Enhanced Growth Performance,

Haemato-biochemical and
Immune Parameters of Asian
Seabass, Lates calcarifer (Bloch,
1790) Fed Dietary Supplementation
with Polygonum chinense
RASHIDAH ABDUL RAZAK1,2, MOHAMED SHARIFF2, FATIMAH MD. YUSOFF3,*, INTAN SAFINAR4
1
Department of Marine Science, Kulliyyah of Science, International Islamic University Malaysia, Kuantan, Pahang, Malaysia
2
Faculty of Veterinary Medicine, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
3
Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Selangor Malaysia
©Asian Fisheries Society 4
Laboratory of Natural Products, Institute of Bioscience, Universiti Putra Malaysia, Serdang, Selangor, Malaysia
ISSN: 0116-6514
E-ISSN: 2073-3720
[Link] E-mail: fatimahyus@[Link] | Received: 26/02/2019; Accepted: 26/03/2019
*

Abstract
The present study evaluated the effects of Polygonum chinense extract (PCE) on the growth performance, haemato-
biochemical and innate immunity parameters of Asian seabass, Lates calcarifer (Bloch, 1790). The fish with an average
weight of 9.5 ± 0.2 g were fed for 60 days using three supplementation diets of PCE at 0.2 %, 0.5 % and 1.0 %, and a
non-supplemented diet as a control. Fish fed on 0.5 % PCE diet revealed significant higher body weight gain, specific
growth rate, protein efficiency ratio, condition factor, and significantly lower feed conversion ratio. Dietary
supplementation with 0.5 and 1.0 % showed a significant increase in red blood cell count and haemoglobin level, but
the white blood cell count significantly increased only in 1.0 % diet. The alkaline phosphatase level significantly
increased in fish group fed 0.2 and 0.5 % diets, but the aspartate transaminase level significantly decreases in 0.2 and
1.0 % groups. Meanwhile, all PCE supplemented diets showed significant increase in total protein, albumin, globulin,
albumin: globulin ratio, total immunoglobulin, lysozyme, respiratory burst and phagocytic activities, and significantly
reduced the alkaline transaminase level. These results suggest that dietary P. chinense supplementation at 0.5 % was
found to be suitable to enhance the growth performance and immune status of Asian seabass.

Keywords: fish; plant extract; growth trial; feed evaluation; haematology; innate immune response

development of resistant fish bacterial pathogens

Introduction (Cabello 2006). With the increasing awareness on the
Asian seabass or commonly known as barramundi is an negative impact of antibiotics usage, researchers have
economically important species in Malaysia and other been inspired to find natural alternatives to overcome
countries in the Indo-Pacific region (Mathew 2009). the disease problems in aquaculture. One of the natural
This euryhaline species is being extensively or solutions for controlling and preventing fish diseases is
intensively cultured in earthen ponds and sea cages the development of immunostimulant diets (Mastan
using fresh, brackish or marine water (Glencross 2006; 2015).
Hutson 2014). Intensification of fish production
together with poor environmental conditions and Immunostimulant diets using natural products and plant
management increases the susceptibility of the fish to extracts have shown promising results in improving the
pathogens, which lead to disease outbreak (Chen 1991). growth and immune response of fish. A large numbers of
Numerous diseases have been reported in Asian plant products have been investigated for growth
seabass, and the causative agents varied from performance and immune response in many fish
parasites, bacteria, fungi, viruses, malnutrition or species: Aloe barbadensis (Adegbesan et al. 2018) and
environmental stresses (Talpur and Ikhwanuddin 2012). Vernonia amygdalina and Carica papaya (Olusola and
These aquatic diseases are being controlled with the Nwokike 2018) in Clarias gariepinus (Burchell, 1822)
use of antibiotics, chemicals, disinfectants and (African catfish); Cissus quadrangularis (Devakumar and
chemotherapeutics (Austin and Austin 2007; Rico et al. Chinnasamy 2017) and Camellia sinensis (Al Ngada et al.
2012, 2013; Reverter et al. 2014). However, the 2017) in Lates calcarifer (Bloch, 1790) (Asian seabass);
indiscriminate use of antibiotics have resulted in the Nigella sativa (Altunoglu et al. 2017), Capparis spinosa

Asian Fisheries Science 32 (2019):19–28 19 19 Asian Fisheries Science 32 (2019):19–28

(Bilen et al. 2016) and Stachys lavandulifolia (Moghanlou protein, 6 % crude fat, 12 % moisture, 5 % crude fibre
et al. 2018) in Oncorhynchus mykiss (Walbaum, 1792) and 10 % crude ash. The feed pellet was coarsely
(rainbow trout). powdered using an electric household blender, and
PCE was added at 0.2 %, 0.5 % and 1.0 % as a
One of the local herbal plants Polygonum chinense used supplement. The non-supplemented basal diet was
for the treatment of enteritis, diarrhoea, hepatitis and treated as a control. The diets were manually pelleted
dysentery (Li 1994; Ren and He 2001; Tang 2003) was using a mould for pressing cakes with the inclusion of
selected for the present study. Polygonum chinense 40 % distilled water to achieve proper pelleting
known as Chinese knotweed belongs to the family consistency. The pellets were dried in an oven
Polygonaceae (Ezhilan and Neelamegam 2012) can be overnight at 35 °C and stored at 4 °C until use.
found in the sub-tropical and warm temperate regions of
Asia such as China, Japan, India, Indonesia, Malaysia and
Vietnam (Maharajan et al. 2012). More recently, Xiao et al. Experimental system and fish
(2013) have proven that P. chinense contained two husbandry
components, ellagic acid and corilagin which contributes
to its anti-diarrheal effect. Moreover, studies on rats that Four hundred and eighty Asian seabass juveniles with
received a diet containing P. chinense extract could an average weight of 9.5 ± 0.2 g were purchased from
significantly prevent gastric ulcer (Ismail et al. 2012) and a local fish farm (Banting, Selangor, Malaysia) and
protect against induced liver injury (Das 2015). transferred to Aquatic Animal Health Unit, UPM,
Furthermore, Maharajan et al. (2012) found that ethanolic Selangor, Malaysia. On arrival, the fish were kept for 2
extract of P. chinense contained antibacterial and weeks in two 1000 L fibre glass tanks for
antifungal properties. Despite the excellent potential of acclimatisation and fed with commercial diet, twice
medicinal properties of this plant, as yet no study has daily. After acclimatisation, the fish were randomly
evaluated its potential use as a growth enhancer and distributed into 200 L aquarium tanks allotted for the
immunostimulant in aquaculture. Therefore, the present three treatments and the control in triplicates (40 fish
study was aimed to investigate the effects of different per tank). The fish were hand fed for 60 days with
doses of P. chinense supplementary diet on the growth their respective diet at 4 % of body weight, twice daily
performance, haemato-biochemical parameters and (9.00 am and 4.00 pm). The fish were maintained at
immune status of L. calcarifer. water temperature of 29.1 ± 0.3 ºC, dissolved oxygen
6.14 ± 0.13 mg.L-1, pH 7.2 ± 0.1, salinity 21.4 ± 0.5 g.L-1
(YSI multi-probe system, YSI Inc., USA) and total
Materials and Methods ammonia 0.68 ± 0.09 mg.L-1 (API® Ammonia Test Kit,
Plant material and preparation USA). Each tank was supplied with filtered seawater,
continuous aeration through air diffuser stones and a
of the plant extract filter and protein skimmer were fixed to maintain
good water quality. The water in the tank was partially
Ten kilograms of fresh leaves of P. chinense were
(40-50 %) replaced daily in the evening throughout the
purchased from a local plant nursery in Johor,
experiment. The Institutional Animal Care and Use
Malaysia. Identification of the plant was carried out by
Committee of Universiti Putra Malaysia approved all
the staff of University Agriculture Park, Universiti
procedures involving Asian seabass treatments
Putra Malaysia (UPM), Serdang, Selangor, Malaysia.
(UPM/IACUC/AUP-R077/2017).
The samples were washed with distilled water, dried
under shade and coarsely powdered using an electric
grinder. The samples were homogenised with 80 %
Growth performance and survival
methanol at a ratio of 1:10 (w.v-1) and shaken for 48 h at
At days 30 and 60, the body weight gain (BWG),
room temperature (Eloff 1998). The crude extracts
specific growth rate (SGR), feed efficiency (FE), feed
were centrifuged at 3000 g for 20 min and filtered
conversion ratio (FCR), protein efficiency ratio (PER),
through a Whatman filter paper (Whatman,
condition factor (CF), thermal growth condition (TGC)
Maidstone, England). These procedures were
and survival of the different groups were calculated
repeated thrice to obtain the maximum extract. The
according to the following equations (Bullerwell et al.
solvent was evaporated using a rotary vacuum
2016; El Basuini et al. 2016):
evaporator (Rotavapor R-215, Buchi, Switzerland) at
BWG = Final body weight (ωf)– Initial body weight (ωi);
30 - 35 °C and the residues obtained were freeze-
SGR = In ωf – In ωi / Number of days (t) × 100;
dried (Labconco, USA) to powder form. The powdered
FE = Final weight of feed (ƒf) – Initial weight of feed (ƒi) /
extracts were stored at -20 °C until use.
number of fish;
FCR = ƒf - ƒi / ωf - ωi;
Diet preparation PER = ωf – ωi / Feed consumed × percentage of protein in
feed;
A commercial basal diet (Star Feed, Star Feedmills (M)
CF = Body weight (g) / [Standard length] 3 (cm) × 100;
Sdn Bhd, Malaysia) was purchased from a local fish 3 3
TGC = (√ωf - √ωi / Temperature × t) × 1000;
pellet supplier in Rawang, Selangor, Malaysia. The
Survival (%) = Final number of fish – Initial number of fish ×
composition of this commercial diet was 42 % crude
100.

Asian Fisheries Science 32 (2019):19–28 20 20 Asian Fisheries Science 32 (2019):19–28

Blood sampling µL) was placed into a microtiter plate well, and 100 µl
of 0.2 % NBT solution was added. The plate was
At day 30 and 60, five fish caught randomly from each incubated at room temperature for 30 min. After
treatment group and anesthetised with MS-222 (0.1 incubation, 50 µL of NBT-blood cell suspension was
mg.L-1) for the collection of blood samples. One taken and added into a glass tube containing 1.0 ml N,
millilitre of blood was drawn from the caudal vein with N-dimethyl formamide (DMF). The suspension was
a 25G needle (Terumo, Australia) and 1 ml syringe centrifuged at 3000 g for 5 min. The optical density of
(Terumo, Australia). For each treatment, blood the supernatant was recorded in a
samples were pooled from the five fish and were spectrophotometer at 540 nm using a glass cuvette.
divided into two halves. One half of the blood sample
was treated with heparin (BD Vacutainer® Lithium Serum lysozyme activity assay
HeparinN (LH), Becton Dickinson, USA) for immediate
analysis of haematological parameters, and An assay based on the lysis of Micrococcus
respiratory and phagocytic activities. The other half lysodeikticus was used for determining serum
was transferred to a plain tube for biochemical and lysozyme activity. The serum lysozyme activity was
immunological studies. The blood specimens were measured according to the method of Ellis (1990) with
left to coagulate at room temperature for 1 h, followed some modifications. Briefly, 0.015% (w.v-1) M.
by overnight at 4 °C, centrifuged at 3000 g for 15 min, lysodeikticus (ATCC No. 4698, Merck, Germany) in 50
and the serum obtained was kept at -80 °C until mM potassium phosphate buffer (pH 6.2) was used as
assayed (Adel et al. 2015). a substrate. Fifty microlitres of fish serum was added
to 950 µL of bacterial suspension, and the reduction
of absorbance at 450 nm was determined after 1 min
Determination of haematological and 5 min of incubation using a spectrophotometer
and biochemical parameters (Shimadzu, Japan). A unit of lysozyme activity was
defined as the amount of sample causing a decrease
The collected blood samples were immediately in absorbance of 0.001 per min.
subjected to haematological analysis. The red blood
cells (RBC), white blood cells (WBC) and haemoglobin Phagocytosis assay
(Hb) were analysed using Cell-Dyn® 3700 Haematology
Analyser (Abbott Diagnostics, USA). Haematocrit (Ht) The phagocytosis activity of fish blood was
or packed cell volume (PCV) was analysed using a determined according to Anderson and Siwicki (1995)
micro-haematocrit reader (Hawksley Micro with some modifications. One hundred microlitres of
Haematocrit Reader, Hawksley and Sons Ltd, UK). The blood was placed into a microtiter plate well, followed
mean cell volume (MCV) and mean cell haemoglobin by 100 µL of Vibrio harveyi (1 x 107 [Link]-1) suspended
concentration (MCHC) were calculated according to in phosphate buffered saline (PBS). The blood-
the following formulae: bacteria suspension was mixed with a pipette, then
the plate was incubated at room temperature for 20
1) MCV (fL) = (PCV × 1000)/RBC min. After incubation, 5 µL of the suspension was
removed and place on a glass slide to make a blood
2) MCHC (g/L) = Hb/PCV smear, and air dried at room temperature. The slide
was fixed with ethyl alcohol (95 %) for 5 mins and air
The blood serum was used to determine the total dried. Then, the slide was stained with 7 % Giemsa’s
glucose (GLU), total protein (TP), albumin (ALB), stain for 10 min. Three smears were made from each
alkaline transaminase (ALT), aspartate transaminase group of fish. A total of 100 cells from each smear
(AST) and alkaline phosphatase (ALP) using automatic were observed under a microscope (Nikon Eclipse
chemistry analyser (Biolis 24i Premium, Tokyo Boeki 80i, Japan) and the percentage of phagocytic activity
Machinery Ltd, Japan) and using optimised tests of was derived by dividing the number of phagocytising
Boehringer Mannheim GmBH by means of cells by 100.
spectrophotometers. The globulin (GLO) was
calculated by subtracting ALB value from the TP Serum total immunoglobulin assay
value, and albumin: globulin ratio (A: G) was calculated
by dividing ALB value with GLO value. The total immunoglobulin was quantified based on
the biuret method by Anderson and Siwicki (1995).
One hundred microliters of serum and 100 µL of 12 %
Determination of immune polyethylene glycol (PEG) were added into a
parameters centrifuge tube. After 2 h of incubation at room
temperature with continuous shaking, the tube was
Respiratory burst activity centrifuged at 5000 g for 10 min. The supernatant was
removed, and the protein concentration was
The respiratory burst activity of the phagocytes was determined. The value obtained from the analysis was
done by nitroblue tetrazolium (NBT) assay following subtracted from the total protein level, to give the
the method of Anderson and Siwicki (1995). Blood (100 total immunoglobulin concentration of the serum.

Asian Fisheries Science 32 (2019):19–28 21 21 Asian Fisheries Science 32 (2019):19–28

Statistical analysis diet supplementations as compared to the control
group on days 30 and 60 (Fig. 1c).
All data obtained were checked for homogeneity of
variances (Levene) and normality (Kolmogorov– Table 1. Effects of feeding Polygonum chinense extract on
Smirnov) prior to statistical analysis. The statistical the growth performance and feed utilisation of Asian
analysis between dietary groups was then analysed seabass, Lates calcarifer over 60 days period.
using one-way variance (ANOVA) followed by Duncan’s
multiple range comparison test using IBM SPSS Experimental diet (%)
Statistics 21.0 (IBM Corporation, 2012) computer 0 0.2 0.5 1.0
software. Body weight gain ([Link]-1)
Day 30 6.10 ± 0.67a 7.97 ± 0.54a 14.89 ± 1.61b 8.93 ± 1.04a

Results Day 60 28.82 ± 1.88a 33.59 ± 2.18ab 47.24 ± 1.59c 38.81 ± 2.36b
Specific growth rate
Growth performance, feed utilisation Day 30 0.57 ± 0.05a 0.74 ± 0.04ab 1.03 ± 0.08c 0.77 ± 0.07b
and survival Day 60 0.86 ± 0.04a 0.96 ± 0.03b 1.14 ± 0.02c 1.03 ± 0.03b
Feed efficiency
The effects of experimental diets containing PCE on Day 30 8.14 ± 0.89a 11.02 ± 0.72ab 18.91 ± 2.15c 11.91 ± 1.39ab
growth performance of Asian seabass are Day 60 22.17 ± 1.44a 26.06 ± 1.68ab 35.80 ± 1.22c 29.86 ± 1.81bc
summarised in Table 1. Fish fed on 0.5 % PCE had Feed conversion ratio
significantly higher BWG, SGR, FE and PER compared Day 30 2.96 ± 0.17c 2.47 ± 0.17abc 1.81 ± 0.32a 2.80 ± 0.39bc
to other experimental diets on day 30 and 60. Day 60 2.07 ± 0.20c 1.7 ± 0.12b 1.17 ± 0.04a 1.47 ± 0.09ab
Similarly, the TGF was significantly higher, and the Protein efficiency ratio
FCR was significantly lower in all treatment diets on Day 30 0.15 ± 0.02a 0.20± 0.01a 0.34 ± 0.04b 0.21 ± 0.02a
day 30 and 60. However, the CF was not significantly Day 60 0.69 ± 0.04a 0.81 ± 0.05ab 1.11 ± 0.04c 0.92 ± 0.06b
different (P > 0.05) in all experimental diets on day 30, Condition factor
but significantly higher in fish fed on 0.5 % PCE on day Day 30 1.36 ± 0.04a 1.36 ± 0.04a 1.38 ± 0.02a 1.40 ± 0.04a
60. After 60 days of feeding, the survival rate was Day 60 1.24 ± 0.02a 1.26 ± 0.03a 1.39 ± 0.02b 1.31 ± 0.03a
significantly higher in 0.2 % and 0.5 % compared with Thermal growth coefficient
the control. Day 30 0.360 ± 0.638 ± 0.732 ± 0.518 ±
0.040a 0.140b 0.066b 0.005b
Day 60 0.650 ± 0.676 ± 0.892 ± 0.801 ±
Haematological and biochemical 0.031a 0.076ab 0.019c 0.003bc
parameters Survival (%)
Day 91.25 ± 97.50 ± 98.75 ± 93.75 ±
On days 30 and 60, the RBC content, Hb and PCV 60 0.40a 0.00b 0.40b 0.40a
levels were significantly higher in fish fed with 0.2 %, Data are mean ± SE (n = 15) and different superscripts within
0.5 % and 1.0 % PCE supplementation diets. The WBC rows indicate significant differences (P < 0.05) between
content was significantly higher in all treatment diets means among dietary treatments but not between
on day 30, but only in 1.0 % PCE supplementation diet parameters.
on day 60. However, the MCV and MCHC levels not
significantly affected by dietary inclusion of PCE on
days 30 and 60 (Table 2). Feeding with 1.0 % PCE
Discussion
significantly reduced the GLU level on days 30 and 60.
Dietary application of medicinal plants have shown
Moreover, the TP, ALB, GLO, A:G ratio and ALP levels
positive effects on the fish growth (Abdel-Tawwab et
significantly increased, but the AST and ALT levels
al. 2010; Yilmaz and Ergün, 2012; Samad et al. 2014;
were significantly reduced in fish fed with all doses of
Giri et al. 2015; Ngugi et al. 2017). In the present study,
PCE diets on day 60 (Table 3).
the growth of Asian seabass was enhanced by the
administration of the methanolic extract of P.
Immune status chinense into the diet. The significant increase of
BWG, SGR, FE, PER, CF, TGC and survival indicated
that diet supplementation with 0.5 % PCE for 60 days
The serum lysozyme activity significantly increased in
promoted the growth and nutrient utilisation in the
fish fed with 1.0 % PCE on day 30 (Fig. 1a). Likewise, on
fish. A similar result was reported by Giri et al. (2015)
day 60, significantly higher serum lysozyme activity
that the BWG and SGR of rohu, Labeo rohita
and total immunoglobulin were noticed in all
(Hamilton, 1822) significantly increased after dietary
treatment groups compared to the control group.
supplemented with 0.5 % Psidium guajava extract for
However, there were no significant variations in the
60 days. Abdel-Tawwab et al. (2010) also
respiratory burst activity and total immunoglobulin of
demonstrated that Nile tilapia, Oreochromis niloticus
all experimental diets on day 30 (Figs. 1b and 1d). The
(Linnaeus, 1758) fed with 0.5 % green tea, Camellia
phagocytic activity increased in 0.2, 0.5 and 1.0 % PCE
sinensis extract had significant higher BWG, SGR,
FCR, PER, feed intake and energy utilisation. The high

Asian Fisheries Science 32 (2019):19–28 22 22 Asian Fisheries Science 32 (2019):19–28

Table 2. Effects of feeding Polygonum chinense extract on Table 3. Effects of feeding Polygonum chinense extract on the
the haematological parameters of Asian seabass, Lates biochemical parameters of Asian seabass, Lates calcarifer
calcarifer over 60 days period. over 60 days period.

Experimental diets (%) Experimental diets (%)

0 0.2 0.5 1.0 0 0.2 0.5 1.0
Red blood cell (× 1012 L-1) Glucose (mmol.L-1)
Day 3.3 ± 0.1a 3.7 ± 0.1b 3.9 ± 0.1c 3.8 ± 0.1bc Day 3.7 ± 0.3b 3.1 ± 0.2ab 3.6 ± 2.5 ± 0.3a
30 30 0.5ab
Day 3.6 ± 0.1a 4.1 ± 0.1b 4.4 ± 0.1c 4.3 ± 0.1bc Day 1.6 ± 0.1b 1.2 ± 0.1ab 1.2 ± 0.1ab 1.0 ± 0.1a
60 60
Haemoglobin (g.L-1) Total protein (g.L-1)
Day 77.5 ± 1.5a 89.5 ± 92.5 ± 89.0 ± Day 45.0 ± 50.6 ± 1.9b 53.6 ± 44.5 ± 1.3a
30 0.3b 3.5b 2.0b 30 0.6a 3.1b
Day 93.0 ± 110.5 ± 120.0 ± 115.0 ± Day 48.5 ± 58.1 ± 0.1b 58.5 ± 58.3 ±
60 1.0a 5.5b 4.0b 4.0b 60 1.8a 0.5b 0.4b
PCV (L.L-1) Albumin (g.L-1)
Day 0.27 ± 0.30 ± 0.32 ± 0.30 ± Day 10.4 ± 0.1a 11.6 ± 0.9a 13.0 ± 11.2 ± 0.4a
30 0.01a 0.01b 0.01c 0.01b 30 0.7a
Day 0.30 ± 0.34 ± 0.37 ± 0.36 ± Day 13.6 ± 18.4 ± 0.3b 19.9 ± 18.9 ± 0.2b
60 0.01a 0.04b 0.01c 0.01bc 60 0.4a 0.4c
MCV (fL) Globulin (g.L-1)
Day 77.5 ± 1.5
a
79.8 ± 81.1 ± 0.7 a
77.5 ± 0.1 a
Day 34.5 ± 39.0 ± 1.1b 40.6 ± 33.3 ±
30 2.4a 30 0.8b 1.9b 0.9a
Day 83.1 ± 81.6 ± 0.7a 82.8 ± 82.9 ± 0.1a Day 35.0 ± 39.7 ± 38.6 ± 39.4 ±
60 0.9a 0.1a 60 1.4a 0.2b 0.3b 0.2b
MCHC (g.L-1) Albumin: globulin ratio (g.L-1)
Day 287.0 ± 303.4 ± 293.6 ± 301.7 ± Day 0.29 ± 0.30 ± 0.31 ± 0.34 ±
30 2.3a 2.0a 0.1a 1.7a 30 0.0a 0.02a 0.01a 0.04b
Day 310.0 ± 330.2 ± 328.7 ± 324.0 ± Day 0.39 ± 0.46 ± 0.51 ± 0.48 ±
60 3.3a 8.7a 6.5a 2.4a 60 0.01a 0.01b 0.01c 0.01b
White blood cells (× 107 L-1) Alkaline phosphate (U.L-1)
Day 2.8 ± 0.1a 3.2 ± 0.1b 3.2 ± 0.1 b
3.6 ± 0.1 c
Day 57.8 ± 68.5 ± 1.2b 62.3 ± 61.8 ± 1.3a
30 30 4.6a
2.6a
Day 2.9 ± 0.3a 3.3 ± 0.5a 3.1 ± 0.3a 5.1 ± 0.7b Day 46.3 ± 67.8 ± 2.3c 57.0 ± 61.3 ± 4.1c
60 60 2.3 a
2.0b
Aspartate aminotransferase (U.L-1)
Data are mean ± SE (n = 3) and different superscripts within Day30 120.0 ± 77.8 ± 3.3a 73.8 ± 71.5 ± 5.7a
rows indicate significant differences (P < 0.05) between 1.6 b
3.4a
means among dietary treatments but not between Day 111.8 ± 72.8 ± 77.8 ± 74.0 ±
parameters. 60 4.5b 3.9a 2.4a 0.4a
Alanine aminotransferase (U.L-1)
amount of bioactive compounds in P. chinense that Day 46.3 ± 40.8 ± 42.5 ± 37.0 ±
possessed antimicrobial properties (Ezhilan and 30 2.5b 1.8ab 1.8ab 0.4a
Neelamegam 2012) may account for the improved fish Day 50.8 ± 41.5 ± 0.6a 34.3 ± 35.0 ±
growth performance. 60 2.3 b
1.7a 2.5a

The evaluation of haematological and biochemical Data are mean ± SE (n = 3) and different superscripts within
parameters in fish blood has been used to monitor rows indicate significant differences (P < 0.05) between
fish health (De Pedro et al. 2005) as the changes in means among dietary treatments but not between
these parameters can be an indicator for stress parameters.
(Kumar and Banerjee 2016; Odedeyi and Odo 2017) or
diseases infection (Aydin et al. 2005; Sebastião et al. properties were reported in catfish, Clarias gariepinus
2011). In the present study, feed supplementation with fed on the extract of Aloe barbadensis (Adegbesan et
PCE did not affect the haematological parameters al. 2018), beluga, Huso huso (Linnaeus, 1758) fed on
except for RBC and WBC counts, and Hb level. Urtica dioica extract (Binaii et al. 2014), C. gariepinus
According to Moghanlou et al. (2018), the increase of fed on Vemonia amydalina and Carica papaya extracts
these haematological parameters indicates the (Olusola and Nwokike 2018), and rainbow trout, O.
immunostimulant effects and anti-infection mykiss fed on Echinacea purpurea extract (Oskoii et al.
properties of the plant. Similar findings of 2012).
immunostimulant effects and anti-infection

Asian Fisheries Science 32 (2019):19–28 23 23 Asian Fisheries Science 32 (2019):19–28

 (a) (b)

0% 0.20% 0.50% 1.00% 0% 0.20% 0.50% 1.00%

450 0.6
b

Respiratory burst (OD at 540 nm)

400 b b b
Lysozyme activity ([Link]-1)

c 0.5
350 a bc
300 a b a
0.4 a a
a a a
250 a
0.3
200
150 0.2
100
0.1
50
0 0
30 60 30 60
Day Day

(c) (d)

0% 0.20% 0.50% 1.00% 0% 0.20% 0.50% 1.00%

50 40 c
c c
a
Total immunoglobulin (g.L-1)

45 35 a
a
Phagocytic activity (%)

40 b
b 30 a
35 b a
c 25
30 b b
25 a 20
a
20 15
15
10
10
5 5
0 0
30 60 30 60
Day Day

Fig. 1. The immunological status of Asian seabass, Lates calcarifer fed with different doses of Polygonum chinense extract. (a)
Lysozyme activity; (b) respiratory burst activity; (c) phagocytic activity; (d) total immunoglobulin. Data are mean ± SE (n = 3) and
the difference in values between groups is indicated by different superscripts (ANOVA, Duncan, P < 0.05).

In the present study, feeding fish with 1.0 % PCE globulin are in agreement with other researchers who
significantly decreased the serum glucose level on observed an increase of similar parameters when
days 30 and 60, but no significant variation was supplementation with Ocimum sanctum extract into L.
noticed in the fish group supplemented with other rohita diet (Das et al. 2015), Zingiber officinale extract
experimental diets. The reduction of glucose level into L. calcarifer diet (Talpur et al. 2013) and Allium
could have been due to the capability of the plant sativum and Z. officinale oil extracts into
extract to reduce the effect of stressors (Baba et al. Dicentrarchus labrax (Linnaeus, 1758) diet (Yilmaz and
2016). Changes in the total protein, albumin and Ergün 2012). These findings might be associated with
globulin levels also can be an indicator in determining a stronger innate immune response by the fish fed
the fish health and nutritional status (Svetina et al. with plant extracts (Wiegertjes et al. 1996), as the
2002; John, 2007). In the present study, the total albumin and globulin are the important components
protein, albumin and globulin levels significantly to maintain the healthy immune system (Jha et al.
increase in Asian seabass fed at all levels of PCE. The 2007).
significant increase in total protein albumin and

Asian Fisheries Science 32 (2019):19–28 24 24 Asian Fisheries Science 32 (2019):19–28

Aspartate aminotransferase (AST) and alanine pathogens (Musthafa et al. 2017). This result is in
aminotransferase (ALT) are the most important liver agreement with studies by Ardó et al. (2008) and
enzymes that can be used as biomarkers for liver Harikrishnan et al. (2010) which showed similar trends
damage (Bhardwaj et al. 2010). These blood serum when Chinese herbs (Astragalus membranaceus and
enzymes are released into the blood plasma if their Lonicera japonica) were fed to Nile tilapia, O. niloticus
cells are injured or infected by disease (Banaee et al. and mixed herbs (Azadirachta indica, Oscimum
2011). The increase of AST and ALT levels indicated sanctum, and C. longa) fed to goldfish, Carassius
abnormal liver function and stress response in fish auratus (Linnaeus, 1758) respectively. Therefore, the
(Tan et al. 2017), and the decreased in ALP activity can mode of action of P. chinense extract on the Asian
be a sign of disturbance of membrane transport seabass immunity was monitored with the activation
system (Oner et al. 2008). However, the dietary P. of the cellular and humoral immune response, i.e.
chinense supplementation increases the ALP activity phagocytosis, respiratory burst, immunoglobulin and
as shown in the present study. The increase of ALP lysozyme with the effective dose of 0.5 % PCE.
indicates that fish fed with PCE enhanced the enzyme Moreover, in the present study, there was a
activity. The increase of ALP activity may be due to significant correlation between phagocytic activity,
increasing of the macrophage cells which and weight gain and specific growth rate implying fish
consecutively produced a higher amount of immunity plays a significant role in the growth of
phosphate enzyme (Pratheepa et al. 2010). Moreover, Asian seabass.
dietary P. chinense inclusion also resulted in a
reduction of AST and ALT activities in the present
study indicating that P. chinense supplementation
Conclusion
may have hepatoprotective effects that prevent liver
In summary, the dietary supplementation of 0.5 and
damage (Deng et al. 2011). Previous studies have also
1.0 % PCE significantly improved the growth
reported similar observations as in the present study
performance and feed efficiency in Asian seabass. In
for ALP, AST and ALT activities in O. mykiss, rainbow
addition, supplementation with 1.0 % significantly
trout fed with extract of propolis (Deng et al. 2011),
increased the RBC and WBC counts, and Hb level, but
rohu, L. rohita fed with Curcuma longa extract (Sahu et
all levels of PCE supplementation significantly
al. 2008), and golden pompano, Trachinotus ovatus
increased the total protein, albumin, globulin,
(Linnaeus, 1758) fed with Taraxacum officinale extract
albumin:globulin ratio, lysozyme, respiratory burst
(Tan et al. 2017) respectively.
and phagocytic activities, and total immunoglobulin.
These results indicate the potential use of P. chinense
Serum immunoglobulin is part of the immune
as a growth promoter and immunostimulant for Asian
response of fish that functions to identify and
seabass.
neutralise the invading microbial pathogens (Ngugi et
al. 2017). In the present study, feeding fish with the
diet supplemented with PCE resulted in statistically
Acknowledgements
increase of total immunoglobulin, which could have
The authors gratefully acknowledge Ministry of
been due to the extract of P. chinense that can
Science, Technology and Innovation, Malaysia for the
actively stimulate the production of immunoglobulin.
grant (Grant number 5532200) provided to complete
Lysozyme is an important defence molecule and is an
this project. The first author is also grateful to the
indicator of the fish innate immune system. It can act
Ministry of Higher Education, Malaysia and
as a defence barrier against foreign bacterial
International Islamic University Malaysia for the
pathogens (Misra et al. 2006), promoting phagocytosis
scholarship for Ph.D. studies.
by activating the leucocytes and macrophages
(Saurabh and Sahoo 2008), and play a role in leucocyte
respiratory burst activity (Biller-Takahashi et al. 2013).
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