Aquaculture 513 (2019) 734388

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Aquaculture
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The effects of Thai ginseng, Boesenbergia rotunda powder on mucosal and T

serum immunity, disease resistance, and growth performance of Nile tilapia
(Oreochromis niloticus) fingerlings
⁎
Hien Van Doana,b, Seyed Hossein Hoseinifarc, , Chanagun Chitmanatd, Sanchai Jaturasithaa,b,
Marina Paoluccie, Ghasem Ashourif, Mahmoud A.O. Dawoodg, Maria Ángeles Estebanh
a
Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand
b
Science and Technology Research Institute, Chiang Mai University, 239 Huay Keaw Rd., Suthep, Muang, Chiang Mai 50200, Thailand
c
Department of Fisheries, Faculty of Fisheries and Environmental Sciences, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
d
Faculty of Fisheries Technology and Aquatic Resources, Maejo University, Chiang Mai 50290, Thailand
e
Department of Sciences and Technologies, University of Sannio, Benevento 82100, Italy
f
Department of Life and Environmental Sciences, Polytechnic University of Marche, via Brecce Bianche, 60100 Ancona, Italy
g
Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, 33516 Kafrelsheikh, Egypt
h
Fish Innate Immune System Group, Department of Cell Biology & Histology, Faculty of Biology, Regional Campus of International Excellence “Campus Mare Nostrum”,
University of Murcia, Spain

A R T I C LE I N FO A B S T R A C T

Keywords: The effects of Thai ginseng (TG), Boesenbergia rotunda, powder on some immune parameters in serum and skin
Boesenbergia rotunda mucus, resistance against pathogenic bacteria, Streptococcus agalactiae as well as growth performance in Nile
Nile tilapia tilapia, Oreochromis niloticus were studied. Experimental diets were prepared to contain 0 (D1), 5 (D2), 10 (D3),
Growth performance 20 (D4), or 40 (D5) g TG kg−1 diet. Fish (17.29 ± 0.11 g) were stocked in fifteen tanks (150 L; 20 specimen per
Mucosal immunity
tank) in triplicates for 8 weeks. Then, fish were challenged with pathogenic bacteria S. agalactiae. The results
Humoral immunity
indicated that feeding with TG significantly enhanced lysozyme and peroxidase activities in tilapia skin mucus
Disease resistance
Streptococcus agalactiae (P < .05), as well as serum lysozyme (SL), serum peroxidase (SP), alternative complement (ACH50), phago-
cytosis index (PI), and respiratory burst activities (RB). These enhancements were more evidenced in fish fed
diets containing 10 g TG kg−1 diet (P < .05). Similar increments were noticed in the immune parameters of fish
with D3 treatment when compared with other treatment group (P < .05). Also, feeding on TG-supplemented
diets were resulted in significantly elevated disease resistance in Nile tilapia against pathogenic bacteria, S.
agalactiae, and highest survival of post-challenged fish was noticed in D3 treatment. Evaluation of growth
performance revealed that TG supplementation to Nile tilapia significantly improved their final weight, weight
gain, specific growth rate, feed intake, and feed conversion ratio (P < .05) with optimum TG level of 10 g kg−1
diet. However, no significant differences in FI and survival rate (SR) were observed in fish fed TG and the control
after 8 weeks post-feeding. These results demonstrated that dietary TG could be used as a beneficial feed additive
aptimally at 10 g kg−1 to improve performance and immune response of Nile tilapia.

1. Introduction
According to FAO statistics, the production rate of Nile tilapia
(Oreochromis niloticus) substantially increased over the past decades and
now this species is among the most cultured fish species around the
world (FAO, 2016; Wang and Lu, 2016). In Thailand, tilapia is among
the main cultured fish species with a production of approximately
337,500 metric tonnes (Global-Aquaculture, 2016). The culture of ti-
lapia under intensive and super intensive systems can pose stressful
condition and disease. Bacterial infection with Streptococcus spp. stated
as one of the main causes of fish mortality in cage culture (Chitmanat
et al., 2016; Mishra et al., 2018). To resolve the issue of disease as well
as decreasing the risk of disease outbreak, farmers used to administer
chemicals and antibiotics in tilapia farming (Tiengtam et al., 2015).
However, it's now generally accepted that overuse of antibiotics will
have negative effects on environment such as emergence of resistant
bacteria as well as environmental issues (Heuer et al., 2009; Sapkota
et al., 2008; Wu et al., 2013; Zhang et al., 2014). Therefore,

⁎
Corresponding author.
E-mail address: hoseinifar@gau.ac.ir (S.H. Hoseinifar).

https://doi.org/10.1016/j.aquaculture.2019.734388
Received 24 March 2019; Received in revised form 5 August 2019; Accepted 5 August 2019
Available online 06 August 2019
0044-8486/ © 2019 Elsevier B.V. All rights reserved.
H. Van Doan, et al. Aquaculture 513 (2019) 734388

development of alternative approaches which can decrease antibiotics Table 1

administration and improve feed utilization is very important. Formulation and proximate composition (on dry matter basis) of the tested diets
The growing demand of novel food products specifically designed to containing different levels of Thai ginseng (TG), B. rotunda.
stimulate well-being and slowing down aging, has attracted global at- Ingredients Diets (g kg−1)
tention on natural alternatives, which are perceived as more respectful
of health and the environment (Henchion et al., 2017). In this context, Diet 1 Diet 2 Diet 3 Diet 4 Diet 5
it would be advantageous to count on a natural food resources of high
Fish meal (60.1%)1 270 270 270 270 270
quality, such as fish products, farmed with natural products and devoid Corn meal 200 200 200 200 200
of any chemical residue. The employment of natural plant extracts or Soybean meal 270 270 270 270 270
phytochemicals has been shown to be effective without side effects on (46%)2
Wheat flour 60 60 60 60 60
fish or surrounding environment (Immanuel et al., 2004). Thus, phy-
Rice bran 150 150 150 150 140
tochemicals have been suggested as substitute for chemical adminis- TG3 0 5 10 20 40
tration in aquaculture (Sivaram et al., 2004). Previous studies revealed Cellulose 30 25 20 10 0
that either different parts (leaf, root or seed) of medicinal plants can be Soybean oil 5 5 5 5 5
used as beneficial feed additive in aquaculture (Abdel-Tawwab, 2016; Premix4 10 10 10 10 10
Vitamin C5 5 5 5 5 5
Awad and Awaad, 2017; Dawood et al., 2018; Van Hai, 2015).
Total 1000 1000 1000 1000 1000
Cultivated ginseng species are widely distributed around the world, Proximate composition of the experimental diets (g kg−1 dry matter basis)
including Korean, South China, and USA (Baeg and So, 2013). Also, Crude protein 322.06 322.09 322.11 322.16 321.44
Boesenbergia rotunda (fingerroot) known as “Thai ginseng (TG)” has Crude lipid 74.75 74.77 74.79 74.82 73.55
Fiber 52.48 52.53 52.58 52.68 51.56
been considered as a health-promoting medicinal plant in Southeast
Ash 106.68 105.73 104.79 102.90 99.75
Asia (Ongwisespaiboon and Jiraungkoorskul, 2017). It has many re- Dry matter 926.80 926.40 925.78 923.20 922.50
medial and therapeutic benefits “anti-allergic, antibacterial, anticancer, GE (cal/g)6 4105 4091 4078 4050 3991
anti-inflammatory, antioxidant, and antiulcer activities”. Its mode of
1
action is depending on the phytochemical components such as alka- Fish meal and 2Soybean meal were bought from R.P.M. Farm and Feed Co.,
loids, essential oils, flavonoids and phenolics (Ongwisespaiboon and Ltd., Company.
3
Jiraungkoorskul, 2017). Previous studies on Nile tilapia, olive flounder, TG: Thai ginseng, Boesenbergia rotunda.
4
Vitamin and trace mineral mix supplemented as follows (IU kg−1 or g kg−1
and white shrimp revealed growth promontory effects of ginseng
diet): retinyl acetate 1,085,000 IU; cholecalciferol 217,000 IU; D, L-a-toco-
powder/extract as well as positive effects on immune responses (Abdel-
pherol acetate 0.5 g; thiamin nitrate 0.5 g; pyridoxine hydrochloride 0.5 g;
Tawwab, 2012; Abdel-Tawwab, 2015; El-Sayed et al., 2014; Liu et al., niacin 3 g; folic 0.05 g; cyanocobalamin 10 g; Ca pantothenate 1 g kg−1; inositol
2011; Won et al., 2008). Thus, the present trial was performed to de- 0.5 g; zinc 1 g; copper 0.25 g; manganese 1.32 g; iodine 0.05 g; sodium 7.85 g.
termine the possible effects of TG on immune parameters in skin mucus 5
Vitamin C 98% 8 g.
and serum, disease resistance, and growth performance of Nile tilapia. 6
GE = gross energy.

2. Materials and methods (5 ± 0.5 mg L−1) were monitored two times a day using
Multiparameter (HI 98196, Hanna, Romania) and maintained at op-
2.1. Preparation of Thai ginseng (TG) timum levels.

Thai ginseng (TG; B. rotunda) tubers were purchased from a local

market in Chiang Mai, Thailand. Tubers were oven dried at 60 °C for
48 h and ground into fine powder (0.2-mm). The fine powder was
stored in refrigerator until preparation of experimental diets.
2.2. Preparation of experimental diets
The non-supplemented (i.e. basal) diet was prepared based on work
of Tiengtam et al. (2015), with minor modifications, and proved to be
suitable for Nile tilapia (O. niloticus). To prepare experimental diets,
various levels of TG, 0.0 (Diet1), 5 (Diet 2), 10 (Diet 3), 20 (Diet 4), or
40 (Diet 5) g kg−1 diet was supplemented. The proximate composition
of diets as well as ingredients are presented in Table 1. The pellets were
manufactured using a floating pellet machine. The prepared diets were
stored sealed polyethylene bags at 4 °C for further use.
2.3. Fish maintenance and experimental design
Nile tilapia fingerlings (17.29 ± 0.11 g) were purchased (Pathana
Farm, Chiang Mai), transported to Department of Animal and Aquatic
Sciences, Faculty of Agriculture, Chiang Mai University, and distributed
into 5 × 5 × 2 m cages and fed a commercial diet (CP9950 – protein
30%, lipid 3%, moisture 12%, and fiber 8%). Then, fifteen 150-L tanks
were acquired (5 treatment in triplicates) and stocked with 20 fish per
tank. Fish were hand-fed ad libitum twice daily up to appearent stati-
tion for eight weeks. Fish were cultured under static water system
equipped with constant aeration and at leat 50% of water in each tank
was changed daily. The water quality parameters including tempera-
ture (27 ± 1 °C), pH (7.8 ± 0.5) and dissolved oxygen
2.4. Growth performance
In the middle and at the end of the experiment, all fish per each tank
were collected, counted, and group-weighed. Parameters of growth
performance and feed utilization were calcaulated based on the fol-
lowing formulae:
Weight gain (g) = W2 − W1;
Specific growth rate (SGR; %g/day) = 100 [Ln W2 (g) − Ln W1
(g)]/T; where W2 is final weight (g), W1 is initial weight (g), and T is the
experimental period (day);
Feed intake (g feed/fish) = the summation of a diet offered to fish
throughout the experiment/fish number;
Feed conversion ratio (FCR) = feed intake/weight gain;
Fish survival (%) = 100 (fish number at final/fish number at in-
itial).
2.5. Immune response
2.5.1. Serum and mucus collection
Blood samples were collected at caudal vain of four fish per tank
using the 1-mL syringe. The individual collected blood sample was
immediately transferred into 1.5 Eppendorf tube, and allowed to
clotted at room temperature for 1 h, then at 4 °C for hours. After in-
cubation the serum from each fish was collected and kept at −80 °C
until further use. The serum from individual fish was used to analyse
serum immue parameters.
Leucocytes were isolated from peripheral blood using the protocols
suggested by Chung and Secombes (1988) with slight modification as

2
H. Van Doan, et al.
mentioned by Van Doan et al. (2016). Briefly, 1 mL of blood sample
from each fish was taken via caudal vain and immediately mixed with
2 mL of RPMI 1640 (Gibthai) in a 15 mL Eppendrof tube. The mixture
was carefully tranfferred on the top of 3 mL of Histopaque (Sigma, St.
Louis, MO, USA) in a 15-mL tube. The tub was then centrifuged at 400g,
for 30 min at room temperature. After the centrifugation, a white buffy
coat of leucocytes present on the top of the Histopaque was collected,
aspirated using a Pasteur pipette, and transferred into a clean 15-ml
tube. The obtained leucocytes washed three times with phosphate
buffer solution (PBS) for removing residual Histopaque, and the re-
quired cell number was prepared for the phagocytosis and respiratory
burst assays.
The skin mucus was sampled from four fish and pooled (i.e. 12 fish
per treatment) as suggested by Hoseinifar et al. (2019a, 2019b). Briefly,
fish were anaesthetized using clove oil at concentration of 5 mL litre−1.
They were then placed into polyethylene bag containing 10 ml of
50 mM NaCl (Merck, Germany), and gently rubbed inside the plastic in
a downward motion for approximately 1 min. Th mucus was im-
mediately poured into 15 mL sterile centrifuge tubes, and centrifuged
1.500 ×g for 10 min at 4 °C. Then 1 mL of obtained mucus was kept in
1.5 mL Eppendroft tube at −80 °C until further use.
2.5.2. Lysozyme activities in serum and mucus
Lysozyme activity of mucus and serum was determined following
the protocol of Parry et al. (1965). Briefly, 25 μL of mucus and serum
loaded into 96-well plate in triplicate, and then 175 μL of Micrococcus
lysodeikticus (Sigma Aldrich - USA) suspension (0.3 mg mL−1 in 0.1 M
citrate phosphate buffer, pH 5.8) was added to each well. After the
rapid mixing, turbidity changing was detected at every 30 s for 5 min at
540 nm and at 25 °C using a micro-plate reader. The equivalent unit of
activity of the sample (compared with the standard) was determined
and expressed as μg mL−1 serum.
2.5.3. Peroxidase activities in serum and mucus
The peroxidase activity determined following the technique sug-
gested by Quade and Roth (1997) and Cordero et al. (2016). Briefly,
5 μL of serum or skin mucus was loaded into 96-well flat-bottom plates
in triplicate. 45 μL of Hank's balanced salt solution (HBSS) without
Ca+2 or Mg+2 was added. After that, 100 μL of solution containing of
40 ml of distilled water, 10 μL of H2O2 (30% - Sigma Aldrich), 1 pill of
3,3′,5,5′-tetramethylbenzidine (TMB; Sigma Aldrich) was loaded into
each well. Later, 50 μL of 2 M H2SO4 was added to stop the colour-
change reaction, and the optical density was read at 450 nm in a plate
reader. Standard samples without serum or skin mucus were used as
blanks. One unit was defined as the amount producing an absorbance
change of 1 and the activity was expressed as units (U) mg−1 serum or
mucus proteins.
2.5.4. The phagocytic activity
The method suggested by Yoshida and Kitao (1991) was adapted for
determination of phagocytosis activity in samples. Briefly, 200 μL leu-
kocyte suspensions containing of 2 × 106 cells mL−1 was placed on
cover slip and incubated for 2 h. After incubation, RPMI 1640 was used
to washed the non-attached cells, and then 200 μL of fluorescence latex
beads (Sigma Aldrich - USA) solution containing of 2 × 107 of beads
mL−1 was dropped on each cover slip, and incubated for 1.5 h at room
temperature (RT). Afterward, the non-phagocyte beads were washed
with RPMI 1640, and the cover slips were fixed with 100% methanol
and stained with Diff-Quick staining dye (Sigma Aldrich - USA) for 10 s.
Excessive stain was washed with PBS (pH 7.4), and the number of
phagocyte cells per 300 adhered cells was counted microscopically. The
phagocytic index (PI) was determined as follows: PI = Average number
of beads per cell / the number of phagocytizing cells.
2.5.5. The respiratory burst activity
The technique suggested by Secombes (1990) was adopted to
3
Aquaculture 513 (2019) 734388
measure the respiratory burst activity in peripheral blood leucocytes.
Briefly, 175 μl leukocytes solution containing of 6 × 106 cells mL−1 in
PBS loaded in the 96-well plate in triplicate. Later, 25 μL of Nitro Blue
Tetrazolium (NBT) at a concentration of 1 mg mL−1 was placed into
each well, and incubated at RT for 2 h. After incubation, the super-
natant in each well was carefully discarded and 125 μL of 100% me-
thanol was added to fix the adherent cells for 5 min. The supernatant
was discarded again and 125 μL of 70% methanol was added to each
well for washing. After washing, the plate was allowed to dry at room
temperature for 30 min. 125 μL of 2 N KOH and 150 μL of DMSO were
then added to each well. After adding the solution, the plate was
measured at 655 nm by a microplate reader. Spontaneous O2– produc-
tion = (Absorbance NBT reduction of sample) − (Absorbance of blank).
2.5.6. Alternative complement pathway activity (ACH50)
The ACH50 was determined following the method of Yano (1992).
Briefly, rabbit red blood cells (R-RBC) were washed with PBS by cen-
trifugation at 3000 rpm, and in 0.01 M ethylene glycol tetra-acetic acid-
magnesium-gelatin veronal buffer (0.01 M – EGTA-Mg-GVB) for twice.
The R-RBC concentration was adjusted to 2 × 108 cells mL−1 in 0.01 M
– EGTA-Mg-GVB bufer. Then 100 μL of the R-RBC suspension was lysed
with 3.4 mL of distilled water. The absorbance of hemolysate was
measured at 414 nm against distilled water as a blank and was brought
to be close to 0.740.
For the ACH50 test, 100 μL of serum was diluted with 400 μL of
0.01 M-EGTA-Mg-GVB and serial two-fold dilution was conducted. The
tubes were performed on ice to retard the reaction of complement until
all tubes were prepared. Consequently, 100 μL of R-RBC suspension was
loaded into each tube and incubated at 20 °C for 1.5 h with occasional
shaking. After incubation, 3.15 mL of cold normal saline solution was
placed into each tube to stop the reaction, and then the tube was cen-
trifuged at 1600 g for 5 min. After centrifugation, 100 μL of supernatant
in each dilution was loaded into 96-well plate and read at 414 nm. The
degree of hemolysis was calculated by dividing the corrected absor-
bance 414 value by the corrected absorbance 414 of the 100% hemo-
lysis control. The degree of hemolysis and the serum volume were
plotted on a log-log paper. The volume of serum that gave 50% he-
molysis was used for calculating the ACH50 using the formula: ACH50
(units/ml) = 1/K x r x ½.
where K is the amount of serum giving 50% hemolysis, r is the re-
ciprocal of the serum dilution, and ½ is the correction factor. The assay
was performed on a ½ scale of the original method.
2.6. The disease resistance test
To determine the disease resistance of treated and control Nile ti-
lapia, fish were subjected to experimental infection with pathogenic
bacteria, S. agalactiae after 8 weeks post-feeding for 15 days. The pa-
thogenic bacterium supplied and the solution prepared as mentioned by
Van Doan et al. (2018). The selected specimens (ten fish per tank) were
interpreatinally injected with 0.1 mL of 0.85% normal saline solution
containing 108 CFU ml−1 of S. agalactiae. The dead fish removed and
the corresponding tanks were recorded every day. The relative per-
centage of survival (RPS) measured based on the following formula
(Amend, 1981):
RPS = 100 − [(test mortality/control mortality) ∗ 100]
2.7. Statistical analysis
The SAS Computer Program (SAS, 2003) was used for data analysis.
To determine statistically significant difference (P < .05) one-way
analysis of variances (ANOVA) and two-way ANOVA were used to de-
termine the relationship between TG levels and feeding periods. Dun-
can's Multiple Range Test was used as a post-hoc test to detemine sig-
nificancy at P < .05.
H. Van Doan, et al. Aquaculture 513 (2019) 734388

Table 2
Serum immunity parameters of Nile tilapia, O. niloticus after 4 and 8 weeks of feeding on experimental diets containing different levels of Thai ginseng (TG), B.
rotunda. Different letters in the same row indicate to significant differences (P < .05).
TG levels (g kg−1 diet) P-value TG levels P-value Periods P-value Interaction

0.0 5.0 10.0 20.0 40.0

Diet 1 Diet 2 Diet 3 Diet 4 Diet 5

SL
4 weeks 4.50 ± 0.35c 6.32 ± 0.19b 7.99 ± 0.18a 6.72 ± 0.41ab 6.02 ± 0.59b 1.09043E−06 3.16779E−09 0.9979
8 weeks 7.15 ± 0.26c 8.70 ± 0.15b 10.46 ± 0.15a 9.24 ± 0.31b 8.46 ± 0.49b

SP
4 weeks 0.11 ± 0.008b 0.17 ± 0.006ab 0.19 ± 0.005a 0.18 ± 0.006ab 0.16 ± 0.10b 3.1833E−05 3.1833E−05 0.6207
8 weeks 0.18 ± 0.008b 0.23 ± 0.02a 0.24 ± 0.01a 0.25 ± 0.02a 0.24 ± 0.006a

ACH50
4 weeks 135.58 ± 5.33b 168.87 ± 0.68a 175.97 ± 3.48a 173.82 ± 4.28a 150.07 ± 4.80b 1.4831E−06 2.7092E−10 0.0142
8 weeks 173.58 ± 8.36c 211.98 ± 8.63b 275.79 ± 11.74a 237.72 ± 5.23b 219.53 ± 12.83b

PI
4 weeks 1.50 ± 0.03c 2.21 ± 0.05ab 2.60 ± 0.011a 2.47 ± 0.08a 1.86 ± 0.21bc 1.28E−06 2.7798E−05 0.0669
8 weeks 1.97 ± 0.09b 2.50 ± 0.08a 2.83 ± 0.10a 2.67 ± 0.06a 2.74 ± 0.07a

RB
4 weeks 0.04 ± 0.003c 0.08 ± 0.008ab 0.09 ± 0.008a 0.09 ± 0.008ab 0.06 ± 0.003bc 2.6368E−06 2.8366E−12 0.3959
8 weeks 0.11 ± 0.003c 0.14 ± 0.003b 0.17 ± 0.10a 0.16 ± 0.008ab 0.14 ± 0.006b

SL = Serum lysozyme activity (μg ml−1); SP = Serum peroxidase activity (μg ml−1); ACH50 = Alternative complement activity (units/ ml); PI = Phagocytosis ac-
tivity (bead cell−1); RB = Respiratory burst activity (OD655).

3. Results

3.1. Serum immune responses

The results revealed that feeding with Thai ginseng (TG) sig-
nificantly increased serum lysozyme (SL), serum peroxidase (SP), al-
ternative complement (ACH50), phagocytosis index (PI), respiratory
burst activities (RB) compared with controls (P < .05); with the up-
permost increase D3 treatment (i.e 10 g TG kg−1 diet). However, the
effects were not dose-dependent and there were no significant differ-
ences among other TG groups (P > .05; Table 2).

3.2. Skin mucus immune parameters

The results of the present study showed in both sampling times (4th
and 8th weeks) there were notable increase of skin mucus lysozyme and
peroxidase activities of fish fed TG-containing diets (P < .05; Table 3),
except for the skin mucus lysozyme activity that remained not altered
after 4 weeks of treatment. Similar to the results of serum immune
parameters, highest increases were observed in fish fed 10 g TG kg−1
diet.
Fig. 1. Survival rate of Nile tilapia, O. niloticus fed on experimental diets con-
taining different levels of Thai ginseng, B. rotunda for 8 weeks and post chal-
lenged against Streptococcus agalactiae for 15 days. Diet 1 (0 – control), Diet 2
(5 g TG kg−1 diet), Diet 3 (10 g TG kg−1 diet), Diet 4 (20 g TG kg−1 diet), and
Diet 5 (40 g TG kg−1 diet).

Table 3
Skin mucus lysozyme and peroxidase activities of Nile tilapia, O. niloticus after 4 and 8 weeks of feeding on experimental diets containing different levels of Thai
ginseng (TG), B. rotunda.
TG levels (g kg−1 diet) P-value TG levels P-value Periods P-value Interaction

0.0 5.0 10.0 20.0 40.0

Diet 1 Diet 2 Diet 3 Diet 4 Diet 5

SMLA
4 weeks 2.06 ± 0.46a 1.82 ± 0.21a 2.67 ± 0.18a 1.96 ± 0.23a 2.47 ± 0.57a 0.00474837 9.4208E−10 0.0378
8 weeks 3.20 ± 0.42c 5.16 ± 0.22ab 5.90 ± 0.08a 4.61 ± 0.18b 4.5 ± 0.27b

SMPA
4 weeks 0.04 ± 0.003c 0.07 ± 0.006b 0.10 ± 0.008a 0.07 ± 0.003b 0.07 ± 0.008b 8.2761E−06 5.1071E−09 0.4979
8 weeks 0.09 ± 0.005c 0.14 ± 0.013ab 0.17 ± 0.006a 0.13 ± 0.005ab 0.11 ± 0.016bc

SMLA (μg ml−1) = Skin mucus lysozyme activity.

SMPA (μg ml−1) = Skin mucus peroxidase activity.
Different superscript letters in the same row indicate to significant differences (P < .05).

4
H. Van Doan, et al. Aquaculture 513 (2019) 734388

Table 4
Growth performances and feed utilization of O. niloticus after 4 and 8 weeks of feeding with experimental diets containing different levels of Thai ginseng (TG), B.
rotunda. Different superscript letters in the same row indicate to significant differences (P < .05).
TG levels (g kg−1 diet) P-value TG levels P-value Periods P-value Interaction

0.0 5.0 10.0 20.0 40.0

Diet 1 Diet 2 Diet 3 Diet 4 Diet 5

IW (g) 17.27 ± 0.12 17.27 ± 0.04 17.26 ± 0.08 17.28 ± 0.07 17.35 ± 0.22
FW (g)
c b a b
4 weeks 50.37 ± 0.28 55.87 ± 1.52 61.56 ± 1.16 57.24 ± 0.88 55.33 ± 1.04b 8.67274E−05 5.23884E−20 0.6619
8 weeks 98.83 ± 0.20b 108.93 ± 3.83a 114.97 ± 1.11a 111.38 ± 3.34a 105.56 ± 1.78ab

WG (g)
4 weeks 33.11 ± 0.37c 38.60 ± 1.96b 44.29 ± 1.20a 39.96 ± 0.81b 37.98 ± 0.83b 9.2707E−05 5.9019E−20 0.6668
8 weeks 81.57 ± 0.29b 91.66 ± 3.86a 97.70 ± 1.12a 94.01 ± 3.36a 88.21 ± 1.86ab

SGR (%g/day)
4 weeks 3.57 ± 0.04c 3.91 ± 0.10b 4.24 ± 0.07a 3.99 ± 0.04b 3.86 ± 0.02b 5.4351E−06 7.51E−16 0.0396
8 weeks 2.91 ± 0.01b 3.07 ± 0.06a 3.16 ± 0.02a 3.10 ± 0.05a 3.01 ± 0.04ab

FI
4 weeks 75.13 ± 0.16c 77.58 ± 0.00b 83.89 ± 0.58a 77.58 ± 0.00b 76.69 ± 0.76bc 0.01175567 3.0438E−21 0.8176
8 weeks 153.42 ± 1.50 160.89 ± 4.98 167.77 ± 1.15 161.03 ± 5.09 156.11 ± 3.39

FCR
4 weeks 1.49 ± 0.01a 1.39 ± 0.04b 1.36 ± 0.02b 1.35 ± 0.02b 1.39 ± 0.02b 0.00025324 8.0533E−06 0.9416
8 weeks 1.55 ± 0.01a 1.48 ± 0.01b 1.46 ± 0.01b 1.46 ± 0.02b 1.48 ± 0.01b

SR (%)
4 weeks 100 ± 0.00 100 ± 0.00 100 ± 0.00 100 ± 0.00 100 ± 0.00 0.67828192 0.11811543 0.6783
8 weeks 100 ± 0.00 96.68 ± 0.10 100 ± 0.00 98.33 ± 0.05 98.33 ± 0.05

IW (g) = Initial weight; FW (g) = Final weight; WG (g) = Weight gain; SGR (%g/day) = Specific growth rate; FI (g) = Feed intake; FCR = Feed conversion ratio; SR
(%) = Survival rate.

3.3. The bacterial challenge test
The survival of Nile tilapia following experimental infection with S.
agalactiae is shown in Fig. 1. The mortality of fish in control and treated
groups were started 5 and 6 days post-challenge, respectively. The in-
fected fish had typical symptom of streptococcosis such as losing ap-
petite, darkness, exophthalmia, pair-fins basal haemorrhage, and pale
liver (data not shown). Like the results of immune parameters, fish in
D3 treatment (i.e 10 g TG kg−1 diet) had the highest survival rate.
However, there were no significant differences between RPS of fish in
D3 and D4 treatments (P > .05). The RPS of D2, D3, D4, and D5 were
31.82, 72.73, 63.64, and 59.09%, respectively (Fig. 1).
3.4. Evaluation of growth parameters
Regardless of inclusion levels, feeding on TG notably improved
growth performance compared to the control diet (P < .05; Table 4).
Fish in D3 and D4 treatments showed the most improvement of final
weight (FW), weight gain (WG), and specific growth rate (SGR)
(P < .05). Similarly, significant decrease in FCR, but increase in feed
intake were noticed in TG treatments (P < .05). However, this de-
crease was not a dose-dependent. Additionally, no significant differ-
ences in survial rate and feed intake were recorded between the control
and supplemented TG diets after 8 weeks post-feeding.
4. Discussion
Aquaculture industry in recent decades has paid great attention to
the application of medicinal plants as alternative strategies for anti-
biotics and chemotherapeutics in diseases prevention and treatment.
Medicinal plants have been demonstrated as the immunostimulants and
growth promoters with minor effects on animals and environment
(Abdel-Tawwab, 2016; Abdel-Tawwab et al., 2018; Awad and Awaad,
2017; Dawood et al., 2018; Harikrishnan et al., 2011; Van Hai, 2015).
Among them, Thai ginseng has been considered as a promise one. The
antioxidative and immunomodulatory effects of Thai ginseng have been
demonstrated, which attributed to its high content in bioactive com-
pounds such as triterpene saponins (ginsenosides), amino acids, alka-
loids, phenols, polysaccharides, polypeptides, vitamins B1 and B2 (Kim,
2012; Lakshmi et al., 2011). Indeed, ginseng contains a considerable
amount of catechins, which have anti-inflammatory, anti-bacterial, and
anti-viral properties (Bulfon et al., 2017). Hence, the present in-
vestigation was conducted to evaluate effects of Thai ginseng (TG), B.
rotunda powder on Nile tilapia growth, immune response, and disease
resistance against S. agalactiae.
As the first layer of defence against pathogens, non-specific immune
system play a vital role in protection fish's health and well-being (Gou
et al., 2018). Lysozyme is an enzyme that can break down pepti-
doglycan cell wall of bacteria, hence, it non-specifically inhibits the
growth of invaded microorganisms (Magnadottir, 2010). The present
study recorded a significant increase in lysozyme activity after 4 and
8 weeks post-feeding, with the highest value in the fish fed 10 g TG
kg−1 diet. Positive effects of medicinal herbs on serum lysozyme ac-
tivity have been observed in rainbow trout, Oncorhynchus mykiss
(Bulfon et al., 2017; Haghighi et al., 2014; Mehrabi et al., 2019; Saeidi
Asl et al., 2017; Sarvi Moghanlou et al., 2018); pacu, Piaractus meso-
potamicus (Zanuzzo et al., 2017); largemouth bass, Micropterus salmoides
(Lin et al., 2017); sea cucumber, Apostichopus japonicus (Dang et al.,
2019); common carp, Cyprinus carpio (Hoseinifar et al., 2019b); Bu-
latmai barbel, Luciobarbus capito (Wu et al., 2019); snakehead, Channa
argus (Li et al., 2019), and zebrafish, Danio rerio (Ahmadifar et al.,
2019). Complement activity is an another vital mechanism in fish's
innate immune system, which can effectively eliminate pahtogens and
stimulate inflammatory reactions (Ellis, 2001; Ichiki et al., 2012). The
present study indicates that fish fed dietary TG notably increased the
alternate complement activity (ACH50). Likewise, significant im-
provement of ACH50 was observed in rain bow trout, O. mykiss (Bulfon
et al., 2017); common carp, C. carpio (Hoseinifar et al., 2019b;
Hoseinifar et al., 2018), and snakehead, C. argus (Li et al., 2019).
Leukocyte respiratory burst activity was determined as an indicator of
mammals' non-specific immune system when the scientists found that
phagocytosis was associated with a high oxygen consumption

5
H. Van Doan, et al.
(Baldridge and Gerard, 1933). Superoxide and hydrogen peroxide are
greatly toxic and represent the basis for a strong antibacterial system
(Klebanoff, 1992). Similarly, fish's respiratory burst is connected with
the releasing of cytokines and inflammatory responses (Neumann et al.,
2000; Rieger et al., 2010). The present study recorded an improvement
in respiratory burst and serum peroxidase activities in fish fed
10 g kg−1 TG diet. Likewise, an elevation of respiratory burst perox-
idase activities was observed in pacu, P. mesopotamicus (Zanuzzo et al.,
2017) Nile tilapia, O. niloticus (Doan et al., 2017; Zahran et al., 2018);
gilthead seabream, Sparus aurata (Beltrán et al., 2018; Fazio et al.,
2017; Guardiola et al., 2018); tambaqui, Colossoma macropomum
(Ribeiro et al., 2018); rainbow trout, O. mykiss (Mehrabi et al., 2019);
sea cucumber, A. japonicus (Dang et al., 2019), and Bulatmai barbel, L.
capito (Wu et al., 2019). Phagocytosis is one of the most important
cellular immune system elements in fish (Magnadottir, 2010). Its role is
to further ensure that no pathogens can attact the fish by recognizing
and binding the pathogens, thus limiting their spread and growth
(Harikrishnan et al., 2011). The present study demonstrated an increase
in phagocytosis, suggesting that dietary TG might activate the immune
response leading to improved tolerance against infectious diseases.
Positive effects of medicinal plants on phagocystosis activity have been
reported in gilthead seabream, S. aurata (Beltrán et al., 2018; Fazio
et al., 2017; Guardiola et al., 2018), and sea cucumber, A. japonicus
(Dang et al., 2019). Although the precise mechanism behind im-
munomodulatory nature of TG remained to be clarified, it may be due
to the presence of bioactive compounds in TG, which included boe-
senbergin, cardamonin, pinostrobin, pinocembrin, panduratin A, and 4-
hydroxypanduratin A (Isa et al., 2013). These substances have been
proven to have antioxidant, antibacterial, antifungal, anti-in-
flammatory, antitumour and anti-tuberculosis activities (Kiat et al.,
2006). Among them cardamonin is one of the bioactive compounds,
classified under polyphenols, which have been demonstrated to be able
to modify the immune function by regulatoring the epigenetic ma-
chenisms (Cuevas et al., 2013). Recent study indicated that cardamonin
possesses a better potency in inhibiting the plasma level of IL-1β and IL-
6 than TNF-α, which resulted in the prohibition of paw edema, me-
chanical alldynia, and thermal hyperalgesia. It may also have directly
suppressed the action of systemic IL-1β and IL-6 in disease progress in
rheumatoid arthritis rats or indirectly through inhibiting the production
of IL-1β and IL-6 from immune cells (Voon et al., 2017). However, more
investigations are needed to clarify the machenism in which TG sti-
mulate serum immunity in fish.
The mucosal immune system of vertebreates comprises of a unique
series of specific and non-specific, as well as molecules that play a
crucial role in protecting the host against pathogens (Gomez et al.,
2013). Skin mucus is a crucial element of fish's innate immune sytem
(Magnadóttir, 2006). It acts as fisrt layer of defence system, which
containing variety of immunological factors that can ensure the host
against pathogen infections (Guardiola et al., 2014; Koshio, 2016). The
present study recorded a significant increase in skin mucus lysozyme
and mucus peroxidase activities with highest value in fish fed 10 g TG
kg−1 diet. Beneficial effects of medicinal plants on skin mucus immune
parameters have been reported in Nile tilapia, O. niloticus fed Cordyceps
militaris mushroom substrate (Doan et al., 2017); common carp, C.
carpio fed Psidium guajava and Mespilus germanica (Hoseinifar et al.,
2017; Hoseinifar et al., 2019b); rainbow trout, O. mykiss fed Myrtus
communis (Mansouri Taee et al., 2017), and Indian major carp, Labeo
rohita fed aloin (Srivastava et al., 2018). It is well-documented that
immunostimulants, such as prebiotics, probiotics, and medical are able
to stimulate fish's mucosal immune system (Caipang, 2015; Abdel-
Tawwab, 2016). Considering as the immunological areas, the mucosal
tissues are able to ascend a robust immune response against pathogen,
which consists of gut-associated lymphoid tissues, skin-associated
lymphoid tissues, and gill-associated lymphoid tissues (Bagni et al.,
2005; Caipang, 2015; Sahlmann et al., 2013; Strand and Dalmo, 1997).
Nonetheless, the exact mechanism in which TG enhances fish's mucosal
6
Aquaculture 513 (2019) 734388
immune response merit further investigations.
The usefulness of TG as immunostimulant in Nile tilapia aqua-
culture is also sustained by the results of the challenging test. Indeed,
the present study observed a reduced mortality of Nile tilapia after S.
agalactiae infection following the TG supplementation. Highest survival
rate was recorded in Nile tilapia fed 10 g TG kg−1 diet. Similarly, Nile
tilapia and rainbow trout fed a diet supplemented with ginseng showed
decreased mortality against Aeromona hydrophila and Yersinia ruckeri,
respectively (Abdel-Tawwab, 2012; Bulfon et al., 2017). Enhanced the
resistance against S. agalactiae infection may be due to the elevation of
mucosal and serum immune responses. Additionally, recent investiga-
tion revealed that bioactive compounds isolated from B. rotunda pos-
sessed antibacterial activity against Escherichia coli, Staphylococcus
aureus, S. epidermidis, and Streptococcus mutants (Sri et al., 2018).
Growth improvement is one of the utmost goals in aquaculture
practice, as it is naturally related to the productivity and profitability of
farmers. Medicinal plants have been used as one of strategies to pro-
mote growth performance and reduce feed conversion ratio of aquatic
farming species (Reverter et al., 2017). The present study noticed a
significant improvement in WG, SGR, and FCR of Nile tilapia after TG
supplementation. Fish fed a diet supplemented with 10 g TG kg−1 diet
showed a significant increase in their growth with a decreased FCR
value. Similarly, significant enhancement of growth performance and
FCR was reported in golden pompano fed dandelion and hawthorn
extracts (Tan et al., 2017b; Tan et al., 2017a); hybrid grouper fed Panax
notoginseng and radix bupleuri extracts (Sun et al., 2017; Zou et al.,
2019); common carp fed white-button mushroom (Hoseinifar et al.,
2019a); sneakehead fish fed flavonoids from Allium mongolicum (Li
et al., 2019). Interestingly, the higher level of TG (20 and 40 g kg−1)
resulted in decreased growth performance in Nile tilapia. The reduction
in fish growth is may be due to the toxic effect of TG to liver cells. Isa
et al. (2012) reported that high dose of boesenbergin A, an active in-
gredient extracted from B. rotunda caused greatly toxic to normal he-
patic cells. The elevation in fish growth performance and FCR seems
probably attributed to TG consting of wide range of bioactive com-
pounds, which able to promote growth and stimulate appetites. Awad
and Awaad (2017) reported that, the increase in WG and FCR by dietary
medicinal herbs is related to the improvement in metabolic parameters
or utilization of nutrients and the activation of the functionality of the
intestinal flora. Moreover, the consumption of ginseng could positively
regulate the intestinal tract microflora balance and trypsin-like enzyme
activities during the digestive processes (Abdel-Tawwab, 2012; Goda,
2008). However, the exact role of ginseng compounds requires further
investigation.
In conclusion, the present study revealed that TG supplementation
could activate the skin mucus mucosal and serum immune mechanisms,
as well as provide disease resistance against pathogenic bacteria S.
agalactiae. Further enhancement in growth performance and feed uti-
lization of Nile tilapia was also observed with an optimum level of 10 g
TG/kg diet.
Acknowledgement
The authors thank the National Research Council of Thailand for
financial assistance. The authors also would like to thank for the staffs
at Central and Biotechnology Laboratories, Faculty of Agriculture,
Chiang Mai University for their kind supports during data analysis
process.
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