Aquaculture Reports 21 (2021) 100798

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Aquaculture Reports
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Effect of Noni, Morinda citrifolia fruit extract supplementation on the

growth performances and physiological responses of the hepatopancreas of
Whiteleg shrimp, Penaeus vannamei Post Larvae
Julia Hwei Zhong Moh a, Khor Waiho a, d, f, Hanafiah Fazhan a, d, Noorbaiduri Shaibani a,
Hidayah Manan a, Yeong Yik Sung b, Hongyu Ma c, d, Mhd Ikhwanuddin a, d, e, *
a
Higher Institution Centre of Excellence (HiCoE), Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu,
Malaysia
b
Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia
c
Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou, 515063, Guangdong, China
d
STU-UMT Joint Shellfish Research Laboratory, Shantou University, Shantou, 515063, Guangdong, China
e
Faculty of Fisheries and Marine, Campus C, Airlangga University, Mulyorejo, Surabaya, 60115, Indonesia
f
Centre for Chemical Biology, Universiti Sains Malaysia, Minden 11900, Penang, Malaysia

A R T I C L E I N F O A B S T R A C T

Keywords: This study focused on the effect of Noni, Morinda citrifolia fruit extract supplementation on the growth and the
Penaeus vannamei hepatopancreas function of Whiteleg shrimp, Penaeus vannamei Post Larvae (PL). The crude extract of M. citrifolia
Morinda citrifolia fruit was obtained through drying and methanol extraction followed by concentration through rotary evapo­
Plant extract
ration. The extracts were incorporated into commercial shrimp pellet at the concentrations of 0%, 1%, 2%, 3%,
Histopathology
Physiology
4% and 5%. Feeding trial was done for 30 days and shrimp samples were collected at 5 days interval for growth
performance determination and physiology analysis on the hepatopancreas. Despite the inconsistency in growth
performances during the early feeding period, the extract supplementation did improved the growth of
P. vannamei PL. Significant differences in the digestive enzymes and antioxidant enzymes activities between
treatments were observed. The correlation results showed only amylase were correlated with M. citrifolia fruit
extract supplementation. In terms of the relationship with growth, amylase and lipase showed correlation with
the BW changes of P. vannamei PL while trypsin otherwise. Antioxidant enzymes were strongly correlated with
the concentration of M. citrifolia fruit extract supplemented and growth of P. vannamei PL. This indicates that the
growth of shrimp was improved with increased antioxidant enzyme activities. The number of hepatopancreatic
cells (B, R & E cells) also increased significantly with the increasing concentration of M. citrifolia supplementation
during the 30-day feeding trial. Similarly, strong correlation was observed between the hepatopancreatic cells
and the growth of P. vannamei PL. The appearance of cell atrophies and haemocytic infiltration in the hepato­
pancreas might indicate the stimulation of innate defense mechanism in the hepatopancreas by the extract
supplementation.

1. Introduction Penaeus monodon that grow up to 1 g per week on average. Besides,

Whiteleg shrimp, Penaeus vannamei is one of the most important
shrimp species in food production sector with an approximation of
228,000 tons of total global production (Ma et al., 2013). Its popularity
among shrimp farmers was due to its fast growing nature at the average
of 1–1.5 g per week which is considerable faster than Black tiger shrimp,
P. vannamei was known to have high tolerance towards extreme condi­
tions such as salinities (0.5− 45ppt) and temperature (as low as 15 ◦ C)
making it a more favourable species choice to be cultured even during
adverse weather condition (Briggs et al., 2004).
Lugert et al. (2014) defined growth as a gradual increase of organ­
isms in the aspect of size and body weight over a period of time.

* Corresponding author at: Higher Institution Centre of Excellence (HiCoE), Institute of Tropical Aquaculture and Fisheries, Universiti Malaysia Terengganu, 21030
Kuala Nerus, Terengganu, Malaysia.
E-mail address: ikhwanuddin@umt.edu.my (M. Ikhwanuddin).

https://doi.org/10.1016/j.aqrep.2021.100798
Received 7 March 2021; Received in revised form 6 July 2021; Accepted 20 July 2021
Available online 27 July 2021
2352-5134/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
J.H.Z. Moh et al.
Meanwhile, growth performance is different among individuals for it is a
non-linear process influenced by various factors such as nutrition,
stocking density (space), water quality, sexual category, genetic varia­
tions and age of the organisms (Lugert et al., 2014). According to Baer
et al. (2011), the economic stability in commercial aquaculture sector
generally depends on the growth performance of the organisms in the
aquaculture system. The improvement of growth performances in
aquaculture species through dietary aspect had been reported previously
and was proven to be beneficial towards their productions in terms of
cost and profitability (Hlophe and Moyo, 2014). The application of plant
compounds in improving of growth performances of fish species had
been well documented (Yilmaz, 2019). The improvement of growth of
aquaculture organisms through dietary supplementation that usually
came along with growth performance include their productivity and
quality as well as their ability to withstand pathogen invasions (Yilmaz,
2019; Tan et al., 2017; Harikrishnan et al., 2011).
Hepatopancreas is the largest organ of shrimps located at the pos­
terior region of the cephalothorax. The structure of this organ is built up
of network of ducts and tubules that plays a role in digestion, nutrient
absorption and storage as well as removal of waste (Silva et al., 2018)
which is similar to the function of liver and pancreas of mammals. In
addition, the hepatopancreas also play a role in other physiological
aspect of the shrimp including immune response and ion transport
(Dugassa and Gaetan, 2018).
The preferences in using plant derived products as immunostimulant
has been increasing as antibiotic or drugs substitutes for this approach is
known to be more cost-effective and environmental friendly (Shabana
et al., 2019). Previous studies reported plant extract potentially
improved the immunity of some aquaculture species including variety of
fish species (Maldonado-Garcia et al., 2019; Bulfon et al., 2017; Hosei­
nifar et al., 2015; Punitha et al., 2008) and shrimp (Kumaran et al.,
2014; Rajasekar et al., 2011). The improvement of the growth and im­
munity of an aquatic organism was reported to be associated to the
enhancement of their digestive enzymes activities (Shabana et al., 2019;
Li et al., 2018; Gabriel et al., 2017; Zahran et al., 2014; Lin et al., 2006).
Although the potential involvement of antioxidant enzymes in
improving defence mechanism and growth of shrimp has not been fully
documented, the hepatopancreas was reported to be an important
metabolic centre in shrimp for both digestive system and detoxification
processes. Besides, crustaceans are known to rely on both cellular and
humoral immune responses in terms of infectious pathogen removal that
often caused oxidative stress. The antioxidant system is therefore
important in protecting the cells from this oxidative stress caused by
immune response besides the effect of Reactive Oxygen Species (ROS)
(De La Fuente and Victor, 2000).
Noni, Morinda citrifolia is a polynesian plants commonly grow in
Southeast Asia (Halim et al., 2018) which known to be used in tradi­
tional medicine in treating diabetes (Algenstaedt et al., 2018; Nerurkar
et al., 2015), arthritis (Kustiarini et al., 2019), hypertension, heart dis­
eases indigestions, headaches and fever (McClatchey, 2002). Previous
studies revealed that M. citrifolia potentially exhibit antibacterial (Zhang
et al., 2016; Sibi et al., 2012) and antifungal (Sibi et al., 2012; Jainkit­
tivong et al., 2009). While the research on the benefits of M. citrifolia is
still limited, this plant was reported to have improved the growth of
cattle (Yancey et al., 2013) and broiler birds (Sunder et al., 2015) as well
as the immune responses of Giant freshwater prawn Macrobrachium
rosenbergii which makes it potent immunostimulant (Halim and Prajitno,
2017).
The purpose of this study therefore is to determine the effectiveness
of M. citrifolia fruit extract in improving the growth P. vannamei (PL)
during culture and its effect on the function of the hepatopancreas in
terms of digestive and antioxidant enzymes activities as well as cell
orientation and functions in the hepatopancreas tissue during supple­
mentation period.
2
Aquaculture Reports 21 (2021) 100798
2. Methodology
2.1. M. citrifolia fruit collection and extraction
A total of 5 kg of M. citrifolia fruits were collected and rinsed with
distilled water. The acquirement of M. citrifolia fruit extract was done
according to the method by Natheer et al. (2012) with slight modifica­
tions. After rinsing, the fruit were chopped into small pieces (1 mm
length ×1 mm width ×5 mm thickness) and dried in the oven at 50 ◦ C for
4–5 days followed by extraction with 70 % methanol at 1:5 ratio for 48 h
with constant stirring. The mixture was then filtered through Whatman
No. 1 filter paper (diameter: 45 mm; thickness: 180 μm; pore size: 11
μm) followed by methanol removal through rotary evaporation under
reduced pressure.
2.2. P. vannamei post-larvae collection and acclimatisation
P. vannamei post larvae of PL 18 were obtained from local private
shrimp hatchery, iSharp, Blue Archipelago, Terengganu branch. The PL
were acclimatised in fibreglass tanks at 28 ◦ C and salinity 30 ppt for 2
weeks before they were subjected for feeding trial with M. citrifolia fruit
extract. Throughout the acclimatisation period, P. vannamei PL were fed
twice daily (0800 h & 2000 h) with formulated feed (Brand: Gold Coin,
903S).
2.3. Incorporation of M. citrifolia fruit extract into shrimp pellet
The crude extract of M. citrifolia fruit was incorporated into com­
mercial shrimp feed (40 % protein, 3% fibre, 7% fat, 12 % moisture and
15 % ash) at the concentration of 1 %, 2 %, 3 %, 4 %, and 5 %, resulting
in five treatments with varying M. citrifolia concentrations. The A con­
trol supplementation (0%) was also prepared without extract added. The
crude extracts of assigned weight were dissolved in sterile distilled water
and mixed evenly into the pellet. The pellet were oven dried at tem­
perature below 40 ◦ C.
2.4. Experimental design and feeding trials
Feeding trial was carried out on P. vannamei PL for 30 days with
supplemented feed. The supplementation groups were divided accord­
ing to concentration of the extracts incorporated into feed, with control
as T0 (0% M. citrifolia concentration), T1 with 1% M. citrifolia concen­
tration, T2 with 2% M. citrifolia concentration, T3 with 3% M. citrifolia
concentration, T4 with 4% M. citrifolia concentration and T5 with 5%
M. citrifolia concentration. Each treatment group was done in triplicates.
Each tank (Dimension: 3.300 m × 1.150 m × 0.750 m) was filled with
approximately 300 shrimps. Feeding was done four (4) times daily, at
0800, 1300, 1900 and 0200. Uneaten feed were removed and minimal
water change was done regularly. The cultures were maintained at 28 ◦ C
and salinity at 30 ppt with continuous aeration. The tanks were covered
with nylon shade netting to ensure minimal light penetration to mimic
the natural habitat and minimise stress of the shrimp. Thirty shrimp
samples were randomly collected from each treatment tank at 5 days
interval for growth (body weight, BW and total length, TL), growth
performance and physiology response determination including diges­
tive, antioxidant enzyme and histopathology of the hepatopancreas.
2.5. Body growth and growth performance of P. vannamei PL
determination
P. vannamei PL growth (BW and TL) and growth performances
including Specific Growth Rate (SGR), Average Daily Growth (ADG)
Feed Conversion Ratio (FCR) and Condition Factor (CF) were deter­
mined at 5 days interval throughout the feeding trial period. The BW of
the shrimps were measured using analytical balance whereas TL was
measured using as standard ruler between the rostrum and the telson.
J.H.Z. Moh et al. Aquaculture Reports 21 (2021) 100798

The calculation of the growth performances were done according to the 2.6.2. Lipase activity assay
formula as followed: Lipase activity assay (Catalog no.: K722-100) was conducted in a 96-
Mean Body Weight (BW) = Mean final BW – Mean initial BW well microplate with glycerol as standard. Hepatopancreas tissue
Gain (g) (approximately 40 mg) was homogenised in 4 volumes of assay buffer
Mean Total Length (TL) = Mean final TL – Mean initial TL (K722-100-1) followed by centrifugation at 13,000 × g for 10 min. 50 μL
Gain (mm)
Specific Growth Rate = [(ln W1 – ln W0)/t] × 100, where W0 = average initial
of supernatant was placed into the well. 100 μL of reaction mix were
(SGR) (%/day) body weight, W1 = average final body weight, and t = added into and the spectrophotometry reading at OD570 nm was taken.
time (days) The reaction mix was prepared earlier as followed:
Average Daily Growth = BW gain / Experimental duration Sample Control
(ADG) (g/day)
Feed Conversion Ratio = Food consumed (g) / BW gain (g) Assay Buffer (K722-100-1) 93 μL 96 μL
(FCR) OxiRed Probe (K722-100-2A) 2 μL 2 μL
Condition Factor (CF) (g/ = BW (g) / [TL (cm)]3 Enzyme Mix (K722-100-4) 2 μL 2 μL
cm3) Lipase Substrate (K722-100-5) 3 μL ——

The standard curve of glycerol for lipase determination in samples

2.6. Digestive enzymes activities assays was as shown in Fig. 1b.

Ten shrimp samples were selected from each treatment tank for the
digestive and antioxidant enzymes activities assays at 5 days interval
throughout the 30 days feeding trial. Three activities of digestive en­
zymes were analysed from the hepatopancreas of P. vannamei PL namely
amylase, lipase and trypsin. The assays were done using the bioassay kit
by Biovision (Catalog no.: K711-100 for amylase, K722-100 for lipase
and K771-100 for trypsin). The assay procedures were performed ac­
cording to the manufacturer protocols.
2.6.1. Amylase activity assay
Amylase activity assay (Catalog no.: K711-100) was carried out with
nitrophenol (K711-100-4) as standard. Each hepatopancreas sample
(100 mg) was extracted with 0.5 ml assay buffer (K711-100-1) and
centrifuged at 16,000 × g for 10 min, after which the supernatant was
collected and assayed directly. The assay was done in a 96-well micro­
plate. 50 μL of sample was placed in each well, followed by 50 μL of
assay buffer and 50 μL substrate mix (K711-100-2) and mixed well.
Spectrophotometry reading was taken at OD 405 nm. The amylase ac­
tivity was calculated as mU/mL in which one unit of amylase equals to
the amount of amylase that cleaves ethylidene-pNP-G7 to generate 1.0
μL mol of nitrophenol per minute. The standard curve of nitrophenol
was as shown in Fig. 1a.
2.6.3. Trypsin activity assay
Trypsin activity assay (Catalog no.: K771-100) was done with p-
nitroaniline (p-NA) (K771-100-4) as standard. Hepatopancreas was
homogenised with 4 volumes of trypsin assay buffer (K771-100-1) and
centrifuged at maximum speed for 10 min. Approximately 25 μL of the
hepatopancreas extract was placed into 96-well microplate and the
volume was adjusted to 50 μL with the assay buffer. The mixture was
then added with 1 μL of 50X chymotrypsin inhibitor (TPCK) solution
(K771-100-6) and incubated for 10 min at room temperature. Subse­
quently, additional 48 μL assay buffer and 2 μL trypsin substrate (K771-
100-2) were added and mixed well. Absorbance was measured at OD
405 nm. The standard curve for p-NA for trypsin activities determination
in samples was as shown in Fig. 1c.
2.7. Antioxidant enzymes activities assays
The antioxidant enzymes activities assays were done using bioassay
kits by EnzyChrom™. Similarly, the assays were carried out according to
the method provided by the manufacturer. The antioxidant enzymes
being analysed include catalase (ECAT-100), glutathione (DIGT-250),
superoxide dismutase (ESOD-100) and thiobarbituric acid (DTBA-100).
2.7.1. Catalase activity assay
Catalase activity was conducted using catalase assay kit (ECAT-100)

Fig. 1. Standard curve of (a) nitrophenol, (b) glycerol and (c) p-NA for digestive enzymes activities determination.

3
J.H.Z. Moh et al.
with H2O2 as standard. Approximately 10 mg of hepatopancreas tissue
was homogenised in 200 μL cold phosphate buffer saline (PBS), centri­
fuged at 14,000 rpm for 10 min, and distributed into a 96-well micro­
plate, with each well consist of 10 μL of clear supernatant. Each well was
added with 90 μL of 50 μM H2O2 substrate to initiate catalase reaction.
The plate was mixed by gentle tap and incubated for 30 min at room
temperature. The mixture was added with 100 μL detection reagent (102
μL assay buffer, 1 μL dye reagent and 1 μL HRP enzyme) and incubated
for another 10 min at room temperature. Optical reading at 570 nm was
taken. The standard curve of H2O2 was as shown in Fig. 2a.
2.7.2. Glutathione activity assay
The glutathione activity assay (DIGT-250) was done using quanti­
tative colorimetric method. Hepatopancreas was washed in cold PBS
followed by homogenisation in 4 vol of cold PBS containing phosphate
and 1 mM EDTA. The homogenate was centrifuged at 14,000 rpm for 15
min at 4 ◦ C. Approximately 120 μL of supernatant was transferred into
clean 1.5 mL microcentrifuge tubes and was mixed with 120 μL Reagent
A followed by centrifugation at 14,000 rpm for 5 min. 200 μL of clear
supernatant was placed into 96-well microplate and was mixed with 100
μL Reagent B. The mixture was incubated for 25 min at room temper­
ature and optical reading was taken at OD 412 nm.
2.7.3. Superoxide dismutase activity assay
The SOD activity assay (ESOD-100) was also done using quantitative
colorimetric method. SOD enzyme was prepared at different concen­
tration for standard curve determination. Hepatopancreas tissue was
rinse with cold PBS and homogenised in 5 mL/g cold lysis buffer (50 mM
potassium phosphate, 0.1 mM EDTA and 0.5 % Triton X-100). The ho­
mogenates were centrifuged at 12,000 g for 5 min at 4 ◦ C. 20 μL of su­
pernatant were transferred into 96-well microplate. 160 μL working
reagent (160 μL assay buffer, 5 μL Xanthine and 5 μL WST-1) and 20 μL
diluted XO enzyme were added into the well. Optical reading at OD 450
nm was taken. The standard curve of SOD was as shown in Fig. 2b.
2.7.4. Thiobarbituric acid reactive substances activity assay
The TBARS activity assay (DTBA-100) was carried out with malon­
dialdehyde (MDA) as standard. Hepatopancreas was homogenised in
cold PBS. Approximately 100 μL of the lysates were placed into 1.5 mL
microcentrifuge tubes and 200 μL of ice cold 10 % trichloroacetic acid
were added. The mixture was incubated on ice for 5 min before trans­
ferring 200 μL of the clear supernatant into new tubes for assay. The
Fig. 2. Standard curve of (a) H2O2, (b) SOD and (c) MDA for antioxidant enzymes activities determination.
4
Aquaculture Reports 21 (2021) 100798
sample was mixed with 200 μL TBA reagent and incubated at 100 ◦ C for
60 min. After cooling to room temperature, 100 μL of the mixture was
loaded into 96-well microplate. Spectrometry reading at OD 535 nm was
taken. The standard curve of MDA for TBARS determination in samples
was as shown in Fig. 2c.
2.8. Histopathology of P. vannamei PL hepatopancreas
The histopathology study of the hepatopancreas of P. vannamei
following the method used by Manan et al. (2015) with slight modifi­
cation to fit the samples condition of the present study. 10 shrimp
samples were also selected from each feeding tank for the histopathol­
ogy analysis of P. vannamei PL hepatopancreas at 5 days interval
throughout the 30 days feeding period. The histology process was
initiated by fixation of the hepatopancreas in Davidson’s solution for 24
h followed by tissue processing in the tissue processor. The tissue pro­
cessing steps include dehydration through a series of alcohol, followed
by clearing in xylene and lastly impregnation in paraffin. Processed
samples were then embedded in paraffin wax and sectioned at 5 μm
thickness on glass slides. The histology process ended with staining with
haematoxylin and eosin (H&E) and mounted with DPX. The prepared
slides were analysed using advance viewing microscope Nikon eclipse 80i
at 100x and 200x magnification. The analysis involved in the identifi­
cation and counting of hepatopancreatic cells presented in the hepato­
pancreas of P. vannamei PL in all supplemented groups.
2.9. Statistical analysis
The effectiveness of the supplementation of M. citrifolia fruit extract
on the growth performance and physiological responses of P. vannamei
PL hepatopancreas were investigated through one-way analysis of
variance (ANOVA). Prior to analysis, the data were checked for homo­
geneity of variance using Levene test. When data failed the homogeneity
test or if the variance were unequal (P < 0.05), ANOVA was then done
with Welch correction at confidence level of 0.05 followed by multiple
comparison between groups through post hoc analysis. Games Howell
comparison method was used when Welch ANOVA was used whereas if
Welch ANOVA was not used during analysis of variance, Tukey’s HSD
and LSD test were used for group comparisons. The significance between
groups were measured at the confidence level of 0.05. Correlation and
regression test were done to determine the relationship between the (i)
extract supplementation and the physiological responses during feeding
J.H.Z. Moh et al.
trial and (ii) growth and physiological responses throughout the feeding
trial.
3. Results
3.1. Body weight (BW) and total length (TL)
BW and TL of P. vannamei PL were relatively inconsistent in regards
of the supplementation during the early feeding period (Fig. 3). BW was
significantly higher in T2 compared to other feeding groups at Day 5
especially T0 and T4 (Fw (5,172) = 4.255, P = 0.002; Posthoc: T0: P =
0.004; T4: P = 0.008). At Day 10, T4 was significantly lower (Fw (5,174)
= 3.897, P = 0.003) especially with T0 (P = 0.026), and T5 (P = 0.023).
Interestingly, the BW growth were similar among treatments at Day 15
and Day 20 (Day 15: F (5,169) = 1.217, P = 0.303; Day 20: F (1,174) =
1.437, P = 0.213). At Day 25, the BW of T2 and T5 feeding groups (P =
0.716) were significantly higher than the others (F (5,174) = 4.918, P <
0.001). At Day 30 the BW of T0 and T1 were significantly lower than the
rest of the feeding groups (Fw (5,174) = 3.270, P = 0.010; Posthoc T0: P
= 0.029, T1: P = 0.027).
No significant difference in TL was observed among feeding groups
at Day 5 (F (5,170) = 0.938, P = 0.458). At Day 10, the TL in T0 and T5
were significantly higher than the rest especially T1 and T4 (F (5,97) =
2.388, P = 0.043; Posthoc: T1: P = 0.012; T4: 0.015). Similar No dif­
ference was found at Day 15 and Day 20 (Day 15: F (5,96) = 1.329, P =
0.258; Day 20: F(5,109) = 0.567, P = 0.725). At Day 25 T1 was
significantly lower compared to T2 and T5 (F (5,148) = 3.717, P =
0.003; Posthoc: T2: P = 0.019; T5: P = 0.002). Similar pattern was
demonstrated at Day 30 (Fw (5,137) = 3.202, P = 0.012; Posthoc: T2: P
= 0.024; T5: P = 0.007).
Correlation between the BW and M. citrifolia fruit extract was only
detected at Day 30 culture period (F (1,4) = 73.793, P = 0.001, r2 =
0.949) (Supplementantary Fig. S1) whereas TL were not correlated with
extract throughout the 30-day feeding trial.
3.2. Analysis of growth performances
Specific Growth Rate (SGR) were increasing with the culture dura­
tion and concentration of M. citrifolia fruit extract supplementation
(Fig. 4a) with slight inconsistency during the early feeding period with
T1 exhibited the highest SGR among all groups. At Day 5, significant
difference in SGR was observed among lower supplemented groups (Fw
(5,172) = 6.129, P < 0.001). T3 was found significantly higher in SGR
than the rest of the feeding groups at Day 15, Day 20 and Day 25 (Day
15: F (5,168) = 8.732, P < 0.001; Day 20: F (5,174) = 8.907, P < 0.001;
Day 25: F (5,174) = 10.335, P < 0.001). At Day 30, significantly lower
SGR was detected in T0 compared to all supplementation groups (F
(5,174) = 11.566, P < 0.001).
Fig. 3. Growth changes in terms of (a) Body Weight and (b) Total Length changes of P. vannamei during supplemented feeding trials with M. citrifolia fruit extract.
Significant differences in growth changes is indicated by italic letters. Identical letters signifies no significant differences between groups.
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Aquaculture Reports 21 (2021) 100798
As for Average Daily Growth (ADG) (Fig. 4b), T2 was significantly
higher especially to T0 and T4 at Day 5 (Fw (5,172) = 4.255, P = 0.002;
Posthoc: T0: P = 0.004; T4: P = 0.008). At Day 10, the ADG in T4 was
significantly lower than other feeding groups (Fw (5,174) = 3.897, P =
0.003). No significant difference was found at Day 15 and Day 20 (Day
15: F (5,168) = 1.229, P = 0.298; Day 20: F (5,174) = 1.437, P = 0.213).
T5 was then found to be significantly higher than the rest at Day 25 and
Day 30 (Day 25: F (5,174) = 4.918, P < 0.001; Day 30: Fw (5,174) =
3.270, P = 0.010).
The FCR was equal among treatments from Day 5 to Day 20 feeding
trial (Day 5: F (5,171) = 2.320, P = 0.045; Day 10: F (5,174) = 1.915, P
= 0.094; Day 15: Fw (5,168) = 2.633, P = 0.030; Day 20: F (5,174) =
1.201, P = 0.311) (Fig. 4c). The FCR in T5 was significantly lower than
the rest of the groups at Day 25 and Day 30 (Day 25: Fw (5,174) = 5.363,
P < 0.001; Day 30: Fw (5,174) = 3.386, P = 0.008).
The condition factor (CF) of the shrimp was presented as Body
Weight (BW) expressed as a percentage of the cube of the Total Length
(TL) of shrimp (Fig. 4d). At Day 5, significant difference was found be­
tween T1 and T4 (Fw (5,172) = 3.009, P = 0.015; Posthoc: P = 0.021). At
Day 10, T1 was significantly higher in CF compared to the rest of the
feeding groups especially T0, T3 and T5 (Fw (5,174) = 5.185, P < 0.001;
Posthoc: T0: P = 0.001; T3: P = 0.017; T5: P = 0.010). At Day 15, T0 was
significantly higher than the others especially T1, T3 and T4 (Fw (5,168)
= 4.455, P = 0.001; Posthoc: T1: P = 0.023; T3: P = 0.001; T4: P =
0.005). At Day 20, T1 was found significantly higher than T0 and T4 (F
(5,174) = 2.851, P = 0.017; Posthoc: T0: P = 0.048; T4: P = 0.009). T1
also significantly higher than T2, T3 and T4 at Day 25 (F (5,174) =
4.200, P = 0.001; Posthoc: T2: P = 0.006; T3: P = 0.001; T4: P = 0.009).
No significant difference in CF was found among all groups at Day 30 (Fw
(5,174) = 1.368, P = 0.245).
Correlation between growth performances of P. vannamei PL and the
M. citrifolia fruit extract supplementation was only detected at Day 30
for SGR, ADG and FCR (SGR: F (1,4) = 15.858, P = 0.016, r2 = 0.799;
ADG: F (1,4) = 73.793, P = 0.001, r2 = 0.949; FCR: F (1,4) = 18.139, P =
0.013, r2 = 0.819). All growth performances were strongly correlated
with BW changes of P. vannamei PL (P < 0.001) (Supplementary Fig. S2).
3.3. Digestive enzyme activities
The activities of amylase in P. vannamei PL showed apparent incre­
ment with increasing culture period as well as increasing concentration
of M. citrifolia fruit extract supplementation (Fig. 5a). At Day 5, signif­
icant difference was divided into two groups where lower feeding groups
(T0, T1 and T2) were significantly lower than higher feeding groups (T3,
T4 and T5) (Fw (5,30) = 6269.927, P < 0.001). Amylase activities in T5
was significantly higher at Day 10, Day15 and Day 20 feeding trial (Day
10: Fw (5,30) = 257.115, P < 0.001; Day 15: Fw (5,30) = 438.018, P <
0.001; Day 20: Fw (5,30) = 176.967, P < 0.001). At Day 25, T0 and T1
J.H.Z. Moh et al. Aquaculture Reports 21 (2021) 100798

Fig. 4. Growth performance of P. vannamei PL in terms of (a) specific growth rate, (b) average daily growth, (c) feed conversion ratio and (d) condition factor during
the 30 days feeding trials with M. citrifolia fruit extract. Significant differences is indicated by italic letters. Identical letters signifies no significant differences in the
growth performance between groups.

Fig. 5. Activities of (a) amylase, (b) lipase and (c) trypsin in P. vannamei PL during 30 days feeding trial with M. citrifolia fruit extract supplementation. Significant
difference in the enzyme activities between groups were represented by italic letters. Identical letters signifies no significant difference between the groups.

were significant lower in amylase than the rest of the groups (Fw (5,30) 155.601, P < 0.001).
= 1072.350, P < 0.001). There was no significant difference in amylase The lipase activities showed inconsistency in the activities in
between T0 and T1 (P = 0.419). At Day 30, the amylase activities in T5 P. vannamei PL throughout the whole feeding period (Fig. 5b). The
was then significantly higher than the rest of the groups (Fw (5,30) = ANOVA results on lipase activities also showed significant difference

6
J.H.Z. Moh et al.
throughout the 30 days feeding trial. At Day 5, significant difference in
lipase activities was detected among all groups except T5 that appear to
be similar to the rest of the groups (Fw (5,24) = 35.728, P < 0.001). T2
were significantly higher than the rest of the feeding groups at Day 10
and Day 15 feeding period (Day 10: Fw (5,24) = 22.249, P < 0.001; Day
15: Fw (5,24) = 173.690, P < 0.001). At Day 20, T3 and T4 were sig­
nificant higher than the rest (Fw (5,24) = 258.273, P < 0.001). At Day 25
and Day 30 on the other hand, T1 was significantly higher than the rest
of the groups (Fw (5,24) = 563.790, P < 0.001).
Similar to that of lipase, trypsin activities were appeared inconsistent
throughout the feeding trial (Fig. 5c) and significant difference in the
activities was also detected throughout the 30- days feeding trial. At Day
5, trypsin activities in T4 was significant higher (Fw (5,36) = 5.727, P =
0.004) especially with T0 (P = 0.005), T1 (P = 0.008) and T3 (P =
0.008). T4 was still significantly higher at Day 10 especially with T1 (Fw
(5,36) = 8.589, P < 0.001; Posthoc: P = 0.048). At Day 15, T5 was
significant higher than the rest of the groups (Fw (5,36) = 1263.788, P <
0.001). T4 was significantly lower at Day 20 (Fw (5,36) = 361.869, P <
0.001). T1 then showed significantly higher trypsin activities at Day 25
(Fw (5,36) = 705.653, P < 0.001) and Day 30 (Fw (5,36) = 65.488, P <
0.001).
Amylase was the only digestive enzyme that showed correlation with
extract supplementation at Day 5 (F (1,4) = 23.721, P = 0.008, r2 =
0.856), Day 10 (F (1,4) = 118.748, P < 0.001, r2 = 0.967), Day 15 (F
(1,5) = 25.555, P = 0.007, r2 = 0.865) and Day 20 (F (1,4) = 28.733, P =
0.006, r2 = 0.878). No correlation was found at Day 25 (F (1,4) = 2.582,
P = 0.183) and Day 30 (F (1,4) = 6.253, P = 0.067). No correlation was
found between the extract supplementation and both lipase and trypsin
(P > 0.05). The absence of the correlation indicated that the activities of
the enzymes were not influenced by the supplementation of the fruit
extract. This may be due to the inconsistency in lipase activities in
P. vannamei PL of all groups throughout the feeding trial causing no
direct relationship were formed between the enzymes activities and the
extract supplementation.
Strong correlation was established between both amylase and lipase
and BW changes of P. vannamei PL throughout the 30-day feeding trial
Fig. 6. Activities and concentrations of (a) CAT (b) GSH (c) SOD and (d) TBARS in the hepatopancreas of P. vannamei post larvae during the 30 days feeding trial
with M. citrifolia fruit extract supplementation. Significant difference were indicated with italic letters where identical letters signifies no significant differences in the
antioxidant activities and concentrations between groups.
7
Aquaculture Reports 21 (2021) 100798
(Correl: Amylase: F (1,34) = 33.862, P < 0.001, r2 = 0.499; Lipase: F
(1,34) = 7.007, P = 0.012, r2 = 0.171) (Supplementary Fig. 3).
3.4. Antioxidant enzymes activities
The ANOVA results of the antioxidant activities and concentrations
throughout the 30 days feeding period with M.citrifolia fruit extract
supplementation was as shown in Fig. 6. From the result, the antioxidant
enzymes activities increased with increasing culture period.
According to the analysis, significant difference in catalase (CAT)
among all feeding groups at all culture period was found with T5
demonstrating the highest activities (Day 5: F (5,36) = 377.612, P <
0.001; Day 10: F (5,36) = 964.974, P < 0.001; Day 15: Fw (5,36) =
1271.763, P < 0.001; Day 20: Fw (5,36) = 5359.046, P < 0.001; Day 25:
F (5,36) = 2239.557, P < 0.001; Day 30: F (5,36) = 7137.737, P <
0.001) (Fig. 6a).
Similar results were found in glutathione (GSH) analysis (Day 5: F
(5,36) = 19205.097, P < 0.001; Day 10: F (5,36) = 9222.278, P < 0.001;
Day 15: Fw = (5,36) = 29893.985, P < 0.001; Day 20: Fw (5,36) =
42449.246, P < 0.001; Day 25: F (5,36) = 25952.638, P < 0.001; Day
30: Fw (5,36) = 29708.176, P < 0.001) (Fig. 6b), superoxide dismutase
(SOD) (Day 5: Fw (5,36) = 10099.483, P < 0.001; Day 10: Fw (5,36) =
7575.009, P < 0.001; Day 15: Fw (5,36) = 18151.365, P < 0.001; Day
20: Fw (5,36) = 39549.294, P < 0.001; Day 25: F (5,36) = 40512.418, P
< 0.001; Day 30: Fw (5,36) = 25773.59, P < 0.001) (Fig. 6c) and thio­
barbituric acid (TBARS) (Day 5: Fw (5,36) = 6043.343, P < 0.001; Day
10: Fw (5,36) = 3622.564, P < 0.001; Day 15: Fw (5,36) = 1688.952, P <
0.001; Day 20: F (5,36) = 583.691, P < 0.001; Day 25: Fw (5,36) =
163.601, P < 0.001; Day 30: Fw (5,36) = 725.622, P < 0.001 (Fig. 6d).
Strong correlation were established between antioxidant enzymes
(catalase, GSH, SOD and TBARS) and the concentration of M. citrifolia
fruit extract supplemented (P < 0.05). Strong relationships were also
found between all antioxidant enzymes and shrimp BW changes (P <
0.001). (Supplemmentary Fig. 4).
J.H.Z. Moh et al.
3.5. Histopathology study of P. vannamei PL hepatopancreas
Evident hepatopancreas tissue changes during M. citrifolia fruit
extract were observed including the appearance of hypertrophies of the
tubule cells (which means the swelling of the cell) in the supplementa­
tion groups during the early feeding period. Abnormal hepatopancreas
tubule lumen shape (narrowing and expansion) and tubule atrophies
(shrinking of tubule) were also observed in the supplementation groups.
Haemocytic infiltration was observed in some groups throughout the 30-
days feeding period. The selected examples of the tissue changes in the
hepatoapancreas in P. vannamei PL from the supplementation groups is
as shown in Fig. 7. The histopathology images of the hepatopancreas of
P. vannamei PL at different feeding period is as referred in Supplemen­
tary Figs. S5–S10.
The results from the histology image showed no morphological dif­
ferences in the control group (T0) at Day 5 when compared to Day
0 culture period whereas the supplementation groups (T1-T5) showed
swelling (hypertrophies) in the tubules. The appearance of abnormal
lumen shape was observed in supplementation groups at Day 5 culture
period. These occurrences appeared to be reduced as the feeding period
increases although they seemed to be emerged at random at different
culture period. For example, the abnormal lumen shape were observed
in T4 and T5 feeding groups at Day 10. At Day 15 on the other hand, this
appearance was found in T1 and T2 feeding groups and in T5 at Day 20.
The supplementation groups also showed signs of atrophy in the tubules
from Day 5 to Day 15 with more evident appearance in T4 and T5. The
occurrence then reduced as the feeding period increased. Throughout
the feeding period, the supplementation groups showed faint signs of
haemocyte infiltration.
3.6. Analysis of hepatopancreas cells
The average number of hepatopancreatic cells including R cells, B
cells and E cells presented in the hepatopancreas of P. vannamei PL
during the 30 days feeding trial with M. citrifolia fruit extract was as
shown in Fig. 8. R cells showed increased in number with culture period
and the concentration of M. citrifolia fruit extract supplemented
(Fig. 8a). At Day 5, the number of R cells in T0 were significantly lower
than the supplementation groups (Fw (5,36) = 28.885, P < 0.001)
especially T2 (P = 0.014), T4 (P < 0.001) and T5 (P = 0.027). At Day 10,
T5 was significantly higher than the rest especially T0 and T1 (Fw (5,18)
= 13.398, P = 0.001; Posthoc: T0: P = 0.030; T1: P = 0.008). At Day 15
and Day 20, T4 and T5 were significantly higher than the lower feeding
groups especially T0 (Day 15: Fw (5,36) = 7.823, P = 0.001; Day 20: Fw
(5,66) = 15.907, P < 0.001). Significantly higher R cells number in T5
was found at Day 25 especially with T0, T1 and T2 (Fw (5,30) = 11.967,
P < 0.001; Posthoc: T0: P = 0.018; T1: P = 0.003; T2: P < 0.001). At Day
30, T4 and T5 were significantly higher than the rest especially T0 and
Fig. 7. An evident changes in the hepatopancreas tubule found in shrimps of the supplementation groups with M. citrifolia fruit extract (200x magnification). The
changes include cell hypertrophies (CH), tubule atrophies (TA), abnormal shape of lumen (ALU) and haemocytic infiltration (HI).
8
Aquaculture Reports 21 (2021) 100798
T1 (Fw (5,66) = 15.013, P < 0.001).
As for B cells, the average number too were increasing with culture
period and the extract supplementation (Fig. 8b). At Day 5, the average
number of B cells in T0 were significantly lower than the supplemen­
tation groups (F (5,66) = 17.183, P < 0.001). The results were similar
for Day 10 and Day 15 where T4 and T5 were significantly higher in B
cells number than T0 (Day 10: F (5,24) = 3.335, P = 0.020; Day 15: F
(5,42) = 5.376, P = 0.001). Similar pattern was observed at Day 25 and
Day 30 (Day 25: F (5,84) = 15.875, P < 0.001); Day 30: Fw (5,84) =
19.679, P < 0.001). T5 was significantly higher at Day 20 especially
with T0 (Fw (5,72) = 27.848, P < 0.001; Posthoc: P = 0.014).
As for E cells, no significant difference were found at Day 5, Day 10
and Day 15 culture period (Day 5: F (5,12) = 1.693, P = 0.211; Day 10: F
(5,24) = 1.641, P = 0.187; Day 15: F (5,42) = 2.223, P = 0.094)
(Fig. 8c). Highest number of E cells was then found in T5 at Day 20, Day
25 and Day 30 (Day 20: F (5,66) = 10.420, P < 0.001; Day 25: F (5,84) =
26.487, P < 0.001; Day 30: F (5,84) = 14.371, P < 0.001).
In the correlation analysis, strong relationships were established
between the extract concentration and all hepatopancreatic cell number
(P < 0.05). Regression results also revealed strong correlation between
all hepatopancreatic cells and P. vannamei PL growth (Correl: R cells: F
(1,34) = 82.417, P < 0.001, r2 = 0.708; B cells: F (1,34) = 85.707, P <
0.001, r2 = 0.716; E cells: F (1,34) = 84.713, P < 0.001, r2 = 0.714)
(Supplementary Fig. S11).
4. Discussion
4.1. Administration of the extract through feed incorporation
Some of the common means of plant extract administration into
aquaculture animals include oral, injection and immersion depending
on the animal size and type, extract type and culture system. The latter
two were reported to be more challenging and labour intensive (Gabriel,
2019) and Syahidah et al. (2015) explained that those approaches often
contribute to stress towards younger and smaller animals compared to
oral. Awad and Awaad (2017) further explained that immersion method
was more tedious for it involved in preparation of large quantity of bath
water. Thus, oral administration of the extract through feed supple­
mentation was chosen in this study as it minimised the risk of stress to
the animal and is suitable for organisms (Selvaraj et al., 2005; Galin­
do-Villegas and Hosokawa, 2004). This approach was also reported to
have effectively improved the bodily function of aquaculture organims
in terms of phagocytic acitivites and bacterial removal (Yoshida et al.,
1995).
4.2. Growth performance of P. vannamei PL
The result showed increased in P. vannamei PL growth in proportion
J.H.Z. Moh et al.
Fig. 8. Graphs show the average number of hepatopancreatic cells namely (a) R cells, (b) B cells and (c) E cells present in each tubule of P. vannamei PL hepato­
pancreas during feeding supplementation with M. citrifolia fruit extract. Significant difference is indicated by italic letters. Similar letters signifies no significant
difference in the average number of hepatopancreatic cells between groups.
with the concentration of M. citrifolia fruit extract supplemented
throughout the feeding trial. Similar pattern were also observed for
ADG. The results in this study was in support of many previous findings
reported the potential of various plant extracts in improving the growth
of shrimp (Munaeni et al., 2019; Sankar et al., 2011; Liu et al., 2009) and
fish species (Kareem et al., 2016; Fallahpour et al., 2014; Punitha et al.,
2008). These evidents indicated that the supplementation of shrimp
with plant extracts or beneficial nutrients often resulted in better
growth.
From the results, the growth performances of P. vannamei PL
increased with M. citrifolia fruit extract supplementation throughout the
feeding trial period. Specific growth rate determined the growth of
shrimp in terms of weight per day and is important during the early
developmental stage of the animal due to their exponential growth
during this time (Lugert et al., 2014). At the end of the culture period,
the SGR of P. vannamei PL recorded were higher in higher supplemen­
tation groups. However, the influence of M. citrifolia fruit extract sup­
plementation on the SGR was difficult to determine due to the
inconsistency in the SGR data during the earlier culture period. Never­
theless, previous studies explained that SGR was highly affected by
external factors such as stocking density (Anand et al., 2019; Moss and
Moss, 2004) and oxygen supply (Susilowati et al., 2014). Therefore, the
limiting space and oxygen supply may be the limiting factor in the
current study that caused the irregularity in SGR in P. vannamei PL as
they grow in which they may need longer period for growth and adap­
tation. The ADG in the same way showed inconsistency however, but
were higher in higher supplemented groups compared to that of control
group. Diet supplementation was proved to increase ADG of P. vannamei
in previous studies (Zhou et al., 2007).
The feed conversion ratio determination is important to measure the
efficacy of an animal to covert feed into meat (Fry et al., 2018). Despite
the occurrence of inconsistency in FCR during the early culture period,
the results then demonstrated decreased in FCR with increasing con­
centration of extract supplemented towards the end of the feeding trial.
This result is in support of the past studies that reported lower FCR in
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Aquaculture Reports 21 (2021) 100798
shrimps and freshwater prawns in different supplementations (El-D­
esouky et al., 2012; Sankar et al., 2011; Liu et al., 2009; Zhou et al.,
2007). While the average FCR for shrimp in culture system was between
1.1–3.0 (Tacon and Metian, 2008), the FCR of P. vannamei PL
throughout the feeding trial in this study was between 0.836− 4.335.
The occurrence of FCR lower than 1.0 is known to be common at the
present day due to the modern diet formula (Torrissen et al., 2011). The
decreased in FCR signified that the animal required lesser food intake to
be converted into meat. This then improved the efficacy of the animal
body system in converting food into meat (Fry et al., 2018) Reduced FCR
thus resulted in reduced waste in the culture (Fourooghifard et al.,
2017).
Similar to that of FCR, the CF decreased with increasing culture
period. Despite the inconsistency in CF during the early culture period,
the CF in control groups appeared to be lower than that of supplemented
groups. However, the correlation results revealed that the CF were not
influenced by extract supplementation. Some of the factors that poten­
tially caused such occurrence include sex, sizes, environmental condi­
tions and the degree of gonad development (Froese, 2006). The study
conducted by Andem et al. (2013) strongly suggested that the CF of
prawn was sex dependant. Previous studies also revealed similar out­
comes in fish species (Fallahpour et al., 2014), prawn (El-Desouky et al.,
2012) and P. vannamei (Samadi et al., 2016). The weight-length rela­
tionship in aquatic animals also depends on both the environmental and
the animals condition themselves. This is because as the aquatic animals
grow, their body weight and body may change due to the changes of
their body dimension depending on seasonal variations (Mazumder
et al., 2016).
The inconsistency of P. vannamei PL growth during the early feeding
period observed in this study may be due to the feeding behaviour of the
animal during the early culture. The addition of M. citrifolia fruit extract
may have caused slow feeding in some of the shrimps for the additional
of the extract may had masked the feed odour which resulted in reduced
feeding. The reduction in feeding thus reduced the body mass of the
shrimp during culture (deVries et al., 2015). The shrimps were
J.H.Z. Moh et al.
cannabilistic during the early growing period therefore, they tend to
prey on each other especially during molting period where weaker in­
dividuals were being eaten (Harpaz, 1997). Hence, the uneven sizes of
shrimps during the early feeding period. Some other factors as explained
by previous studies that caused uneven growth include genetic differ­
ences and sexual dimorphism (Cheng and Chen, 1990). Nevertheless,
Widanarni et al. (2019) then reported that the increased in the growth of
shrimp were associated to the improved growth performances in terms
of SGR, ADG and the FCR. Therefore, the result in this study revealed
that the supplementation of P. vannamei PL with M. citrifolia fruit extract
potentially improved the growth performance of the shrimp thus,
enhanced their growth during the 30 days culture period.
4.3. Digestive enzyme activities
The improvement of growth performance of P. vannamei PL in this
study may be associated to the improved of digestive enzymes induced
by M. citrifolia fruit extract. Previous studies proved that the application
of plant extract improved growth through improvement the digestive
system (Sankar et al., 2011). Other studies also discussed that presence
of active compounds in plant extract stimulated the secretion of diges­
tive enzymes, resulted in improved appetite, food consumption and
digestion in animals hence enhanced their growth performances (Sha­
bana et al., 2019; El-Desouky et al., 2012; Harikrishnan et al., 2012; Nya
and Austin, 2009). The increased in growth performaces is also associ­
ated to the therapeutic effect of the extract as anti-pathogenic and re­
duces stress in the animal (Tan et al., 2017).
Amylase showed increasing activities in P. vannamei PL with
increasing concentration of M. citrifolia fruit extract. Similar results were
reported previously with improved amylase activities in shrimps
(Akbary et al., 2017) and fish species (Zahran et al., 2014) of supple­
mented groups. The increased preference of shrimps towards carbohy­
drates were reported as they grow (Gamboa-delgado et al., 2003). Hence
the increased production of amylase as the growth of P. vannamei PL
increases during the feeding trial. The results also showed that lipase
and trypsin activities did not correlate with extract supplementation.
This outcome inferred that the activities of lipase and trypsin may not be
influenced by the extract supplementation and growth of the shrimp.
Such occurrence was also reported in previous study explaining that the
lipase activities were not affected by the supplementation with micro­
bial lysozyme (Javahery et al., 2019). Lipase activity was reported to be
region specific depending on the organisms. For example, higher activity
was reported in the anterior gut of milkfish, cod, gilthead seabream and
red porgy fish, whereas that of turbot was found in the anterior gut re­
gion (Izquierdo et al., 2000). Therefore, in this study, there is a possi­
bility that lipase was distributed at different region of P. vannamei PL of
the digestive tract instead of the hepatopancreas which resulted in the
inconsistency of its activity. Lipid are stored as energy reserved in small
amount in crustaceans with only 8% of the total lipids being stored at
triacyglycerides (Asaikkutti et al., 2016). This may explain low lipase
activities in P. vannamei PL throughout the feeding trial period.
The changes in protein digestion is associated to the life stage de­
velopments such as maturation (Sunde et al., 2004) and moulting (Klein
et al., 1996; Van Wormhoudt et al., 1995). Therefore, due to uneven
moulting and cannibalism during early culture period, trypsin activities
among shrimps may differ causing inconsistency of trypsin activities.
Similar to lipase, trypsin may be secreted at different region. According
to previous findings, higher trypsin activity was observed in the stomach
and duodenum of tilapia compared to liver during supplementation with
aloe vera (Gabriel et al., 2017). Since the hepatopancreas is analogous to
the liver, trypsin may have been highly distributed in stomach or the
instestine of P. vannamei PL instead of the hepatopancreas. The corre­
lation results showed that trypsin activity was not influenced by the
growth of P. vannamei PL during the 30 days culture period. According
to previous study, bigger shrimps do not utilise protein as effectively as
smaller shrimps (Gamboa-delgado et al., 2003). Therefore, along with
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Aquaculture Reports 21 (2021) 100798
the irregularity in growth among P. vannamei PL during the feeding trial,
the differences in protein preferences among the shrimps may occur in
the culture system.
4.4. Antioxidant enzyme activities
The overall results showed increased in activities of antioxidant
enzyme with increasing concentration of M. citrifolia fruit extract during
the feeding trial period. While the role of antioxidant enzymes in shrimp
growth has not been fully discovered, the purpose of the measurement of
antioxidant enzymes activities was often to determine the capacity of
the defense mechanism of an organism associated to oxidative stress (Liu
et al., 2011). The results in this study is in par with the results reported
previously where plant extract supplementation were proven to have
improved antioxidative mechanism in P. vannamei (Liu et al., 2011).
The increased in CAT activities showed that the extract of the fruit of
M. citrifolia potentially improved the health of P. vannamei PL during
supplementation. Similar results were reported in previous studies on
both shrimp (Pacheco et al., 2011), and fish species (Amer et al., 2018;
Giannenas et al., 2012). Similar results were also observed for GSH, SOD
and TBARS. GSH activity was described previously to be dose dependant
in which the activity increases with the increase of supplement con­
centration (Amer et al., 2018; Dandapat et al., 2000). These results may
explain the increasing glutathione activities in P. vannamei PL with
increasing M. citrifolia fruit extract concentration supplemented in cur­
rent study. The increased GSH in P. vannamei was also reported previ­
ously to be responsible in increasing the activities of gluthatione
peroxidase (GPx) and SOD, two of the most important antioxidant en­
zymes (Xu et al., 2012). The correlation results of the current study is
similar to previous studies done by Amer et al. (2018) and Giannenas
et al. (2012) which concluded that the growth of animal is in direct
correlation to the activity of antioxidant enzymes. TBARS activity
determination was done to measure the MDA production. MDA is a
secondary production from lipid peroxidation are used to determine the
oxidative stress level of an organism (Liu et al., 2011). The capability of
lipid recovery by the hepatopancreas was due to its high lipid content
(Takeungwongtrakul et al., 2012). The increased in MDA level indicated
increased in lipid content in the hepatopancreas. However, the results
obtained for TBARS activities in the result of this study may not be useful
for the process of lipid peroxidation and its secondary production take
place only during storage (Shi et al., 2017; Papastergiadis et al., 2012;
Takeungwongtrakul et al., 2012).
Although not being conducted in the current study, the antioxidant
enzymes activities were reported to be influenced by several factors such
as pH and temperature (Arun et al., 2003), sex (Mourente and Día­
z-Salvago, 1999) and species (Mourente and Díaz-Salvago, 1999).
Antioxidant enzymes in shrimps are known to be released in response to
stress due to escalation of radicals in the cells. These radicals are usually
being scavanged to protect the cell from oxidative damages. This
response is recognised as an adaptive mechanism to ensure their survival
(Li et al., 2008). An increase in antioxidant enzymes in crustaceans is
also an indication of their adaptation whenever there were changes in
their habitat condition, even at low pollution condition (Tavar­
es-Sánchez et al., 2004). Therefore, the increase in antioxidant enzymes
activities indicated increased defense mechanism in shrimp. In this
study, the introduction of M. citrifolia fruit extract may have caused
changes in the natural condition of the shrimp and the culture envi­
ronment. The increased in antioxidant enzymes activities may also
indicate that M. citrifolia fruit extract supplementation became a stress
detection inducer to P. vannamei PL. The immune response may
increased shrimp resistance towards environmental stress such as tem­
perature, dissolved oxygen, salinity and pH fluctuation which therefore
did not affect the healthy growth of shrimps throughout the culture
period.
The potential of M. citrifolia fruit extract in ensuring healthy growth
of P. vannamei was mainly due to the presence of potential bioactive
J.H.Z. Moh et al.
compounds. Although this aspect has not been widely discussed, the
bioactive analysis reported from previous studies concluded M. citrifolia
fruit contained excellent sources of antioxidant and polyphenols
including alkaloids, flavonoids, tannins and anthocyanins with potential
anti-radical activities towards stress factors such as pathogen (Sina et al.,
2021). Strong antioxidant potential in M. citrifolia fruit was also reported
previously due to the presence of potential immunostimulant bioactive
component such as hexanoic acid, octanoic acid, n-Decanoic acid,
n-hexadecanoic acid and may more as discussed by Rivera et al. (2012).
4.5. Histopathology on the Hepatopancreas of P. vannamei PL
The histology images of the hepatopancreas of P. vannamei PL during
the early feeding period with M. citrifolia showed atrophies in the tu­
bules accompanied by the appearance by the appearance of desqua­
mation of B cells with more evident occurrence in shrimp of higher
supplementation groups. This occurrence was acknowledged as part of a
normal mechanism for epithelial cell renewal where the damaged cells
are compensated by the mitotic activity of E cells (Boudet et al., 2015).
However, there may be possibilities that the extract supplementation
that cause stress during the early feeding period that caused the onset of
necrosis (Saptiani et al., 2017). The hepatopancreas have the ability of
adaptation towards short term stress factor which may clarify the
appearance of swollen tubules, changes in lumen shapes and damaged
cells that were gradually being restored as the culture period increased
(Díaz et al., 2010). The possible stress that the supplementation of
M. citrifolia fruit extract as foreign substance may have prompted
hepatopancreas tubule and cell damage during the early culture period
in which required the tissue function for cell repair. This may have also
been the cause of inconsistency in the growth performance of
P. vannamei PL during the early culture period.
The appearance of haemocytes infiltrating the hepatopancreas tissue
was observed in the histology image of the hepatopancreas. These were
recognised to be circulating haemocytes and were known to be involved
in cellular defence especially towards pathogens (Yogeeswaran et al.,
2012). Circulating haemocytes were reported to be play a crucial role in
immune responses in many crustaceans (Lv et al., 2014; Ding et al.,
2012; Yogeeswaran et al., 2012) and mollusc species (Troncone et al.,
2015; Pipe, 1990). Haemoctyes involved in the innate immune systems
in invertebrates due to their nature in which they lack adaptive immune
system for effective removal of harmful material (Matozzo and Marin,
2010). According to another study, these circulating haemocytes were
also involved in the foreign particle and metabolic debris removal
through phagocytosis (Factor and Beekeman, 1990). The engagement of
haemocytes in immune responses were then responsible to the reactive
oxygen intermediates production (Troncone et al., 2015). The produc­
tion of haemocytes were also described to be associated to the digestive
system in which increased haemocytes in the hepatopancreas were re­
ported to have higher level of digestive enzymes which in turns
increased the resistance of the animal towards pathogen (Wang et al.,
1999). In the current study, higher digestive and antioxidant enzymes
activities were recorded in shrimp of the supplemented groups, hence
the appearance of haemocytic infiltration in these groups.
4.6. Hepatopancreatic cells analysis
The nutritional condition of crustaceans are determined by the
presence of R cells in the hepatopancreas, R cells are known as storage
cells in which nutrients are stored in lipid and/or glycogen droplets
(Wang et al., 2014). The results showed increasing in R cells number
with increasing concentration of M. citrifolia fruit extract supplemented
throughout the feeding trial period. Previous studies revealed that
nutrient reserved in R cells is important for the physiology of decapods
during starvation, moulting and reproduction (Ribeiro, 2016). There­
fore, the increase in the number of R cells in the hepatopancreas indi­
cated increased in lipid/glycogen storage which then lead to improved
11
Aquaculture Reports 21 (2021) 100798
growth in P. vannamei PL during feeding trial period with M. citrifolia
fruit extract supplementation. B cells are the most abundant hepato­
pancreas cells developed from F cells (Silva et al., 2018). These cells are
highly vacuolated, each at different functional stages making them to
appear at different sizes and shapes (Silva et al., 2018; Ribeiro et al.,
2016). B cells involved in intracellular digestion and nutrient absorption
through protein and digestive enzymes synthesis (Ribeiro et al., 2016).
Nutrients are stored in the vacuoles before being released with the
remaining enzymes into the lumen as well as for R cell reabsorption (Hu
and Leung, 2007). Due to these evidence, the results from the current
study may revealed that the supplementation of P. vannamei PL with
M. citrifolia fruit extract potentially improved the conversion of F cells to
B cells where the increased in B cells then led to improved intracellular
digestion and nutrient absorption and thus, improved the growth of the
animal. E cells are actively dividing cells and are important for cell
renewal for tubular growth and optimum health of the hepatopancreas
(Silva et al., 2018; Franceschini-Vicentini et al., 2009). The increased in
the number of E cells in this study indicated the increased in mitotic
division of the cell (Bhavan and Geraldine, 2000), hence showed that the
supplementation the shrimp with M. citrifolia fruit extract improved cell
renewal and growth of the hepatopancreas of P. vannamei PL.
5. Conclusions
The feeding supplementation of P. vannamei PL M. citrifolia fruit
extract had proven to have improved their growth performances in
terms of body weight, total length, average daily growth, feed conver­
sion ratio and condition factor despite the inconsistency in the values
during the early feeding period. The relationships between the digestive
enzymes activities and the growth of the shrimp showed that the
improved in the digestive function of the shrimp indirectly improved
their growth. Similarly, the enhanced antioxidant enzymes activities
indicated improved in their immune response, thus increased their
resistance towards stress allowing a healthy growth of P. vannamei PL
during culture. In terms of histopathology, the supplementation of
shrimp with M. citrifolia fruit extract improved the number hep­
atopancreatic cells (R, B & E cells) which also aided in improved growth
of the shrimp throughout the feeding trial period. The appearance of cell
atrophies, abnormal change in lumen shape and haemocytic infiltration
were also associated to the effect of the extract supplementation that
stimulate the enhancement of cell renewal and cell defense against stress
factors.
Author statement
Julia Hwei Zhong Moh: Conceptualization, methodology, investi­
gation, formal analysis, writing – original draft.
Mhd Ikhwanuddin: Conceptualization, supervision, validation,
writing – review & editing.
Khor Waiho: Writing – Review & editing, formal analysis,
validation.
Hanafiah Fazhan: Writing – review & editing, validation.
Noorbaiduri Shaibani: Methodology, Investigation.
Hidayah Manan: Writing – review & editing, validation.
Yeong Yik Sung: Supervision, validation, writing – review &
editing.
Hongyu Ma: Validation.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
J.H.Z. Moh et al.
Acknowledgements
This study is a part of the Higher Institution Centre of Excellence
(HICoE) Program by the Ministry of Higher Education, Malaysia awar­
ded to the Institute of Tropical Aquaculture and Fisheries (AKUATROP),
Universiti Malaysia Terengganu (Vot No. 56046).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.aqrep.2021.100798.
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