indicating high stress and anxiety.
After each trial, the tank water was changed. Six identical test tanks were
used for the simultaneous testing of six fish, therefore performing all the
observations during a short period of time.

Statistics
All the data were previously tested for normality using the Kolmogorov-
Smirnov test. Data for growth parameters and survival activity index were
normally distributed and were analyzed using one-way analysis of variance
(ANOVA), followed with Duncan’s multiple range tests. However, behavioral
data were not normally distributed, and Kruskal-Wallis H test was per-
formed. The optimum level of MOS on zebrafish growth and behavior was
determined using polynomial regression analysis (Yossa and Verdegem
2015). All given P values were based on the two-tailed tests at the significance
level of 0.05. The statistical analyses were carried out using SPSS (IBM
Statistics, version 22.0).

Results
Growth performances
No fish mortality was recorded in the present trial due to treatments.
Table 1 summarizes mean growth parameters for every treatment group.
There was no significant difference in the initial body weight of the fish
among treatments (P > 0.05). However, at the end of the experiment, fish
fed with 4 g MOS kg−1 diet were significantly larger than that of the other
treatment groups (P ≤ 0.001). Analysis of variance for BWI (P ≤ 0.001),
PBWI (P ≤ 0.01), and SGR (P ≤ 0.01) at the end of the feeding trial also
showed significant effect on the growth performances of the fish fed with
4 g MOS kg−1 feed. However, differences in CF were not significant
(P > 0.05). Also, the regression analyses revealed that the concentration
of 4–4.1 g MOS kg−1 diet is optimum for growth parameters of zebrafish
juveniles (Figure 1).
Table 1. Growth performance of zebrafish juveniles fed with diets containing different levels of Agrimos® MOS for six weeks.*
Dietary MOS levels (g kg−1 diet)
Growth parameters** 0 2 4 6 8
Initial weight (g) 0.046 ± 0.021 0.051 ± 0.018 0.048 ± 0.017 0.046 ± 0.017 0.047 ± 0.014
Final weight (g) 0.152 ± 0.075a 0.186 ± 0.032b 0.221 ± 0.036c 0.190 ± 0.044b 0.153 ± 0.041a
BWI (g) 0.105 ± 0.074a 0.135 ± 0.039b 0.173 ± 0.041c 0.143 ± 0.048b 0.106 ± 0.045a
PBWI (%) 282.43 ± 189.61a 321.29 ± 191.41ab 430.64 ± 244.16c 368.58 ± 223.87ab 262.71 ± 169.97a
SGR 2.88 ± 1.31a 3.19 ± 1.05ab 3.75 ± 1.00c 3.44 ± 1.07ab 2.84 ± 1.04a
CF 4.53 ± 3.80 4.16 ± 2.29 3.66 ± 1.34 3.73 ± 1.80 4.64 ± 2.61
Survival (%) 100 100 100 100 100
*Values are presented as the mean ± SD; values with a different superscript letter are statistically different at P ≤ 0.05.
**BWI: Body weight increase; PBWI: percent body weight increase; SGR: specific growth rate; CF: condition factor.
JOURNAL OF APPLIED AQUACULTURE
189
190 M. N. FORSATKAR ET AL.

Figure 1. Second-degree polynomial relationships between the growth parameters and the
dietary MOS levels in zebrafish juveniles. Arrows show the optimum level of MOS after six-
week feeding experiment.

Larvae survival activity index

Results revealed a significant increase in larvae survival activity index (SAI) for
zebrafish fed with 4 g MOS kg−1 diet after six weeks of feeding (P < 0.05). Also,
fish fed with 6 g MOS kg−1 diet had more tolerance to starvation than the control
treatment. No significant differences were found between the remaining dietary
treatments (Figure 2A). Second-degree polynomial fit of SAI data to dietary
MOS levels yielded maximum larvae survival at inclusion of 4.1 g MOS kg−1
basal diet (Figure 2B).

Figure 2. (A) Larvae survival activity index (SAI; mean ± SD) of zebrafish after six weeks of
feeding with different levels of MOS. (B) The polynomial relationship between SAI and dietary
MOS levels. Bars with different letters are significantly different at P ≤ 0.05.
 JOURNAL OF APPLIED AQUACULTURE 191

Behaviors
The results of the behaviors involved in the intensity of stress and anxiety
of zebrafish at the end of feeding trials are presented in Figure 3. The
Kruskal-Wallis H test showed that there was a statistically significant
difference in time spent in the upper portion of the tank between the
different dietary treatments, χ2(4) = 9.97, P = 0.041, with a mean rank
score of 40.80, 59.45, 63.43, 42.98, and 45.85, for 0, 2, 4, 6, and 8 g kg−1
inclusion of MOS to the basal diet respectively (Figure 3). However, no
significant influences of MOS were observed for the other accounted
behaviors, including time to reach the upper portion of the tank, χ2
(4) = 4.24, P = 0.37 (Figure 2), number of entries to the upper half, χ
2

(4) = 4.57, P = 0.33 (Figure 2), number of freezing bouts, χ (4) = 3.17,
2

P = 0.52 (Figure 2), and freezing duration, χ2(4) = 4.69, p = 0.32 (Figure 2)
respectively. Regression analyses on latency to upper half, transition to
upper half, time in upper half, freezing bouts, and freezing duration data
against dietary MOS levels showed 3.9, 2.2, 4, 2.5, and 4 g MOS kg−1 diet
respectively as the best concentration to reduce anxiety in zebrafish
juveniles. The relationships were described by the following regression
equations:

Latency to upper half: y ¼ 1:375x2 7:825x þ 67:73;R2 ¼ 0:565

Transition to upper half: y ¼ 0:3214x2 þ 1:3586x þ 8:03;R2 ¼ 0:625
Time in upper half: y ¼ 5:1714x2 þ 30:559x þ 54:06;R2 ¼ 0:627
Freezing bouts: y ¼ 0:0393x2  0:1807x þ 0:95;R2 ¼ 0:673
Freezing duration: y ¼ 1:0763x2  6:4311x þ 17:27;R2 ¼ 0:544

Figure 3. Mean (±SD) of the anxiety-like behaviors in zebrafish juvenile after six weeks of
feeding with different levels of MOS. Asterisk (*) indicates significant difference at P ≤ 0.05.
192 M. N. FORSATKAR ET AL.

Discussion
The results of the present study strongly suggested that dietary supplementa-
tion with 4 g MOS kg−1 diet had significant effects on growth performance
and survival of zebrafish juveniles. Similarly, administration of several dif-
ferent prebiotics resulted in enhancement of growth performance in red
drum (Zhou et al. 2010). MOS-supplemented diet had significant effects on
growth and survival in hybrid striped bass (Li and Gatlin 2004), marron
(Sang et al. 2009), Atlantic salmon (Refstie et al. 2010), and Pacific white
shrimp (Zhang et al. 2012). All the previous studies have shown nutritional
benefits of prebiotics for aquatic animals. However, some disparities in the
experiments have also been observed (Piccolo et al. 2013), mainly in the
structural differences of the MOS used, dose supplemented, time of supple-
mentation, culture conditions, fish species, or age (Torrecillas et al. 2014).
The nonsignificant growth effect of higher inclusions of dietary MOS, i.e., 8 g
MOS kg−1 diet, when compared to control treatment, suggests that high
levels of MOS are not practically advantageous for growing zebrafish.
These results are in agreement with some previous studies that reported
ineffective influences of higher levels of prebiotics on fish growth (Akrami
et al. 2013; Hoseinifar et al. 2011). It seems that in higher inclusion levels,
intestinal microbiota is not able to ferment the prebiotic, which per se may
result in accumulation of nonfermentable material in the intestine that has
negative effects on both the gut microbiota and the histomorphology of the
intestine (Hoseinifar et al. 2011).
Survival activity index (SAI), which is expressed as a function of tolerance
to starvation of larvae, is defined as an index for larval quality in fish (Furuita
et al. 2000; Hedayati Rad, Forsatkar, and Huang 2014). Similarly to the
results from growth performance, SAI data showed that six weeks feeding
zebrafish with a diet containing 4 g MOS kg−1 feed increased the quality of
juveniles regarding their tolerance to starvation. These results are interesting,
as any natural mortality was not observed during the feeding trials.
Therefore, the higher quality observed in fish fed 2–6 g MOS kg−1 diet
reflects its nutritional benefits. MOS supplementations have been shown to
ameliorate intestinal morphology, enhance the digestive enzyme activities,
and increase villi length and microvilli density in several fish species
(Anguiano et al. 2013; Salze et al. 2008; Zhou et al. 2010). The improvement
in gut morphology may lead to improved nutrients absorption and therefore
to better energy and feed utilization, thereby improving starvation tolerance.
Unfortunately, we did not measure the mentioned digestive indicators, mak-
ing it impossible to accurately associate the SAI data with digestive function.
Recently, much attention has been given to the welfare of aquatic animals,
especially those species undercultured and/or used in research programs
(Ashley 2007). The health status and well-being of the research animal may
 JOURNAL OF APPLIED AQUACULTURE 193

influence research results (Johansen et al. 2006). In this study, following

completion of feeding trials, fish were subjected to a stressor and then tested
to see if diets containing different levels of MOS had some effects on their
behaviors. Using the novel tank test, subjects fed with 4 g MOS kg−1 diet
showed lower anxiolytic behaviors than the other treatments, but only the
behavior time spent in the upper half of the tank in the 4 g MOS kg−1 diet
group was significantly higher than that of the control fish. The lack of
significant improvement in relief from the effects of stress in fish fed MOS
may be due to the (1) lack of effect of dietary MOS on zebrafish stress and
welfare, and (2) short duration of supplementation. The second option seems
more likely because the lower incidence of anxiety-like behaviors were
observed in dietary treatments supplemented with lower levels of MOS, i.e.,
4 g MOS kg−1 diet. This is in accordance with the previous studies that
suggested the modulatory action of MOS in response mechanisms to stressful
condition (Torrecillas et al. 2012). It is worth noting that prebiotics promote
growth of beneficial gut microorganisms (Torrecillas et al. 2014) that may
produce B vitamins (Gibson and Roberfroid 1995), which in turn might
reduce stress. However, such effects are not yet documented in fish and
other aquatic animal species, but their scientific investigation would be
informative. Also, an insignificant effect of higher levels of MOS was
observed on the behavioral parameters of the experimental fish. Hence,
higher concentrations of a prebiotic may not always lead to the better fish
performance and welfare (Akrami et al. 2013; Zhang et al. 2012), and there is
evidence showing the benefits of low prebiotic levels on stress responses in
fish (Zhang et al. 2015).

Acknowledgments
The authors thank M. Hedayati Rad for helpful assistance during the experiments and the
editor and anonymous reviewers for their constructive and insightful comments.

Funding
This study was supported by grants from University of Tehran. All of the authors declare that
they have no conflict of interest.

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