Celia
Celia
Celia
a r t i c l e i n f o a b s t r a c t
Article history: The aim of this study was to evaluate the effect of encapsulated synbiotic (Bacillus sp. NP5 and oligo-
Received 24 June 2014 saccharide) dietary at different dosages on growth performance, survival rate, feed conversion ratio, and
Accepted 6 April 2015 immune responses of Litopenaeus vannamei against Vibrio infection. The shrimps of the main treatments
Available online 11 December 2015
were fed by the diet that contained three different dosages of encapsulated synbiotic [0.5% (A), 1% (B),
and 2% (C) (w/w)] with feeding rate of 5% of shrimp biomass (4 times a day). The shrimps of two control
KEYWORDS:
treatments (negative control and positive control) were fed only by commercial feed without supple-
dosage,
mentation of encapsulated synbiotic. The growth, feed conversion ratio, and survival rate were observed
Litopenaeus vannamei,
microencapsulation,
after 30 days of encapsulated synbiotic dietary. The shrimps were then challenged by injection of Vibrio
synbiotic, harveyi (6 log colony forming units/mL) 0.1 mL/shrimp, excluded the negative control treatment. Af-
vibriosis terward, the survival and immune responses were observed for 9 days after experimental infection. The
shrimps treated with 2% encapsulated synbiotic (treatment C) in the diet showed the highest growth
performance (2.98 ± 0.42%), feed conversion ratio (1.26 ± 0.19), and better immune responses i.e. total
hemocyte counts, differential hemocyte count, phenoloxidase, and intestine bacteria observation
compared to those of positive control treatment.
Copyright © 2016 Institut Pertanian Bogor. Production and hosting by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
http://dx.doi.org/10.1016/j.hjb.2015.10.007
1978-3019/Copyright © 2016 Institut Pertanian Bogor. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://
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164 A. Zubaidah, et al
marked by rifampicin resistancy (50 mg/mL). Sweet potato as pre- The experiment was conducted in a completely randomized
biotic source used in this study was obtained from sweet potato design. Significant differences of regressions of survival rate (SR),
research center (Balitkabi), East Java. The preparation of prebiotic SGR and FCR were tested by analysis of variance. The results which
according to the method of Marlis (2008) was conducted at showed differences were tested by least significance different test.
Nutrition Laboratory, and the probiotic was prepared at Fish Health The immune responses are showed in graphs and analyzed
Laboratory, Department of Aquaculture, Faculty of Fisheries and descriptively.
Marine Sciences, Bogor Agriculture University. Synbiotic microen-
capsulation process was done at SEAFAST Center Laboratory, Bogor 3. Results
Agricultural University. The selected materials used for microen-
capsulation process were whey protein and maltodextrin. SR of shrimp was observed for 30 days before and 9 days after
The shrimps (L. vannamei) with initial weight of 2.43 ± 0.26 g V. harveyi infection (Figure 1). The encapsulated synbiotic dietary
were obtained from Situbondo Brackishwater Aquaculture Devel- before challenge test did not show a significant different effect on
opment Center (BBAP), East Java, Indonesia. The shrimps were SR. However, after challenge test it showed a significant
cultured in plastic aquaria (each with a dimension of difference (p < 0.05) among shrimps fed with encapsulated syn-
60 40 35 cm3). This experiment was conducted in a completely biotic dietary and infected with V. harveyi i.e. treatment A
randomized design with five treatment diets including three dos- (93.33 ± 5.77%); B (93.33 ± 5.77%); and C (93.33 ± 11.55%); and
ages of encapsulated synbiotic diet [0.5% (A), 1% (B), and 2% (w/w) shrimps without encapsulated synbiotic dietary and infected with
(B), as well as positive control (PC), and negative control (NC)]. Each V. harveyi (PC), that is 63.33 ± 5.77%. NC treatment showed the
treatment was conducted in four replications. The initial densities highest SR (100 ± 0%).
were 10 shrimps of each tank. Each tank containing 35 L of sea No mortality occurred during encapsulated synbiotic dietary (30
water and kept under controlled conditions (temperature was days before challenge test) for all treatments (Figure 2). Mortality
ranging from 28 to 29 C, salinity was from 33 to 34 ppt, total started to occur from 32nd day (1st day after V. harveyi infection) at
ammonia nitrogen was ranging from 0.02 to 0.66 mg/L, dissolved PC treatment and continued until 36th day. The mortality of
oxygen was ranging from 5 to 6.5 mg/L, and pH was from 7.6 to 7.9). shrimps fed with encapsulated synbiotic dietary (treatment A, B,
Faeces and uneaten food were sucked out at the same time of water and C) occurred at 33rd day and until 35th day.
replacement, which was done every day (after the first feeding) up SGR and FCR were observed after encapsulated synbiotic dietary
to 10 L. (30 days before infection). The result of feeding, growth perfor-
The experimental diets were prepared by adding the encapsu- mance (SGR), is presented in Figure 3. The result clearly showed the
lated synbiotic (0.5%, 1%, and 2% g/kg) to the diet. The control beneficial effects of encapsulated synbiotic dietary on SGR of
treatment diets were added only with egg white and without L. vannamei. The shrimps that supplemented with encapsulated
supplementation of encapsulated synbiotic. Feed used in this study synbiotic have significant increase of SGR in comparison to the
was commercial shrimp feed pellet (containing 36% protein, 5% fat, control treatments (both negative and PC) (p < 0.05). The experi-
4% fiber, 12% moisture and 15% ash). Feed mixing process was mental treatments of this study were significantly different for all
sprayed manually for each treatment. Encapsulated synbiotic treatments. The greatest effect was obtained in treatment C [2% (w/
weighed according to the treatment and then egg white added as w) encapsulated synbiotic dietary] which had a value of
the binder [2% (v/w)] from the total feeding rate (Wang 2007). Feed 2.98 ± 0.42%, and then treatment B (2.69 ± 0.3%), treatment A
that has been weighed according to the feeding rate was then put in (2.23 ± 0.16%), NC treatment (2.12 ± 0.31%), and PC treatment
the mixture homogenously. Feeding was done four times a day (at (2.09 ± 0.23%). Both NC and PC control treatments before chal-
07.00; 11.00; 15.00 and 19.00) for 30 days. lenged test were fed by the same treatments, so there was no sig-
After 30 days of encapsulated synbiotic dietary, specific nificant difference between them.
growth rate (SGR) and feed conversion ratio (FCR) were observed. The FCRs of shrimps fed by encapsulated synbiotic were lower
On 31st day, shrimps in the treatments A, B, C, and PC were compared with the control treatment (p < 0.05) (Figure 4). The
challenged by injecting 0.1 mL/shrimp with V. harveyi intramus- lowest FCR value was showed by treatment C (1.26 ± 0.19), followed
cularly in the cell density of 6 log colony forming unit (CFU)/mL, by treatment B (1.56 ± 0.25), then treatment A (1.89 ± 0.08),
whereas treatment NC was only injected by phosphate buffer PC (1.97 ± 0.27), and NC (1.99 ± 0.25).
saline 0.1 mL/shrimp. V. harveyi used in this study was genetically Immune response of L. vannamei was observed at the pre-
marked by rifampicin resistancy (50 mg/mL). Observations of challenge test of V. harveyi (30th day), 1 day after challenge test
immune response parameters included the total hemocyte counts (32nd day), and 9 days after challenge test (40th day). Observations
(THCs), the differential hemocyte count (DHC) according to the
method of Hai and Fotedar (2009) and the phenoloxidase (PO)
according to the method of Liu and Chen (2004). Immune re-
sponses were observed on 30th, 32nd and 40th day after the initial
treatment, while intestine bacteria (total plated) was observed on
0, 30th, 32nd, 35th and 40th day.
Shrimp intestine was isolated and weighed (g) and then put in a
microtube that contained 1 mL of phosphate buffer saline (NaCl
0.8%, K2HPO4 0.15%, Na2HPO4 0.02% and KCl 0.02%). After homog-
enization, mixture was serially diluted and plated by performing
total plate count (Li et al. 2009). The media used for total plate count
were sea water complete (SWC) (bacto peptone 0.5%, yeast extract
0.1%, glycerol 0.3%, bacto agar 2%, sea water 75%, and distilled water
25%) without rifampicin for total viable bacterial count, SWC with Figure 1. SR of L. vannamei. Different letters over each treatment bar (mean ± standard
error) indicate significant difference (p < 0.05). A: 0.5% encapsulated synbiotic dietary;
rifampicin (50 mg/mL) for counting the Bacillus NP5 which resis- B: 1% encapsulated synbiotic dietary; and C: 2% encapsulated synbiotic dietary. ( )
tant to Rifampicin (RfR) and thiosulphate citrate bile-salt sucrose Before V. harveyi infection, ( ) after V. harveyi infection. NC ¼ negative control;
(TCBS Criterion, USA) for counting the V. harveyi RfR. PC ¼ positive control; SR ¼ survival rate.
Dietary supplementation of encapsulated synbiotic at different dosages to prevent vibriosis in white shrimp, Litopenaeus vannamei 165
increase also occurred after the challenge test (32nd day) in all
treatments. However, at the 40th day, total hemocytes decreased in
all treatments.
PO values of encapsulated synbiotic dietary (30th day) showed
improvement, especially in treatment C (Figure 6). These results
indicated that encapsulated synbiotic dietary was able to stimulate
the shrimp immune system by increasing the activity of PO. After
challenge test the PO value also increased in all treatments,
including control treatments, except for treatment C. Before
V. harveyi infection, the treatment of encapsulated synbiotic dietary
resulted in higher granular cells compared with control treatments
(Figure 7). The same results were showed at the time after chal-
lenge test and at the end of observation (40th day).
The intestinal bacterial population were observed at 0 (before
treatment), 30th, 32th, 35th, and 40th day (the last day of treatment).
Figure 2. Mortality rate after the challenge test of L. vannamei at differential dosages of
encapsulated synbiotic dietary. A: 0.5% encapsulated synbiotic dietary; B: 1% encap- The observation included bacterial abundance/TVBC, V. harveyi RfR
sulated synbiotic dietary; and C: 2% encapsulated synbiotic dietary. NC ¼ negative count, and Bacillus NP5 RfR count. The number of bacteria in the
control; PC ¼ positive control. intestine was ranging from 7 up to 9 log CFU/g (Figure 8). Intestinal
bacterial populations increased with the encapsulated synbiotic
dietary treatments (30th day), but did not occur in control treat-
ments. The intestinal bacterial population also increased after
challenge test. However declined intestinal bacterial population
was noted at 35th day and 40th day in treatments A, B, and C, but not
in the PC. The lowering of the intestinal bacterial population was
most probably caused by the decreasing population of Bacillus NP5
RfR and V. harveyi RfR in the intestine (Figure 9). Bacillus NP5 RfR
populations in the intestine were relatively low on 35th and 40th
day, but the B and C treatments of Bacillus NP5 RfR population were
still higher than A treatment. On the other hand, the population of
V. harveyi RfR at all shrimps treated with encapsulated synbiotic
diet was not found in intestines (Figure 9).
4. Discussion
Figure 4. FCR of L. vannamei. Different letters over each treatment bar Figure 5. THC of L. vannamei. A: 0.5% encapsulated synbiotic dietary; B: 1% encapsu-
(mean ± standard error) indicate significant difference (p < 0.05). A: 0.5% encapsulated lated synbiotic dietary; and C: 2% encapsulated synbiotic dietary. ( ) Before V. harveyi
synbiotic dietary; B: 1% encapsulated synbiotic dietary; and C: 2% encapsulated syn- infection (the 30th day), ( ) 1 day after V. harveyi infection (the 32nd day); ( ) 9 days
biotic dietary. FCR ¼ feed conversion ratio; NC ¼ negative control; PC ¼ positive after V. harveyi infection (the 40th day). NC ¼ negative control; PC ¼ positive control;
control. THC ¼ total hemocyte count.
166 A. Zubaidah, et al
showed that growth rate increased with supplementation of pro- Chiu et al. (2007) showed that the probiotics was capable of
biotic Bacillus NP5 at a dose of 8 log CFU/mL. The increase of growth increasing the THC value as well as enhancing the immune
rate was assumed because of enzymatic activity in shrimp intestine. response during the period of stress because of pathogen infection.
Probiotic Bacillus NP5 that were used in this study were isolated Hemocyte cell count decline is an effect of the body's defense
from tilapia intestine which is capable of secreting amylase enzyme mechanisms such as the infiltration of the networks of infected
(Putra et al. 2015) and has been adapted to SWC medium to survive hemocytes, and hemocyte cell death due to apoptosis mechanism
when exposed to sea water and in shrimp intestine. The amylase (Costa et al. 2009). Pro-PO activity system and other humoral body
enzyme acts as an exogenous enzyme (Taoka et al. 2007; Wang defense mechanisms also affect the number of hemocyte cells
2007). This enzyme was suggested to have capability to stimulate (Huang et al. 2013; Tassanakajon et al. 2013).
endogenous enzyme that is produced by shrimps (Saeed et al. The increase and decrease of THC were due to the increase and
2006), so feed that is absorbed in shrimp intestine can be decrease components of its hemocyte cells. Hemocyte consists of
degraded effectively, then the optimum nutritional absorption can three types of granules in the cytoplasm, i.e. the hyaline, granular
be reached. Besides that, prebiotic also provided as the additional and semigranular hemocytes. The percentage of granular cells and
nutrients for probiotic bacteria (Evivie 2013). semigranular in this study was made into one group, namely the
The lower FCR value in this study indicated that the shrimps fed percentage of granular cells. The hyaline and granular cells
by encapsulated synbiotic dietary showed the effectivity in contributed to destroy the antigen at shrimp body through
nutrient digestibility. Similar result reported by Nurhayati et al. phagocytosis, encapsulation, nodule formation and produced hu-
(2015) that supplementation of synbiotic SKT-b gave a significant moral components. Humoral components are stored in
effect on the growth and feed conversion of shrimp (L. vannamei). granule hemocyte which include anticoagulant protein, aglutinin,
In observation of immune responses, THC value in this study PO enzymes, antimicrobial peptides, and protease inhibitors
showed an increase after infection of V. harveyi for all treatment and (Jiravanichpaisal et al. 2006).
then declined. This means a rapid reaction of shrimp immunity to PO is an enzyme responsible for melanization process in crus-
infection. The treatment C showed no significant different before taceans as response to antigen and for pigmentation (Zufelato et al.
and after infection of V. harveyi. This suggested that supplementa- 2004). PO system can be activated by several microbe poly-
tion of encapsulated synbiotic with optimum dosage have the saccharides and specific pattern recognition proteins, such as LPS
capability to stimulate the production of hemocyte cell, therefore, (lippo polysaccharides) and b-1, 3-glucan-binding protein and
the infection did not affected the THC value. A study presented by peptidoglycan-binding proteins (Wang 2007). The treatment C
Figure 7. Differential hemocyte count: (A) hyaline count; and (B) granular count. A: 0.5% encapsulated synbiotic dietary; B: 1% encapsulated synbiotic dietary; and C: 2% encap-
sulated synbiotic dietary. ( ) Before V. harveyi infection (the 30th day), ( ) 1 day after V. harveyi infection (the 32nd day); ( ) 9 days after V. harveyi infection (the 40th day).
NC ¼ negative control; PC ¼ positive control.
Dietary supplementation of encapsulated synbiotic at different dosages to prevent vibriosis in white shrimp, Litopenaeus vannamei 167
Figure 9. (A) Bacillus NP5 RfR count; and (B) V. harveyi RfR count in the intestine of L. vannamei. A: 0.5% encapsulated synbiotic dietary; B: 1% encapsulated synbiotic dietary; and C:
2% encapsulated synbiotic dietary (the 0 day); ( ) before V. harveyi infection (the 30th day), ( ) 1 day after V. harveyi infection (the 32nd day); ( ) 4 days after V. harveyi infection (the
35th day); ( ) 9 days after V. harveyi infection (the 40th day). NC ¼ negative control; PC ¼ positive control.
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