Fish & Shellsh Immunology 36 (2014) 582e589

Contents lists available at ScienceDirect

Fish & Shellsh Immunology

journal homepage: www.elsevier.com/locate/fsi

Full length article

Effects of dietary katuk (Sauropus androgynus L. Merr.) on growth,

non-specic immune and diseases resistance against Vibrio
alginolyticus infection in grouper Epinephelus coioides
Agus Putra A. Samad a, Urip Santoso b, Meng-Chou Lee c, Fan-Hua Nan a, *
a
Department of Aquaculture, National Taiwan Ocean University, Keelung 20224, Taiwan, ROC
b
Department of Animal Husbandry, University of Bengkulu, Bengkulu 38371, Indonesia
c
Department of Aquaculture, College of Agriculture, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan, ROC

a r t i c l e i n f o a b s t r a c t

Article history: This study was conducted to investigate the effect of katuk (Sauropus androgynus L. Merr) on growth,
Received 15 September 2013 non-specic immune and diseases resistance against Vibrio alginolyticus in grouper (Epinephelus
Received in revised form coioides). Grouper juveniles (mean weight 10.97
1.99 g, and length 9.67
0.33 cm) were separated into
18 November 2013
four groups and cultivated in 100-L tank. Each group was fed with diets containing 0, 1.0, 2.5 and 5.0 g/kg
Accepted 22 November 2013
Available online 1 December 2013
herbs diet twice daily. Fish were sampled for non-specic immune parameters at 0, 1, 2, 4, 7, 14 and 30
days. Results showed that sh received S. androgynus at 1.0 and 2.5 g/kg diets affected the growth and
non-specic immune responses. Weight gain, specic growth rate, respiratory burst activity, phagocy-
Keywords:
Epinephelus coioides
tosis and reactive oxygen species signicantly increased in sh administered with 1.0 and 2.5 g/kg
Plant extract S. androgynus diets. The mortality rate after V. alginolyticus challenge decreased in sh fed with 1.0 g/kg
Respiratory burst activity S. androgynus extract. Thus, this study indicated that administration of grouper with S. androgynus
Sauropus androgynus supplemented diets can affect the growth performances, diseases resistance and enhances non-specic
Vibrio alginolyticus immune responses.
2014 Elsevier Ltd. All rights reserved.

1. Introduction resistant pathogen, bioaccumulation and environment pollution

Recently, aquaculture is recognized as a fast growing food
production sector in the world. A rapid expansion of aquaculture
activities has been practiced in many countries including:
Indonesia, Thailand, China, Japan and Taiwan [1e3]. Some marine
species such as: grouper, sea bass, mullet and sea bream are
cultured intensively [1,4] to support good quality seeds and to
improve productivity. However, in intensive systems, sh are
reared in high stocking density in a limited area; therefore such
conditions often negatively affect the sh immune system and
consequently increase susceptibility to disease [5e7]. Further-
more, when the diseases occur, sh farmers are still dependent
on antibiotics and chemotherapeutics to treat sh disease.
However, this condition is believed as an improper way and not
environmentally friendly, since it has a risk of generating
* Corresponding author. Tel.: 886 2 24622192x5231; fax: 886 2 24635441.
E-mail address: fhnan@mail.ntou.edu.twe (F.-H. Nan).
[8e10]. Besides that, although vaccine is considered more
effective to treat sh diseases, however it specic against
particular pathogens and the price are not affordable by farmers
[11e14]. Therefore, one of the most promising methods of con-
trolling diseases is by strengthening the sh defense
mechanisms.
Application of plants materials is believed to contribute the
increasing of immune responses against pathogen and increasing
appetites to accelerate recover from disease. A large number of
plants products have been investigated for immune response ac-
tivity in sh such as: Nyctanthes arbortristis in Oreochromis mos-
sambicus [5]; Lactuca indica in Epinephelus bruneus [15]; Astragalus
radix and Ganoderma lucidum in Cyprinus carpio [16]; Allium sat-
ivum in Labeo rohita [17]; Nigella sativa in Oncorhynchus mykiss
[18]; Prunella vulgaris in Paralichthys olivaceus [19]; Lonicera
japonica and G. lucidum in Oreochromis niloticus [20]; Achyranthes
aspera in Catla catla [21]; Andrographis paniculata in C. carpio [22];
Psidium guajava, A. paniculata and Piper betle in Pangasius hypo-
phthalmus [23]; and Picrasma javanica in Osphronemus gouramy

1050-4648/$ e see front matter 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fsi.2013.11.011
 A.P.A. Samad et al. / Fish & Shellsh Immunology 36 (2014) 582e589
[24]. Moreover, components such as polysaccharides, lectins,
proteins and peptides present in plants have been shown to
stimulate the immune system [25].
In the present study, we use katuk (S. androgynus L. Merr.) as a
feed additive in sh diets. Katuk (family: Euphorbiaceae) is widely
known as vegetable. This plant is mostly grown in South Asia and
Southeast Asia. Additionally, it is commonly used as traditional
medicine to purifying the blood, fever, ulcer, urinary problem,
earache and frambesia [26,27]. Recent studies showed that sup-
plementing S. androgynus in terrestrial animals diets could affect
the growth performance, egg production and bacterial inhibitation.
S. androgynus extract could stimulate body weight, feeding ef-
ciency and feed conversion ratio [28,29], reduced cholesterol and
enriched amino acid [30,31], improved egg quantity and quality
[32,33], increased milk production [34], and inhibited the growth of
Salmonella aureus, Salmonela typhosa and Escherichia coli [35].
Furthermore, it mentioned that S. androgynus is a high nutritive-
value plant contained several important materials such as: multi-
vitamin [36], metabolic compounds [37], essential oils [38], alka-
loids and non alkaloids [28] and active compounds [29]. Even
though many trials have been applied in terrestrial animal as a
result of benecial substances contain in this plant, the efcacy of
S. androgynus in aquaculture is still unknown. Thus, this study is
conducted to investigate the effect of dietary supplementation of
S. androgynus on growth performances, non-specic immune
response and diseases resistance against Vibrio alginolyticus infec-
tion in grouper Epinephelus coioides.
2. Materials and methods
2.1. Experimental sh and bacterial preparation
Grouper (E. coioides) weighed 10.97
1.99 and 59.45
4.53 g
body weight for experiments on growth performances and im-
mune responses activities, respectively, were obtained from
Aquatic Animal Center, National Taiwan Ocean University and then
acclimated in the hatchery of the Department of Aquaculture for 2
weeks prior to experimentation. Fish were reared and fed twice
daily by feeding commercial diet. Fish of each group were distrib-
uted into 100-L total water volume (60  50  35 cm). Well aerated
water was provided from a storage berglass. Water quality pa-
rameters were maintained at temperature 28.0
1  C; pH 8.0
1
and salinity 34
1 ppt.
The gram-negative bacteria used in this study were isolated from
V. alginolyticus infected grouper. Samples were collected from the
liver and the spleen for inoculation onto tiosulphate citrate bilesalt
sucrose agar (TCBS, Difco), selective to vibrionaceae. Plates were
incubated for 24 h at 30  C and the yellow colonies produced from
TCBS were isolated on tryptone soy agar (TSA, Difco) to obtain a
pure culture for identication with the API 20E (Biomeriux) kit
following the instructions of the manufacturer. Pure culture
V. alginolyticus was then grown in TSA medium with 1% NaCl and
incubated in 28  C for 24 h [39]. Then, the obtained bacteria were
diluted using Phosphate Buffer Saline (PBS, Bio Basic Inc.) and were
prepared to a nal concentration of 1.0  109 cfu/ml before intra-
peritoneally injection. Healthy and disease free juveniles of
E. coioides were treated in challenge test. The juveniles injected with
PBS (20 mL) served as control group, whereas, the challenge test was
conducted by the injection of 20 mL of bacterial suspension
(1.0  109 cfu ml1) resulting in 2.0  107 cfu sh1. Furthermore,
reisolation and identication of the bacteria from kidney and the
ascites of moribund grouper after bacterial challenge were con-
ducted with TSA (1% NaCl) and TCBS agar plates [39]. In bacterial
challenge, each group (11 sh/groups) was fed their respective diets
583
for 1, 4, or 7 days before infection. During this trial, the diseases
symptoms and mortality were monitored for 2 weeks.
2.2. Experimental design
For growth performances and immune responses trials, the
experimental facility was composed of 24 tanks (100-L each).
Water was provided from storage tanks, ltered and supplied to the
system. The water system was equipped with mechanical lter
(spongy), UV light, automatic heater and supplied with compressed
air via air-stones from air pumps. Water ows in experimental tank
were measured and adjusted before the experiment in order to be
proportional to the sh density and to ensure sufcient water
circulation.
Groupers were randomly distributed into cultivating system in
triplicate (at a density of 25 sh/tank). The feeding trial was divided
into four treatment groups: Control group (C) were fed the diet
without S. androgynus extract; SAA were fed 1.0% of S. androgynus
mixed diet; SAB were fed 2.5% of S. androgynus mixed diet and SAC
were fed 5.0% of S. androgynus mixed diet. During experimentation,
sh were hand fed twice daily (08:00 and 17:00) at 3% of the total
biomass. All sh were received their respective diets twice daily for
70 days and 30 days for the growth performances and immuno-
logical trials, respectively. For bacterial challenge test, sh were
cultivated in non-circulating system, with ve-day intervals water
exchange. All sh were fed their respective diets before injection
using V. alginoliticus.
Proximate analyses (crude protein, total lipid, moisture and ash)
of diets were examined following the standard of AOAC methods
[40]. Data on growth rate was recorded regularly every 2 weeks by
weighed and measured individual sh from each tank. On each
sampling day, each of individual sh was caught from tank using a
small net. Then the sh were quickly weighed and measured. The
body wet weight was measured using an analytical balance (Ohaus
Navigator, no.4120, Canada) and the total length using digital
caliper (Mitutoyo, Absolute Digimatic, Japan). Immediately after
measurements, the sh were carefully returned to its original tank.
Growth performances were calculated as following: specic
growth rate (SGR, %/day) 100  (lnW2  lnW1)/T; where: W1 and
W2 are initial weight and nal weight, respectively and T is the
number of days in the feeding periods; weight gain (WG,
%) 100  [(nal weight (g)  initial weight (g)]/initial weight (g)
[41,42]; and condition factor (K) [(105  weight of sh (g)/(length
of sh)3(cm)] [43]. In experiment on survival rate, all treatments
were observed daily and the data was calculated by the following
formula: survival rate (SR, %) (nal no. of sh/initial no. of
sh)  100 [44]. Viscerosomatic index (VSI) was examined by
sacricing ve experimental sh from each replication to weigh the
visceral. VSI was measured using formula: (VSI, %) 100  [weight
of visceral (g)/weight of sh (g)]. Hepatosomatic index (HSI) was
carried out by dissecting ve experimental sh from each replica-
tion to weigh the liver. HSI was calculated using formula: (HSI,
%) 100  [weight of liver (g)/weight of sh] [45,46].
2.3. Dietary preparation
Fresh katuk leaves (S. androgynus) have been collected from
Bengkulu, Indonesia. The leaves were cleaned and shadow dried for
three days. After drying, all specimens were ground using an
electrical blender and then ltered using 20 m mesh to obtain the
powder. Then, the powder was extracted with 70% ethanol using
soxhlet apparatus. This extraction was operated with gently
shaking at room temperature for 24 h [47]. The obtained extract
was then ltered and condensed using rotary vacuum evaporator
under reduced pressure at 45  C [48].
584 A.P.A. Samad et al. / Fish & Shellsh Immunology 36 (2014) 582e589

The extract was then examined using Gas Chromatography Mass Rad, Hercules, CA, USA) with Hanks Balanced Salt Solution (HBSS).
Spectrometry (GCMS) to analyze the active compounds. Instrument: The cell suspension was transferred to the tubes containing 3 mL of
Agilent Technology 6890 Gas Chromatograph with auto sampler 34e51% Percoll (GE Healthcare, Buckinghamshire, UK). The tubes
and 5975N Mass Selective Detector and Chemstation data system; were centrifuged at 400  g for 40 min at 4  C, and the leukocytes
ionization mode: electron impact; electron energy: 70 eV, column: on the interface of the 34% and 51% Percoll were collected and
HP Ultra 2. Capilarry coloumn length (m) 30  0.25 (mm) ID  0.25 transferred into HBSS. The leukocytes were centrifuged three times
(mm) lm thickness; carrier gas: helium; column model: constant at 500  g for 10 min at 4  C for complete removal of supernatant.
ow; ow column: 0.9 mL/min; injection volume 5 mL; methods le: The cell pellets were resuspended with Leibovitzs L-15 medium
BAHALAM. (SigmaeAldrich), and the viable cells were counted with 0.4% try-
Furthermore, S. androgynus extract was prepared in three pan blue solution (SigmaeAldrich) [49].
different concentrations (1.0%, 2.5% and 5.0% of total ingredients)
before mixed into experimental diets (Table 1). The ingredients of 2.5. Measurement of the non-specic immune parameter
each diet were mixed together for 30 min for pelletization. Control
diet was treated similarly with the supplemented diets, but no Phagocytic activity assays was measured using the methods
extract was added. The diets were then dried in a forced-air drier at described by Fujiki and Yano [50]. Briey, 50 mL of leukocytes
room temperature for 24 h. After drying, the pellets were stored in (5  106 cells) was placed on a glass slide, and allowed to adhere for
plastic bags and stored at 4  C for further use. 20 min at 25  C in a moisture incubation chamber. Then, 50 mL of
latex beads (107 beads/mL, SigmaeAldrich) was added to the leu-
2.4. Sample preparation kocytes monolayer, and incubated for 30 min at 25  C. The per-
centage of phagocytes ingesting beads (PR) and the number of
For serum, blood samples from specimens in dietary adminis- beads ingested per phagocyte (PI) were calculated by enumerating
tration were withdrawn from caudal veins in the remaining 100 phagocytes under a microscope. Phagocytic activity was
anaesthetized sh into blood collecting tubes or Eppendorf tubes expressed as the phagocytic index (PI) [51]. The phagocytic rate
without anticoagulant in the syringe. Blood samples in Eppendorf (PR) and phagocytic index (PI) were determined as followed:
tubes were allowed to clot for 2 h at room temperature in a slanting
position. The tubes were kept at 4  C overnight and were then PR (Phagocytosing cell/Total cell)  100
centrifuged at 2500  g for 15 min and the supernatant serum was
collected. The serum was stored at 80  C until used for lysozyme PI (Total phagocytosed beads/ Phagocytosing cell)  100
activity analysis. The sh then used for the separation of head
kidney leukocytes and the liver samples. Respiratory burst activity produced by phagocytes in the head
The head kidneys and spleens of E. coioides were excised from kidney was measured according to the methods described by
bled sh (n 5), and passed through a 100 mm nylon mesh (Bio- Cheng et al. [52]. In brief, 100 mL of leukocytes (5  106 cells) was
placed in 96 wells and incubated for 1 h at 37  C. Then, the non-
adherent cells were removed by washing the wells with Hanks
Table 1 Balanced Salt Solution (HBSS). 100 mL of zymosan (SigmaeAldrich)
Composition of experimental diets.
at 1 mg/mL HBSS was added to half of the wells, and 100 mL HBSS
Materials Formulation (g/100 g diets) was added to another half. 100 mL of nitroblue tetrazolium (NBT)
Control SAA SAB SAC was added and incubated at 25  C for 30 min. Then the HBSS was
discarded and the reaction was stopped by adding methanol. After
Fish meal 67.2 67.2 67.2 67.2
Fish oil 10.0 10.0 10.0 10.0
washing with methanol, the formazan formed in each well was
a-starch 6.64 6.64 6.64 6.64 dissolved by adding 120 mL of 2 M Potassium hydroxide (KOH) and
Vitamin premixa 2.00 2.00 2.00 2.00 140 mL of dimethyl sulphoxide (DMSO). The NBT reduction was
Mineral premixb 3.00 3.00 3.00 3.00 measured using an ELISA microplate reader at 630 nm. Cells from
Vitamin C 0.05 0.05 0.05 0.05
each sh were in triplicate wells. Respiratory burst activity was
Choline chloride 0.50 0.50 0.50 0.50
Sauropus androgynus extractc 0.00 1.00 2.50 5.00 expressed as NBT-reduction.
Carboxymethylcellulose, CMC 2.00 2.00 2.00 2.00 Reactive Oxygen Species was measured by measurement of the
Cellulose 8.57 7.57 6.07 3.57 chemiluminescent response. 100 mL leukocytes suspension was
Crude protein 45.08 45.86 45.91 45.80 placed into 96 wells, add 100 mL 1 mM luminal suspension liquid
Crude lipid 14.02 13.98 14.02 14.01 and 100 mL of 1 mg/mL zymosan in HBSS. Respiratory burst induced
Crude ber 3.19 2.71 2.66 2.32 by phagocytosis of zymosan particles was measured in relative
Ash 14.37 14.60 14.39 14.88
luminescence unit (RLU) per second.
Moisture 8.94 7.71 7.79 8.49
NFE 23.34 22.85 23.02 22.99 The SOD assay was conducted using the Ransod kit (Randox
Calculated energy 399.86 400.66 401.90 401.25 Laboratories, Crumlin, UK) following the manufacturers instruc-
P/E ratio 112.74 114.46 114.23 114.14 tion. In brief, 850 mL of the reaction substrate containing xanthine
Abbreviations: SAA: feed mixed with 1.0% S. androgynus extract; SAB: feed mixed and INT 2-(4-iodophenyl)-3-(4-nutrophenol 3-5-phenlte-
with 2.5% S. androgynus extract; SAC: feed mixed with 5.0% S. androgynus extract. trazolium) were mixed with 25 mL of liver tissue solution obtained
NFE (nitrogen free extract): 100  (crude protein crude lipid crude ber ash from sh fed with the test diets, or with 25 mL of HBSS as a control,
Calculated energy: crude protein (4 kcal/g), crude lipid (9 kcal/g) and carbohydrate
followed by the addition of 125 mL of xanthine oxidase (XOD).
(4 kcal/g). P/E ratio: (crude protein/calculated energy)  1000.
a
Vitamin premix (mg/g): thiamin hydrochloride, 2.5; calcium pantothenate, 2.5; During the reaction, xanthine was reduced by XOD to produce uric
riboavin, 10; nicotinic acid, 37.5; folic acid, 0.75; inositol, 100; pyridoxine hydro- acid and superoxide radicals, and further reacted with INT to pro-
chloride, 0.25; menadione, 2; retinol acetate, 1; alphatocopheryl acetat, 20; chole- duce formazan dye. The SOD in the sample solution would compete
calciferol, 0.0025; biotin, 0.25; vitamin B12, 0.05. with INT for the superoxide radicals, thus the SOD activity could be
b
Mineral premix (mg/g): Ca(H2PO4)2H2O, 134; calcium lactate, 333; ferric citrate,
33; MgSO4$7H2O, 133; NaH2-PO4$2H2O, 83; K2HPO4, 234; ZnCl2, 20; AlCl3$6H2O, 7;
determined based on its ability to inhibit formazan dye formation.
MnSO4$H2O, 7; KI, 6; CuSO4$5H2O,10. The rate of formazan formation was measured by detecting the
c
Sauropus androgynus was extracted using ethanol 70%. absorbance at 505 nm at 30 and 210 s after the initiation of reaction.
 A.P.A. Samad et al. / Fish & Shellsh Immunology 36 (2014) 582e589 585

The rate of formazan formation inhibition was calculated by Table 3

comparing the formazan formation rate of the liver tissue solution Components in S. androgynus extracted using ethanol 70%.

treated groups with the HBSS treated control group. The specic Retention Compounds Molecular Peak area
activity was dened as a unit of SOD that could cause a 50% time formula (%)
reduction in the rate of formazan dye formation. 28.372 9,12,15-Octadecatrienoic acid C18H30O2 31.75
Lysozyme activity was measured based on turbidimetric assay 25.738 Hexadecanoic acid CH3(CH2)14 CO2H 12.14
according to methods described by Ellis [53]. A standard suspension 33.957 2,8-Dimethyl-2-(4,8, C27H46O2 9.33
12-Trimethyltridecyl)-6-
(0.2 mg/mL) of Micrococcus lysodeikticus (SigmaeAldrich) was pre-
Chromanol
pared in 0.05 M sodium phosphate buffer (pH 6.2). Lyophilised hen 23.552 1H-Purine-2,6-dione,3, C8H10N4O2 6.19
egg white lysozyme was used as a standard. A new standard curve 7-dihydro-1,3,7-trimethyl-
was prepared for each assay. Standard solutions as well as samples 28.462 9,12,15-Octadecatrienoic acid C18H36O2 4.50
16.085 Methylparaben C8H8O3 4.46
were added to the bacterial suspension and the decrease in absor-
19.477 Propylparaben C10H2O3 4.12
bance at 530 nm was recorded after 1 and 6 min. The results were 28.607 Hexadecanamide C16H33NO 2.70
expressed as mg/mL equivalent of hen egg white lysozyme activity. 44.790 3,7,11,15-Tetramethyl-2- C20H40O 2.33
hexadecen-1-OL
7.521 1,2,3-Propanetriol C3H8O3 2.27
2.6. Statistical analysis 35.033 2,5,8-Trimethyl-2- C28H48O2 1.64
(4,8,12-Trimethyltridecyl)-
Data were analyzed using one-way analysis of variance (ANOVA). 6-chromanol
When overall differences were signicant at less than the 5% level, 32.495 13-Docosenamide C22H43NO 1.50
52.002 Pyridine-3-carboxamide, C13H10F3N3O 1.35
Tukeys test was used to compare the means between individual
oxime, N-(2-triuoromethylphenyl)-
treatments. Statistical analysis was performed using the SAS soft- 27.979 9,12,15-Octadecatrienoic acid, C20H34O2 1.33
ware (SAS Inc. Cary, NC, USA), and the differences between experi- ethyl ester, (Z,Z,Z)-
mental data were determined to be signicant at P value <0.05. 29.882 9-Octadecenamide C18H35NO 1.26
10.961 2-Methoxy-4-Vinylphenol C9H10O2 1.01

3. Results
3.1. Growth performances
The effects of supplementary katuk (S. androgynus) diets on
growth responses are provided in Table 2. During the whole
experimental periods, all groups increase the weight and length
steadily every week. The greatest improvements in weight gain
(WG) was observed in sh received 1.0 g/kg katuk diets (SAA),
maintaining a signicantly higher (P < 0.05) than other groups.
Result on the WGs and SGRs indicated that sh treated with 1.0 and
2.5 g/kg katuk extract (SAB) could enhance relative growth rate
compare with control sh. However, statistical analyses showed
that there were no signicant different in specic growth rate (SGR)
between control and sh received 5.0 g/kg katuk diets (SAC) and
the value of viscerosomatic index (VSI) and hepatosomatic index
(HSI) in all groups.
3.2. Administration of S. androgynus in diets enhanced the non-
specic immune responses
Extraction from dried S. androgynus leaves powder using 70%
ethanol was obtained around 25.6 g/100 g of dried S. androgynus
extract. Immunomodulatory compounds in the extract were
analyzed using Chromatography Mass Spectrometry (GCMS) and
those compounds are presented in Table 3, based on the GCMS
results with database W8N08. L.
This study revealed the presence of 16 immunomodulatory
compounds. Analyses using GCMS identied those active
compounds were mainly fatty acid, chlorophyll, alkaloid and glyc-
erol. The major components detected were linolenic acid (31.75%),
palmitic acid (12.14%), chlorol (9.33%), benzoic acid (8.58%) and
alkaloid (7.2%). Other components were also detected in the plant
extract such as: stearic acid (4.50%), oleic acid amide (3.96%) and
vitamin (1.64%).
To analyze the stimulating ability of S. androgynus toward
E. coioides immune cells, sh leukocytes were induced with various
concentrations of S. androgynus extract (0e10 mg/mL), and a res-
piratory burst was assayed. Superoxide production analysis of
E. coioides leukocytes tends to enhance after incubated with katuk
extract at 1.0e5.0 mg/mL. Based on their ability to enhance innate
immunity, the plant extracts were then used as supplementary feed
in in vivo experiment. The effect of S. androgynus extract on respi-
ratory burst (RB) activities producing superoxide anion (Fig. 1)
showed that SAA signicantly enhance RB on day 7 and 14; while,
SAB reached the highest RB production on day 4. In SAC, groupers
leucocytes exhibited a signicant response on day 2, followed by
steadily decreasing until the end of experiment.
The Superoxide dismutase (SOD) enzyme activity seemed to be
higher in sh fed with S. androgynus mixed diets. Fish received
1.0 g/kg S. androgynus diets (SAA) showed signicantly higher on
day 2 and day 7 compare with control. However, statistical analyses
showed that no signicant difference was observed in all groups
after 14 days of experiment (Fig. 2).
The rate of phagocytic activity of grouper fed with test diets is
shown in Fig. 3. Phagocytic rate (PR) of SAA increased steadily from
day 1 to day 4, followed by decreasing afterward. While, SAB showed

Table 2
Mean (
S.D.) initial and nal length, initial and nal body weight, weight gain (WG), specic growth rate (SGR), viscerosomatic index (VSI) and hepatosomatic index (HSI) of
grouper fed experimental diets.

Treatments Length (cm) Weight (g) WG (%) SGR (%) VSI (%) HSI (%)

Initial Final Initial Final

Control 9.67
0.33a 15.31
0.37c 10.97
1.99a 59.27
4.61b 440
42c 2.41
0.11b 11.19
0.49a 3.18
0.37a
SAA 9.67
0.33a 17.25
0.55a 10.97
1.99a 89.52
6.49a 716
59a 3.00
0.11a 11.25
0.47a 2.85
0.23a
SAB 9.67
0.33a 16.58
0.31ab 10.97
1.99a 75.01
5.29ab 584
48b 2.74
0.10a 11.85
0.60a 3.25
0.32a
SAC 9.67
0.33a 15.53
0.57bc 10.97
1.99a 59.99
5.23b 447
48c 2.42
0.12b 10.99
0.48a 2.85
0.30a

Data in the same column with different letter are signicantly different (p < 0.05) among treatments. Values are means of triplicate groups
S.D.
586 A.P.A. Samad et al. / Fish & Shellsh Immunology 36 (2014) 582e589

Control SAA SAB SAC Control SAA SAB SAC

70.0
2.00 a
a
1.80 a
a 60.0
1.60 a a
ab
a
Superoxide (O.D. 620)

Phagocytic Rate (%)

b
1.40
a 50.0 ab
1.20 b a
ab ab ab a a a
1.00 b b b ab b b a
b 40.0 b a a
b b
0.80 c
a a c
a c a
0.60 c c c
a a a a c a a 30.0 a a a a
b ab b
0.40
0.20
20.0
0.00
0 1 2 4 7 14 30
Days 10.0
0 1 2 4 7 14 30
Days
Fig. 1. Superoxide anion (O
2)
production ratio in the macrophage of grouper fed with
experimental diets. Different superscripts at the end of each bar chart indicate sig-
Fig. 3. Phagocytic rate (%) of groupers leucocytes fed with experimental diets.
nicant different (P < 0.05) among treatments.
Different superscripts at the end of each bar chart indicate signicant different
(P < 0.05) among treatments.

the highest point on day 1; however, signicant enhancement was

found only on day 7. Even though PR activity seemed to be higher in 3.3. Administration of S. androgynus in diets effect against V.
all treated groups compare with control group, still no meaningful alginolyticus challenge test
signicant different was found after 7 days. The number of phago-
cytic index (PI) in leukocytes of E. coioides fed with test diets is shown After experimental infection, the juveniles infected by
in Fig. 4. SAA gained the highest value on day 1. Signicantly dif- V. alginolyticus showed symptoms of disease, such as hemorrhagic
ference between treated groups and control group occurred on day 2 ns and ulcers associated with high mortality. Survival rate (SR) of
to day 4. Wherein, SAB attained a higher PI at day 2 compare with treated groups and control group of grouper challenged with
other groups and showed signicantly different with control and V. alginoliticus are shown in Fig. 5. Survival rate of E. coioides offered
SAC groups on day 30. with 1.0% of S. androgynus extract was higher than those offered
Inclusion at different dosage of the plant extracts can induce with 2.5% and 5.0% of S. androgynus extract. However, no signicant
phagocyte reactive oxygen species (ROS). It was detected by difference was found between the 5.0% with the control group
chemiluminescent reactions method. Supplemented diets with during the challenge test. SR was increased in S. androgynus sup-
S. androgynus showed signicantly enhance the immune response plemented group, particularly when sh received 1.0% and 2.5% of
system. SAB groups signicantly increased in chemiluminescent herb extract. It is indicated that the use of 1.0% and 2.5%
response from day 7e14 and able to maintain this enhancement S. androgynus mixed diets was effective in facing the pathogenic
until the end of experiment with the highest point obtained on day invasion and increase survival rate.
7. Furthermore, sh fed with S. androgynus mixed diets showed a
higher immune response than control sh (Table. 4). However, it 4. Discussion
found that SAC attained the lowest value on day 2 and day 14.
Variance in serum lysozyme activity is as seen on Table 5. There This study was conducted to examine the efcacy of dietary S.
was a signicant difference in serum lysozyme activity on day 1 and androgynus in stimulating the growth and immune system of
2, when sh received 5.0 g/kg katuk diets and 2.5 g/kg katuk diets, grouper (E. coioides) against V. alginolytycus. Although studies on
respectively. Moreover, inclusion of 2.5 g/kg katuk diet showed the S. androgynus in sh are still unknown, it is expected that some
highest value among all treatment groups on day 2. While, in sh information about the application of this materials in poultry and
fed with 1.0 g/kg katuk diets showed gradually increase the serum mammalian will have similar effect in sh. The results of this study
lysozyme activity until day 2, however, it was gradually declining have shown the possible effect of S. androgynus as a growth-
until the nal day of experiment. stimulating and stimulated sh appetence.
In this study, groupers received 1.0% of S. androgynus extract
mixed diets showed increased in the weight gain, however no

Control SAA SAB SAC

Control SAA SAB SAC
11.0
3.0
10.0 a
a
Phagocytic Index ( beads/cell)

9.0 a 2.5 a
a a a a
8.0 ab ab ab a
a a 2.0 a
a ab a a a a a
SOD (U/mg)

a a
7.0 a a a a a ab a ab ab ab ab
a b
a 1.5 b b
6.0 b a a a aa b b
a b
b b
5.0 a a a a
1.0
4.0
0.5
3.0

2.0 0.0
0 1 2 4 7 14 30 0 1 2 4 7 14 30
Days Days

Fig. 2. Superoxide dismutase (SOD) activity (U/mg protein) of groupers liver fed with Fig. 4. Phagocytic index (beads/cell) of groupers leucocytes fed with experimental
experimental diets. Different superscripts at the end of each bar chart indicate sig- diets. Different superscripts at the end of each bar chart indicate signicant different
nicant different (P < 0.05) among treatments. (P < 0.05) among treatments.
 A.P.A. Samad et al. / Fish & Shellsh Immunology 36 (2014) 582e589 587

Table 4
Chemiluminescent response (RLU/s) of groupers macrophages fed with experimental diets.

Treatments Dose g/kg diets Days

0 1 2 4 7 14 30
4
Value  10

Control 0 4.639
0.205a 8.992
0.626b 6.400
0.482c 22.500
0.705ab 21.791
1.295d 36.368
1.772c 41.187
1.342c
SAA 1.0 4.639
0.205a 13.120
0.808a 12.372
0.512a 24.785
1.217a 92.524
6.287c 77.032
4.123b 65.259
2.335a
SAB 2.5 4.639
0.205a 12.701
0.957a 11.290
0.393b 25.4643
3.222a 143.580
8.185a 88.887
5.752a 50.181
4.775b
SAC 5.0 4.639
0.205a 15.528
0.482a 3.476
0.267d 19.015
1.798b 115.208
6.329b 33.038
2.173c 43.553
1.859bc

Data in the same column with different letter are signicantly different (p < 0.05) among treatments. Values are means of triplicate groups
S.D.

Table 5
Lysozyme activity (mg/ml) of groupers serum fed with experimental diets.

Treatments Dose g/kg diets Days

0 1 2 4 7 14 30

Control 0 4.095
0.337a 4.333
1.673b 4.571
0.337b 4.810
0.668a 4.571
0.337a 4.573
0.338b 3.619
0.337a
SAA 1.0 4.095
0.337a 6.952
1.010ab 7.669
0.671ab 7.429
1.011a 6.238
0.681a 5.763
0.658ab 5.048
0.339a
SAB 2.5 4.095
0.337a 5.048
1.014ab 9.095
1.345a 5.286
0.675a 6.952
1.017a 7.905
1.015a 5.762
0.672a
SAC 5.0 4.095
0.337a 7.667
1.347a 6.238
0.598ab 6.952
1.013a 5.534
0.413a 4.810
0.658a 3.619
0.335a

Data in the same column with different letter are signicantly different (p < 0.05) among treatments. Values are means of triplicate groups
S.D.

signicant different in the WG and SGR was observed between
control and SAC. This nding showed that administered
S. androgynus in diets meet the maximum value in grouper species.
Analyses of nal body weight and weight gain showed that appli-
cation of 1.0% S. androgynus extract in total diets meet the optimal
growth of the sh. Similar requirement of S. androgynus extract was
reported in chicken [28,54] and sheep [34], found that low dietary
S. androgynus in diets could affect a better growth and increase milk
production. It is suggested that supplemented 1.0% of S. androgynus
in diets is the optimal dosage of this grouper. This may be due to
individuals acquiring of nutrition and feeding quantity. It has been
demonstrated that appropriate usage of plant extract depends on
species, age, size and any stress factors during cultivation periods
[54]. Therefore experiments on different concentration of
S. androgynus need to be examined in each species for efciency in
increasing protability.
When sh received experimental diets with higher dietary
S. androgynus, it seemed to reduce growth performances. In this
study, sh offered 2.5% and 5.0% of S. androgynus showed lower
growth rate compare with sh received 1% of S. androgynus. It is
assumed that higher dosage of herb extracts has resulted in acti-
vation of enzyme inhibitors due to most plants contain inhibitors to
protect their major components from unintended degradation.
Some studies reported that antinutrients substances contained in
Control SAA
100.0
a a
90.0
80.0 a b
70.0
Survival Rate (%)
plants such as protease inhibitors, amylase and lipase, tannins,
saponins, lectins and antivitamins [55] may cause disturbance in
the gastrointestinal tract [56]. This condition could affect the
digestive processes and feeding efciency. Thus, consumed nutri-
ents cannot be utilized for growth maximization. Even though no
specic experiment was carried out to determine the antinutrients
in S. androgynus leaf extract and the effect of those inhibitors, but
observation found that different percentage of S. androgynus
extract play a big role for the growth and feeding efciency in
experimental sh.
In the present study, dietary administration of S. androgynus was
shown to be effective in defending the pathogenic invasion and
increasing survival rate. Dietary administration of S. androgynus in
poultry animal was reported can improve the balance of microor-
ganisms and decrease the pathogenic microorganisms such as E.
coli and Salmonella sp [57], Staphylococcus aureus and salmonella
typhosa [35]; and increase the benecial microorganisms such as
Bacillus subtilis and Lactobacillus sp. [57]. Moreover, traditional drug
and medical herbs have been proven to be effective when applied in
sh, and some plants have also been documented for their anti-
microbial properties. Therefore, in this study we observed the
enhancement innate immune responses and showed the positive
effects on some immune parameters in grouper against
V. alginolyticus infection to examine its immunostimulatory effect.
We also demonstrated that S. androgynus could signicantly
enhance the non-specic immune responses of E. coioides. The
SAB SAC
extract had screened for its ability to enhance the non-specic
immunity after incubation with the leukocytes of E. coioides in
respiratory burst activity in superoxide production analysis. A se-
ries of concentration level were examined and found that 1.0e
b

5.0 mg/l was adequate to achieved leukocyte stimulation. Further

60.0
b
study, we administered the extract by supplementing it in sh diet
50.0
c in order to provide an optimal dietary level and duration for katuk
40.0
d c
c extract to activate the non-specic immune responses.
30.0
c c Recently, plant materials are currently used in commercial
20.0
aquaculture as growth promoting substances, anti-microbial agents
10.0
and nutrient sources [58]. The application of herbs as a dietary
0.0
1 4 7 supplement in sh diets is acceptable and it can affect sh growth
Days [59]. In the present study, administration of S. androgynus in sh
diets signicantly increased the weight gain (WG) and specic
Fig. 5. Survival rate (%) of grouper challenged with V. alginoliticus. Different super-
scripts at the end of each bar chart indicate signicant different (P < 0.05) among growth rate (SGR) of grouper juveniles. Similar studies on the effect
treatments. of plant extracts for sh growth and survival have been conducted
588 A.P.A. Samad et al. / Fish & Shellsh Immunology 36 (2014) 582e589
in some species, such as: A. aspera in common carp C. carpio [60], A.
sativum in L. rohita [17], Cynodon dactylon in Catla catla (Ham.) [49],
Massa medicate, Crataegi fructus, Artemisia capillaries and Cnidium
ofcinale in Pagrus major [61], and Viscum album and Zingiber of-
cinale in O. mykiss [62]. Comparing to those studies, in this study,
dietary S. androgynus which could signicantly affect the WGs and
SGRs occurred when sh received 1.0 and 2.5 g/kg S. androgynus
diets. Presumably, these concentrations were suitable for this
species. Moreover, observation found that sh offered
S. androgynus diets acted aggressively to catch the feed and
consumed more feed than control sh. It is assumed that the
respective experimental diets effect on the sh appetite. Citarasu
[63] mentioned that the active compounds such as alkaloids, a-
vonoids, pigments, phenolics, terpenoids, steroids and essential oils
in plants are also have characteristics to improve the immune
system, anti stress and appetite stimulation. It is suggested that,
even though plant extracts could increased the growth perfor-
mances, appropriate dietary level is needed. It could also be
affected by different species, sh size, environmental condition and
feeding strategy.
In this experiment, a signicant increase of phagocytic rate (PR)
was measured in all dietary S. androgynus groups, whereas at 2.5%
dosage of S. androgynus (SAB) showed the highest point on day 1
and day 7, and SAA attained the highest PR on day 4. Increasing
dosage of S. androgynus seemed to reduce the ability in PR of
E. coioides. The rates of phagocytic activity in sh treated with
S. androgynus were signicantly higher than control until 7 days.
This similar nding was also reported by Dugenci [62] in rainbow
trout after feeding with Z. ofcinale; Nile tilapia when offered
Astragalus extract [11]; and E. coioides when administered using
sodium alginate [52].
Phagocytosis and the production of oxygen free radicals via the
respiratory burst are important events in bactericidal pathways in
sh, but mechanism are not well established [64]. Oxygen-
dependent killing mechanisms as mediated by reactive oxygen
species (ROS) can be detected by the chemiluminescent and the
Nitroblue tetrazolium (NBT) test [59]. In this study, we obtain two
main methods to measure reactive oxygen activity; there were the
superoxide production analysis by NBT test and reactive oxygen
species production by chemiluminescent. Intracellular respiratory
burst activities in leukocytes of E. coioides fed with S. androgynus
was signicantly enhanced. The low dosage of S. androgynus at
1.0 g/kg and 2.5 g/kg diet can stimulate the superoxide production,
and this activity was maintained on day 14 and day 4, respectively.
It was also reported that administration of sodium alginate at 1e
2 g/kg in E. coioides [65], and dietary administration of Astragalus
membranaceus and L. japonica extract 0.1% in O. niloticus [11]
showed signicantly enhance the respiratory burst activity.
The respiratory burst activity was also studied by a chemilumi-
nescent method. Respiratory burst induced by phagocytosis of
zymosan particles was measured in relative luminescence unit (RLU)
per second. Verlhac et al. [66] demonstrated a stimulatory effect of
vitamin C mixed with glucan in enhancement of chemiluminescent
response of rainbow trout at 2 and 4 weeks after feeding. Enhance-
ment of chemiluminescent also found in juvenile oil ounder (P. oli-
vaceus) after dietary intake 1% of Paecilomyces japonica [67]. In the
present study, respiratory burst activity was signicantly enhancing
in all treatment groups with different time of induction and different
RLU/s value. Among the group received S. androgynus extract, sh
received 1.0 g/kg extract was signicantly stimulate the chemilumi-
nescent response. Signicant increase was observed from day 7e14,
whereas, the highest point obtained on day 7.
A number of non-specic humoral factor contribute to the sh
natural resistance to infections. They act in a variety of different
ways to inhibit the growth and spread of pathogenic organisms
[68]. It was observed that the lysozyme activity was seen in Pseu-
dosciaena crocea when treated with Chinese herbal extracts [69], C.
carpio [70], and O. niloticus [11]. As shown in this result,
S. androgynus signicantly increase the lysozyme activity. Inclusion
of 2.5 g/kg S. androgynus diet showed the highest value among all
treatment groups on day 2.
Superoxide dismutase (SOD) is metalloenzymes that play major
roles in protection of cells against oxidative damage [71]. A signif-
icant different of SOD activity were observed in juvenile of
E. coioides [65] and Epinephelu fuscoguttatus [72] using dietary so-
dium alginate. The result showed enhancement of SOD activity in
both species. In the present study, dietary administration of
S. androgynus at 1.0 g/kg showed a signicant enhancement in SOD
activity on day 2 and day 7 compare with control. It can be corre-
lated that SOD tends to be higher after that inducement of reactive
oxygen species go to resting phase.
In conclusion, we have demonstrated that incorporating
S. androgynus in sh diets have the ability to enhance the non-
specic immune responses of E. coioides. Supplementing this herb
in sh diet has exhibited growth performances and diseases
resistance against V. alginolyticus infection. Moreover considering
its low cost and immunostimulative effect, S. androgynus could be
recommended to be used for farmed sh to decrease mortalities
caused by pathogenic microorganisms.
References
[1] Liao IC, Su HM, Chang EY. Techniques in nsh larviculture in Taiwan.
Aquaculture 2001;200:1e31.
[2] Rimmer MA, McBride, Williams KC. Advances in grouper aquaculture. Can-
berra: Australian Center for International Agricultural Research. ACIAR
Monograph; 2004.
[3] Sadovy Y. Regional survey of fry/ngerling supply and current practices for
grouper mariculture: evaluating current status and long-term prospects for
grouper mariculture in Southeast Asia. Final report to the Collaboration APEC
grouper research and development network (FWG 01/99). Hongkong: Uni-
versity of Hongkong; 2000.
[4] Sorgeloos P, Dehasque M, Dhert P, Lavens P. Review of some aspects of marine
sh larviculture. International Council for the exploration of the Sea. Marine
Sci Symp 1995;201:138e42.
[5] Kirubakaran CJW, Catherine PA, Michael RD. Enhancement of non-specic
immune responses and diseases resistance on oral administration of Nyc-
tanthes arbortristis seed extract in Oreochromis mossambicus (Peter). Aquac Res
2010;41:1630e9.
[6] Kajita Y, Sakai M, Atsuta S, Kobayashi M. The immunomodulatory effects of
levamisole on rainbow trout, Oncorhynchus mykiss. Fish Pathol 1990;25:93e8.
[7] Rottmann RW, Francis-Floyed R, Durborow R. The role of stress in sh disease.
Southern Regional Aquaculture Center; 1992. p. 474.
[8] Farag RS, Dawz ZY, Hewedi FM, El-Barotyl GS. Antimicrobial activity of some
Egyptian Spice essential oils. J Food Prot 1989;52:665e7.
[9] Citarasu T, Babu MM, Sekar RJR, Marian PM. Developing Artemia enriched
herbal diet for producing quality larvae in Penaeus monodon, Fabricius. Asian
Fish Sci 2002;15:21e32.
[10] Sagdic O, Ozcan M. Antibacterial activity of Turkish spice hydrosols. J Food
Cont 2003;14:141e3.
[11] Ardo L, Yin G, Xu P, Varadi L, Szigeti G, Jeney Z, et al. Chinese herbs Astragalus
membranaceus and Lonicera japonica and boron enhance the non-specic
immune response of Nile tilapia Oreochromis niloticus and resistance against
Aeromonas hydrophila. Aquaculture 2008;275:26e33.
[12] Leong JC, Fryer JL. Viral vaccines for aquaculture. Annu Rev Fish Dis 1993;3:
225e40.
[13] Murray AL, Pascho RJ, Alcorn SW, Fairgriev WT, Shearer KD, Roley DD. Effects
of various feed supplements containing sh protein hydrolysate or sh pro-
cessing by-products on the innate immune functions of juvenile coho salmon
(Oncorhynchus kisutch). Aquaculture 2003;220:643e53.
[14] Gopalakannan A, Arul V. Immunomodulatory effects of dietary intake of chitin,
chitosan and levamisole on the immune system of Cyprinus carpio and control of
Aeromonas hydrophila infection in ponds. Aquaculture 2006;255:179e87.
[15] Harikrishnan R, Kim JS, Kim MC, Balasundaram C, Heo MS. Lactuca indica
extract as feed additive enhances immunological parameters and disease
resistance in Epinephelus bruneus to Streptococcus iniae. Aquaculture
2011;318:43e7.
[16] Yin G, Ardo L, Thompson KD, Adams A, Jeney Z, Jeney G. Chinese herbs
(Astragalus radix and Ganoderma lucidum) enhance immune response of carp,
Cyprinus carpio, and protection against Aeromonas hydrophyla. Fish Shellsh
Immunol 2009;26:140e5.
 A.P.A. Samad et al. / Fish & Shellsh Immunology 36 (2014) 582e589
[17] Sahu S, Das BK, Mishra BK, Pradhan J, Sarangi N. Effect of Allium sativum on the
immunity and survival of Labeo rohita infected with Aeromonas hydrophila.
J Appl Ichthyol 2007;23:80e6.
[18] Dorucu M, Ozesen SC, Ispir U, Altinterim B, Celayir Y. The effect of Black Cumin
seed, Nigella sativa, on the immune response of Rainbow trout, Oncorhynchus
mykiss. J Med Aqua 2009;2:27e33.
[19] Harikrishnan R, Kim JS, Kim MC, Balasundaram C, Heo MS. Prunella vulgaris
enhances the non-specic immune response and disease resistance of
Paralichthys olivaceus against Uronema marinum. Aquaculture 2011;318:61e
6.
[20] Yin G, Ardo L, Jeney Z, Xu P, Jeney G. Chinese herbs (Lonicera japonica and
Ganoderma lucidum) enhances non-specic immune response of tilapia,
Oreochromis niloticus, and protection against Aeromonas hydrophila, pp. 269e
282. In: Bondad-Reantaso MG, Mohan CV, Crumlish M, Subasinghe RP, editors.
Diseases in Asian aquaculture VI. Manila, Philippines: Asian Fisheries Society;
2008. Fish Health Section.
[21] Rao YV, Chakrabarti R. Stimulation of immunity in Indian major carp Catla
catla with herbal feed ingredients. Fish Shellsh Immunol 2005;18:327e
34.
[22] Taukhid, Suharni I, Supriyadi H. Efektivitas ekstrak daun sambiloto (Androg-
raphis paniculata) bagi pengendalian penyakit Koi Herpes Virus (KHV) pada
ikan mas (Cyprinus carpio). Jurnal Riset Akuakultur 2007;2:411e8.
[23] Giyarti D. Efektivitas ekstrak daun jambu biji (Psidium guajava L), sambiloto
(Andrographis paniculata) dan sirih (Piper betle L) terhadap infeksi bakteri
Aeromonas hydrophyla pada ikan patin (Pangasius hypophthalmus). In: Pro-
gram Studi Budidaya Perairan, Fakultas Perikanan dan Ilmu Kelautan. Bogor:
IPB; 2000. Thesis.
[24] Supriyadi H, Maharani F, Priono B, Kusrini E, Sugiani D. Penggunaan beberapa
materi bahan alami bagi upaya penanggulangan penyakit ikan gurami
(Osphronemus gouramy). In: Seminar Nasional Tahunan III Hasil Penelitian
Perikanan dan Kelautan. Yogyakarta: Universitas Gajah Mada; 2006.
[25] Bafna AR, Mishra SH. Immunomodulatory activity of methanol extract of roots
of Cissampelos pareira Linn. Ars Pharmaceutica 2005;46:253e62.
[26 Astuti Y, Wahjoedi B, Winarno MW. Efek diuretic infuse akar katuk terhadap
tikus putih. Warta Tumbuhan Obat 1997;3(3):42e3.
[27] Benjapak N, Swatsitang P, Tanpanich S. Determination of antioxidant capacity
and nutritive values of Pak-Wanban (Sauropus androgynus L. Merr). Khon
Kaen Univ Sci J 2008;36(4):279e89.
[28] Santoso U, Suteki T, Fenita Y. Effects of supplementation of alkaloid and non
alkaloid from Sauropus androgynus leaves on egg production and lipid prol in
layer chicken. Animal Prod 2009;12(3):184e9.
[29] Suprayogi A, Meulen UT. The inuence of sauropus androgynus (L.) merr.
leaves on the body weight changes and calcium - phosphorus absorption in
lactating sheep. Gakuryoku 2006;XII(3):50e5.
[30] Santoso U, Sartini. Reduction of fat accumulation in broiler chickens by Sau-
ropus androgynus (Katuk) leaf meal supplementation. Asian-Aust J Anim Sci
2001;14:346e50.
[31] Subekti S, Piliang WG, Manalu W, Murdiati TB. Utilization of Katuk (Sauropus
androgynus L Merr) meal and extract as ration substitution to produce low
chollesterol Japanese Quail product. JITV 2006;11(4):254e9.
[32] Wiranda G, Piliang WG, Astuti DA, Widya H. Pengkayaan produk puyuh
melalui pemanfaatan pakan lokal yang mengandung antioksidan dan mineral
sebagai alternatif penyediaan protein hewani bergizi tinggi. In: Prosiding
Seminar Hasil-hasil Penelitian IPB 2009. pp. 27e39.
[33] Santoso U, Setianto J, Suteky T. Effect of Sauropus androgynus (Katuk) extract
on egg production and lipid metabolism in layers. Asian-Aust J Anim Sci
2005;18(3):364e436.
[34] Suprayogi A. Meningkatkan produksi susu kambing melalui daun katuk
(Sauropus androgynus (L. Merr). Agrotek 1993;1(2):61e2.
[35] Darise M, Sulaeman. Ekstraksi komponen kimia daun katuk asal Sulawesi
Selatan berbagai metode serta penelitian daya hambat terhadap bakteri uji.
Warta Tumbuhan Obat 1997;3(3):37e8.
[36] Nahak G, Sahu RK. Free radical scavenging activity of multi-vitamin plant
(Sauropus androgynus L. Merr). Researcher 2010;2(11):6e14.
[37] Agustal A, Harapini M. Chairul. Analisis kandungan kimia ekstrak daun katuk
(Sauropus androgynus L Merr) dengan GCMS. Warta Tumbuhan Obat
1997;3(3):31e3.
[38] Malik A. Tinjauan tokimia, indikasi penggunaan dan bioaktivitas daun katuk
dan buah trengguli. Warta Tumbuhan Obat 1997;3(3):39e40.
[39] Liu PC, Lin JY, Hsiao TP, Lee KK. Isolation and characterization of pathogenic
Vibrio alginolyticus from diseased Cobia Rachycentron canadum. J Basic
Microbiol 2004;44(1):23e8.
[40] AOAC. Ofcial methods of analysis. 15th ed. Virginia, USA: Association of
Ofcial Analytical Chemists, Inc.; 1990.
[41] Hepher B. Nutrition of pond shes. UK: Cambridge University Press; 1988.
[42] Zhao Y, Zhang W, Xu W, Mai K, Zhang Y, Liufu Z. Effects of potential probiotic
Bacillus subtilis T13 on growth, immunity and disease resistance against Vibrio
splendidus infection in juvenile sea cucumber Apostichhopus japonicas. Fish
Shellsh Immunol 2012;32(5):750e5. http://dx.doi.org/10.1016/j.fsi.2012.01.027.
[43] Fulton TW. The sovereignty of the sea: an historical account of the claims of
England to the dominion of the British seas and of the evolution of the ter-
ritorial waters, with special reference to the rights of shing and the naval
salute. Edinburgh: William Blackwood and Sons; 1911.
589
[44] Khan MHK, Ang KJ, Ambak MA, Saad CR. Optimum dietary protein require-
ment of Malaysian freshwater catsh, Mystus nemurus, (Cuvier). Aquaculture
1994;112:227e35.
[45] Hopskin KD. Reporting sh growth: a review of the basics. J World Aquac Soc
1992;23:173e9.
[46] Ghanawi J, Roy L, Davis DA, Saoud IP. Effects of dietary lipid levels on growth
performance of marbled spinefoot rabbitsh Siganus rivulatus. Aquaculture
2011;310:395e400.
[47] Chopra RN, Nair SL, Chopra JC. Glossary of Indian medicinal plants. New Delhi:
Council of Scientic and Industrial Research Publication; 1992.
[48] Kaleeswaran B, Ilavenil S, Ravikumar S. Dietary supplementation with Cyn-
odon dactylon (L.) enhances innate immunity and diseases resistance of Indian
major carp, Catla catla (Ham.). Fish Shellsh Immunol 2011;31:953e62.
[49] Kuan YC, Fuu S, Lee GC, Tsai MW, Hung CL, Nan FH. Administration of re-
combinant Reishi immunomodulatory protein (rLZ-8) diet enhances innate
immune responses and elicits protection against nervous necrosis virus in
grouper Epinephelus coioides. Fish Shellsh Immunol 2012;32:986e93.
[50] Fujiki K, Yano T. Effect of sodium alginate on the non-specic defense system
of Common carp (Cyprinus carpio L). Fish Shellsh Immunol 1997;7:417e27.
[51] Matsuyama H, Yano T, Yamakawa R, Nakao M. Opsonic effect of the third
complement component (C3) of Carp (Cyprinus carpio) on phagocytosis by
neutrophils. Fish Shellsh immunol 1992;2:69e78.
[52] Cheng AC, TU CW, Chen YY, Nan FH, Chen JC. The immunostimulatory effect of
sodium alginate and iota-carrageenan on orange-spotted grouper Epi-
nephelus coioides and its resistance against Vibrio alginolyticus. Fish Shellsh
Immunol 2007;22:197e205.
[53] Ellis AE. Lysozome assay. In: Stolen JS, Anderson BS, Robertson BS, editors.
Techniques in sh immunology. Fair Heaven, NJ: SOS Publication; 1990.
[54] Bidura IGNG, Candrawati DPMA, Sumardani NLG. Pengaruh daun katuk
(Sourupus androgynus) dan daun bawang putih (Allium Sativun) dalam ransum
terhadap penampilan ayam broiler. Majalah Ilmiah Peternakan 2007;10:1.
[55] Francis G, Makkar HPS, Bekker K. Antinutitional factors in plant derived
alternate sh ingredients and their effects in sh. Aquaculture 2001;199:
197e227.
[56] National Research Council (NRC). Nutrient requirements of sh and shrimp.
Washington DC: The National Academies Press; 2011.
[57] Santoso U, Suharyanto. Handayani E. Effects of Sauropus androgynus (Katuk)
leaf extract on growth, fat accumulation and fecal microorganisms in broiler
chickens. JITV 2001;6:220e6.
[58] Galina J, Yin G, Ard L, Jeney Z. The use of immunostimulating herbs in sh. An
overview of research. Fish Physiol Biochem 2009;35:669e76.
[59] Sakai M. Current research status of sh immunostimulants. Aquaculture
1999;172:63e92.
[60] Chakrabarti R, Srivasyava PK, Kundu K, Khare RS, Banerjee S. Evaluation of
immunostimulatory and growth promoting effect of seed fractions of Achyr-
anthes aspera in common carp Cyprinus carpio and identication of active
constituents. Fish Shellsh Immunol 2012;32(5):839e43. http://dx.doi.org/
10.1016/j.fsi.2012.02.006.
[61] Ji SC, Takaoka O, Jeong GS, Lee SW, Ishimaru K, Seoka M, et al. Dietary me-
dicinal herbs improve growth and some non-specic immunity of red sea
bream Pagrus major. Fisheries Science 2007;73:63e9.
[62] Dugenci SK, Arda N, Candan A. Some medicinal plants as immunostimulant
for sh. J Ethnopharmacol 2003;88:99e106.
[63] Citarasu T. Herbal biomedicine: a new opportunity for aquaculture industry.
Aquac Inter 2010;18:403e14.
[64] Sharp GJ, Secombes CJ. The role of reactive oxygen species in the killing of the
bacterial pathogen, Aeromonas salmonicida by rainbow trout macrophages.
Fish Shellsh Immunol 1993;3:119e29.
[65] Yeh SP, Chang CA, Chang CY, Liu CH, Cheng W. Dietary sodium alginate
administration affects ngerling growth and resistance to Streptococcus sp
and iridovirus, and juvenile non-specic immune responses of the orange-
spotted grouper, Epinephelus coioides. Fish Shellsh Immunol 2008;25:19e
27.
[66] Verlhac V, Gabaudan J, Obach A, Schuep W, Hole R. Inuence of dietary glucan
and vitamin C on non-specic immune responses of rainbow trout (Onco-
rhynchus mykiss). Aquaculture 1996;143:123e33.
[67] Lee CH, Paek NS, Kim DS, Kim K. Effects of a Paecilomyces japonica supple-
mented diet on the chemiluminescent response of phagocytes and growth in
juvenile olive ounder (Paralichthys olivaceus). Aquaculture 2002;208:51e7.
[68] Manning MJ. Immune defense system. In: Black KD, Pickering AD, editors.
Biology of farmed sh. England: Shefeld Academic Press; 1998. pp. 180e211.
[69] Jian J, Wu Z. Effect of traditional Chinese medicine on non-specic immunity
and diseases resistance of large yellow croaker, Pseudosciaena crocea
(Richardson). Aquaculture 2003;218:1e9.
[70] Jian J, Wu Z. Inuences of traditional Chinese medicine on non-specic immu-
nity of Jian carp (Cyprinus carpio var Jian). Fish Shellsh Immunol 2004;16:1e9.
[71] Metaxa E, Deviller G, Pagand P, Alliaume C, Casellas C, Blancheton JP. High rate
algal pond treatment for water reuse in a marine sh recirculation: water
purication and sh health. Aquaculture 2006;252:92e101.
[72] Chiu ST, Tsai RT, Hsu JP, Liu CH, Cheng W. Dietary sodium alginate
administration to enhance the non-specic immune responses and disease
resistance of the juvenile grouper Epinephelus fuscoguttatus. Aquaculture
2008;277:66e72.