Aquaculture Reports 24 (2022) 101114

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Aquaculture Reports
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Dietary treatment of Nile tilapia (Oreochromis niloticus) with aquatic fern

(Azolla caroliniana) improves growth performance, immunological
response, and disease resistance against Streptococcus agalactiae cultured in
bio-floc system
Chompunut Lumsangkul a, Nguyen Vu Linh a, Fapailin Chaiwan b, Mohsen Abdel-Tawwab c,
Mahmoud A.O. Dawood d, e, Caterina Faggio f, Sanchai Jaturasitha g, *, Hien Van Doan a, *
a
Department of Animal and Aquatic Sciences, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand
b
Department of Plant and Soil Science, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand
c
Department of Fish Biology and Ecology, Central Laboratory for Aquaculture Research, Agriculture Research Center, Abbassa, Abo-Hammad, Sharqia, Egypt
d
Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, Egypt
e
The Center for Applied Research on the Environment and Sustainability, The American University in Cairo, Cairo 11835, Egypt
f
Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Italy
g
Science and Technology Research Institute, Chiang Mai University, 239 Huay Keaw Rd., Suthep, Muang, Chiang Mai 50200, Thailand

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

Keywords: The present study was performed to examine the effects of aquatic fern (Azolla caroliniana) (AQF) on growth
Aquatic fern performance, skin mucus and serum immunities, and disease resistance of Nile tilapia reared in a biofloc system.
Nile tilapia Three hundred Nile tilapia fingerlings (average 10.48 ± 0.56 g fish-1) were distributed into 15 glass tanks (150
Growth rate
liters tank− 1) at a concentration of 20 fish tank− 1. A completely randomized design was used in triplicates,
Innate immunity
Disease resistance
where the control group was fed 0 g kg− 1 AQF (AQF1), while AQF2, AQF3, AQF4, and AQF5 were fed 25, 50,
100, and 200 g kg− 1 AQF, respectively. Growth and immunological specimens were collected every 4 weeks,
while disease resistance was conducted after 8 weeks post-feeding. The results showed that dietary adminis­
tration of AQF significantly promoted the growth rate and feed efficiency of Nile tilapia, with the maximum level
noticed in fish fed 100 g kg− 1 AQF. However, no significant differences were observed between the AQF1 and
AQF5 diets. Similarly, incorporation of AQF resulted in significantly higher skin mucus and serum immunities
than the control, except for AQF5. The highest values were observed in fish fed the AQF5 diet. The challenge test
showed that in contrast to the control (26.67%), the AQF administrated diets led to significantly higher sur­
vivability by 60% (AQF2), 66.67% (AQF3), 83.33% (AQF4), and 53.33 for AQF5. The relative percent survival
(RPS) was 45.45%, 54.55%, 77.27%, and 36.36% in AQF2, AQF3, AQF4, and AQF5, respectively. The highest
RPS against S. agalactiae was noted in the AQF4 diet. In conclusion, dietary supplementation of 100 g kg− 1 AQF
can be potentially used as a growth promoter and immunostimulant in Nile tilapia aquaculture.

1. Introduction
Aquaculture is being increased as a major food supplier to ensure its
sustainability for future generations (Ahmad et al., 2021; Osmundsen
et al., 2020; Reverter et al., 2020). However, under extensive farming
circumstances, the production and profit of raising aquatic organisms
are insufficient to have a feasible aquaculture activity Nguyen et al.,
2021). Therefore, unconventional technologies are required to increase
and intensify production using available resources (Hisano et al., 2021).
The recirculating Aquaculture System (RAS) has been employed in
aquaculture practice for many years (Holan et al., 2020). Nonetheless,
energy consumption, greenhouse gases, and the high cost of treating and
purifying the effluent are major drawbacks to expanding this technol­
ogy, particularly for tilapias (Ahmed and Turchini, 2021; Wambua et al.,
2020). Additionally, the farming intensive and recirculating system
techniques have stressfully impaired the performance of aquatic

* Corresponding authors.
E-mail addresses: ja.sanchai@gmail.com (S. Jaturasitha), hien.d@cmu.ac.th (H. Van Doan).

https://doi.org/10.1016/j.aqrep.2022.101114
Received 3 March 2022; Received in revised form 28 March 2022; Accepted 2 April 2022
Available online 6 April 2022
2352-5134/© 2022 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/).
C. Lumsangkul et al. Aquaculture Reports 24 (2022) 101114

organisms (Liu et al., 2020; Romano and Sinha, 2020). In this sense, Table 1
bio-floc technology (BFT) is practiced in several countries to recycle the The formulation and proximate composition of the experiment (g kg− 1).
water in the farming units and utilize the nitrogen emissions produced Ingredients Diets (g kg¡1)
from fish feces to form microbial flocs (Castro et al., 2021). Under these
AQF1 AQF2 AQF3 AQF4 AQF5
circumstances, aquaculture units can operate with a low water exchange
rate (less than 10%) and less artificial aquafeed consumption (Liu et al., Fish meal 150 150 150 150 150
Corn meal 200 200 200 200 200
2021). BFT is successfully applied to farm several aquatic organisms, Soybean meal 390 385 379 368 347
including tilapia, carp, and shrimp (Kim et al., 2020; Xu et al., 2021). Wheat flour 70 70 70 70 70
Nile tilapia (Oreochromis niloticus) is a well-documented candidate for Rice bran 150 135 121 87 13
BFT technology, owing to its omnivorous feeding behavior, fast growth, AQFa 0 25 50 100 200
Cellulose 20 15 10 5 0
tolerance to stressful conditions, and high market demand (Elayaraja
Soybean oil 5 5 5 5 5
et al., 2020; Sarsangi Aliabad et al., 2022; Suárez-Puerto et al., 2021). Premix b
10 10 10 10 10
Therefore, the development of sustainable methods for tilapia farms is Vitamin Cc 5 5 5 5 5
an urgent issue. Proximate composition of the experimental diets (g kg¡1 dry matter basis)
Aquatic fern, or Azolla, is a water-floating weed that belongs to the Crude protein 302.29 306.71 309.73 307.25 306.41
Crude lipid 55.39 57.92 58.49 56.14 55.37
Azollaceae family and exists in tropical and subtropical regions world­ Fiber 56.49 58.78 59.68 57.34 59.07
wide (Farahpour-Haghani et al., 2017; Maity and Patra, 2008; Prabina Ash 91.29 93.23 94.45 92.70 95.34
and Kumar, 2010). Azolla is composed of eight species, one of which, Dry matter 913.15 905.67 907.89 914.55 916.69
Azolla caroliniana, has a high nutritional value. Azolla is capable of ni­ GE (cal/g)d 3987 3821 3798 3861 3976
trifying both water and atmospheric nitrogen as well as producing a
AQF = Aquatic fern.
valuable protein in their leaves (Azab and Soror, 2020; Brouwer et al., b
Vitaminand trace mineral mix supplemented as follows (IU kg-1 or g kg-
2014; Kollah et al., 2016). There are a variety of nutrients in Azolla 1diet): retinyl acetate 1,085,000 IU; cholecalciferol 217,000 IU; D, L-α-toco­
meals, such as proteins, vital amino acids, minerals, and vitamins (Azhar pherolacetate 0.5 g; thiamin nitrate 0.5 g; pyridoxine hydrochloride 0.5 g; niacin
et al., 2018; Mosha, 2018). In addition, it has antioxidant and immu­ 3g; folic 0.05 g; cyanocobalamin 10 g; Ca pantothenate 1 g kg-1; inositol 0.5 g;
nostimulant roles attributed to its phenol content, carotenoids, flavo­ zinc 1 g; copper 0.25 g; manganese 1.32 g; iodine 0.05 g; sodium 7.85 g.
c
Vitamin C 98% 5 g.
noids, and tannins (Brouwer et al., 2018; Hassan et al., 2020; Tran et al., d
GE: gross energy.
2020). Thus, many trials have been conducted to replace plant protein
ingredients partially or totally with Azolla meals without affecting fish
performance (Magouz et al., 2020; Mosha, 2018; Mosha et al., 2020).
Recent research indicates that including 20–25% Azolla meal into fish 2.2. Experimental animal and cultured water conditions
diets has no negative effects on growth, feed efficiency, immune
response, or health status (Magouz et al., 2020). However, the effects of Three hundred healthy Nile tilapia were purchased from a local farm
dietary Azolla as a functional feed supplement for Nile tilapia raised in (Chiang Mai, Thailand) and adapted to experimental conditions by
the bio-floc system have not been investigated. Therefore, this study receiving a basal diet twice daily for 14 d before being fed the experi­
aimed to investigate whether dietary treatments of Nile tilapia with mental diets. Experimental fish were kept in the 120-liter glass tanks.
different doses of AQF diets can improve growth performance, immu­ Prior to performing the trials, 10 fish were subjected to bacterial and
nological response, as well as disease resistance against Streptococcus parasitic examinations to verify their health. Temperature, pH, and
agalactiae. dissolved oxygen (DO) levels in the water were measured and kept
within acceptable values for Nile tilapia under the experimental settings.
2. Materials and methods Water parameters were determined using a HI98196 meter (Hanna
Romania), which was in the range of temperature (28 ± 0.85◦C), pH
2.1. Preparation of diets (5.92 ± 0.24), and DO (5 mg/L). The protocol for animal usage was
approved by Chiang Mai University (Approval No. 2562/AQ-0004).
Aquatic fern was freshly provided by the Department of Plant and
Soil Science, Faculty of Agriculture, Chiang Mai University. Aquatic fern
2.3. Bio-floc water preparation
(AQF) was dried and powdered. The powder was stored at 4 ◦ C after
sifting using a 100 µm mesh screen. The AQF levels used in this study
Bio-floc inoculants were prepared 3 weeks before starting the
were modified from the previous study (Fiogbé et al., 2004). The
experiment by adding wheat flour (2 g), salt (400 g), dolomite (5 g), and
following quantities of AQF powder were added to the basal diet to
molasse (5 g). The ratio of C:N (15:1) was maintained by daily adding
produce the experimental dietary treatments: 25 (AQF2), 50 (AQF3),
molasses (40% C). It was determined schematically using the residual
100 (AQF4), 200 (AQF5), and 0 g kg− 1 as the control (Table 1). The feed
nitrogen content of each tank and the diet contribution (Cardona et al.,
materials were crushed to a fine powder and completely blended with
2016). Molasses and probiotics (PondPlus, Bayer) were added to main­
soybean oil and water to form a dough. Then, feed pellets were produced
tain bio-floc concentrations at 9.45 ± 0.71 mL, while NH3 concentra­
using a pellet extruder and dehydrated at 50 ◦ C to a moisture level of
tions remained at 0.13 ± 0.02 mg/L.
10%. The pellets were then kept at 4 ◦ C until needed.
Proximate compositions of the diets were analyzed using AOAC
(1995), where dry matter (DM) was determined via oven-drying at 2.4. Experimental design and challenge test
95–100 ◦ C for 5 h (method 934.01). Ash was determined following the
complete combustion of samples in a muffle furnace at 600 ◦ C for 2 h 2.4.1. Experimental design
(method 942.05). Crude protein (CP) content was determined via the Two weeks after acclimation, 300 mono sex Nile tilapia (10.48 ±
macro-Kjeldahl technique (method 2001.11) and calculated as nitrogen 0.56) were randomly distributed into 5 feeding treatment groups: AQF2
× 6.25. Ether extract (EE) was extracted via Soxhlet extraction for 16 h (25 g kg− 1), AQF3 (50 g kg− 1), AQF4 (100 g kg− 1), AQF5 (200 g kg− 1),
using dichloromethane as a solvent (method 920.39). CF (crude fat) was and AQF1 (0 g kg− 1) as the control. The experimental treatment was
determined following method 962.09, and gross energy (GE) was scaled performed in triplication, with 20 fish per tank. The feeding trials were
using an adiabatic bomb calorimeter (CAL2k oxygen bomb calorimeter, conducted for 60 days. Daily feedings occurred at 8:30 a.m. and 4:30 p.
Digital Data Systems, South Africa). m.

2
C. Lumsangkul et al.
2.4.2. Challenge test
Streptococcus agalactiae (S. agalactiae) strain ANS1 was kindly pro­
vided by Dr. Worawit Maneepitaksanti (Department of Animal and
Aquatic Sciences, Faculty of Agriculture, Chiang Mai University,
Thailand). Detailed preparation of S. agalactiae was described in the
previous study by Van Doan et al. (2018). Briefly, S. agalactiae was
cultured in Tryptic Soy Broth and incubated at 30 ºC for 24 h in the
rotation shaker at a speed of 110 rpm. The sub-culture was obtained
from the stock. Then, 5 mL of the stock solution was transferred into a
50 mL flask containing Tryptic Soy Broth and incubated at 30 ºC for 24 h.
The sub-cultures were raised in duplicate under similar conditions for
the experiment. Growth was evaluated by the optical density of 560 nm
(0.75% NaCl was used to adjust bacterium concentration) and then by
plate counting in Tryptic Soy Agar. The calibration curves, relating
optical density (OD) at 560 nm with plate counts, were collected by
measuring the OD of consecutive one-half dilution series in triplicate
each, before determining the cell density by classic plate count methods
(107 CFU mL− 1 of S. agalactiae = 0.8465 OD + 1.6187, r2 = 0.91).
Eight weeks post-feeding, ten fish from each tank were randomly
retrieved for testing. The fish were intraperitoneally injected with 0.1
mL of 0.85% saline solution containing 107 CFU mL− 1 of S. agalactiae
(Wang et al., 2016). The clinical signs and lesions of disease were
observed, and dead fish were removed daily. We computed the tilapia’s
mortality rates, in percentages, for each treatment, 15 days after the
challenge; as well as the relative percentage of survival (RPS), through
the following equation of Amend (1981): RPS = [1 – (% mortality in
challenge/% mortality in control)] × 100.
2.5. Growth performance analysis
To investigate the productive growth and feed utilization in Oreo­
chromis niloticus supplemented with various concentrations of AQF after
4- and 8-week feeding, the growth parameters were calculated using the
following equations:
Weight gain (WG) = final weight (g) – initial weight (g)
Specific growth rate (SGR %) = 100 x (final weight - initial weight)/total
duration of experiment
Feed conversion ratio (FCR) = feed given (dried weight)/weight gain (wet
weight)
Survival rate (%) = (final fish number/initial fish number) x 100
2.6. Innate immune response analysis
2.6.1. Sample collection
Serum was collected from blood as protocol described in Van Doan
et al. (2019). Briefly, blood samples were collected at caudal vein of
three fish per tank using the 1-mL syringe. The individual collected
blood sample was immediately transferred into 1.5 Eppendorf tube, and
allowed to clot at room temperature for 1 h, then at 4 ◦ C for 4 h. After
incubation the serum from each fish was collected and kept at − 80 ◦ C
until further use. The serum from individual fish was used to analyze
serum immune parameters.
Leukocyte were isolated from blood using Chung and Secombes
(1988), schedule with some modifications as mentioned in Van Doan
et al. (2019). Briefly, 1 mL of blood sample from each fish was taken via
caudal vein and immediately mixed with 2 mL of RPMI 1640 (Gibthai) in
a 15 mL Eppendorf tube. The mixture was carefully transferred on the
top of 3 mL of Histopaque (Sigma, St. Louis, MO, USA) in a 15-mL tube.
The tub was then centrifuged at 400 g, for 30 min at room temperature.
After the centrifugation, a white buffy coat of leukocytes present on the
top of the Histopaque was collected, aspirated using a Pasteur pipette,
3
Aquaculture Reports 24 (2022) 101114
and transferred into a clean 15-mL tube. The obtained leukocytes
washed three times with phosphate buffer solution (PBS) for removing
residual Histopaque, and the required cell number was prepared for the
phagocytosis and respiratory burst assays.
Mucus collected from fish skin (3 fish per tank) was followed the
previous method of Subramanian et al. (2007), with modifications
stated in Van Doan et al. (2019). Briefly, fish were anaesthetized using
clove oil at concentration of 5 mL litre− 1. They were then placed into
polyethylene bag containing 10 mL of 50 mM NaCl (Merck, Germany),
and gently rubbed inside the plastic in a downward motion for
approximately 1 min. Th mucus was immediately poured into 15 mL
sterile centrifuge tubes, and centrifuged 1.500g for 10 min at 4 ◦ C. Then
1 mL of obtained mucus was kept in 1.5 mL Eppendorf tube at − 80 ◦ C
until further use.
2.6.2. Activity of lysozyme in serum and skin mucus
Serum and mucus lysozyme levels were determined in accordance
with method Parry et al. (1965) with some variations. Briefly, 25 μL of
undiluted serum and 100 μL of skin mucus from each fish were loaded
into 96-well plates in triplication. Micrococcus lysodeikticus (175 μL,
0.3 mg mL− 1 in 0.1 M citrate phosphate buffer, pH 5.8) was then added
to each well. The contents were rapidly mixed, and any changes in
turbidity were measured every 30 s, for 5 min, at 540 nm, 25 ◦ C, via a
microplate reader. The sample’s equivalent unit of activity was deter­
mined and compared with the standard curve, which was generated
from the reduction of OD value vs. the concentration of hen egg-white
lysozyme ranging from 0 to 20 μL mL− 1 (Sigma Aldrich, USA), and
expressed as μg mL− 1 serum.
2.6.3. Activity of peroxidase in serum and skin mucus
Serum and skin mucus peroxidase levels were detected using pro­
tocol of Quade and Roth (1997), with slight modifications. Briefly, 5 μL
of serum or skin mucus was loaded into 96-well flat-bottom plates in
triplicate. 45 μL of Hank’s balanced salt solution (HBSS) without Ca2+ or
Mg2+ was added. After that, 100 μL of solution containing of 40 mL of
distilled water, 10 μL of H2O2 (30% - Sigma Aldrich), 1 pill of 3,3′ ,5,
5′ -tetramethylbenzidine (TMB; Sigma Aldrich) was loaded into each
well. Later, 50 μL of 2 M H2SO4 was added to stop the color-change
reaction, and the optical density was read at 450 nm in a plate reader.
Standard samples without serum or skin mucus were used as blanks. One
unit was defined as the amount producing an absorbance change of 1
and the activity was expressed as units (U) mg− 1 serum or mucus
proteins.
2.6.4. Phagocytic activity
Phagocytic assay was determined according to the protocols as pre­
viously described (Yoshida and Kitao, 1991). Briefly, 200 μL of leuko­
cyte suspension (2 × 106 cells mL− 1) was added to the cover slip and
incubated for 2 h, followed by washing with RPMI 1640 and incubating
with 200 μL of fluorescence latex beads (2 × 107 beads mL− 1) for 1.5 h at
room temperature. The cover slips were washed and fixed with 100%
methanol and stained with Diff-Quick staining dye (Sigma Aldrich -
USA) for 10 s. Excessive stain was washed with PBS (pH 7.4), and the
number of phagocyte cells per 300 adhered cells was counted micro­
scopically. The phagocytic index was calculated as follows: PI = Average
number of beads per cell/phagocytizing cell numbers.
2.6.5. Respiratory assay
Blood leukocytes respiratory burst activity was determined as the
method of Secomebs (1990), with slight modification. Briefly, 175 μL
leukocytes solution containing of 6 × 106 cells mL− 1 in PBS loaded in the
96-well plate in triplicate. Later, 25 μL of Nitro Blue Tetrazolium (NBT)
at a concentration of 1 mg mL− 1 was placed into each well and incubated
at RT for 2 h. After incubation, the supernatant in each well was care­
fully discarded and 125 μL of 100% methanol was added to fix the
adherent cells for 5 min. The supernatant was discarded again and 125
C. Lumsangkul et al. Aquaculture Reports 24 (2022) 101114

μL of 70% methanol was added to each well for washing. After washing, Table 2
the plate was allowed to dry at room temperature for 30 min 125 μL of 2 Growth indices and feed utilization (mean ± SE) on Nile tilapia 4- and 8-week
N KOH and 150 μL of DMSO were then added to each well. After adding post-feeding.
the solution, the plate was measured at 655 nm by a microplate reader. AQF1 AQF2 AQF3 AQF4 AQF5
Spontaneous O2– production = (Absorbance NBT reduction of sample) IW (g) 10.52 ± 10.47 ± 10.47 ± 10.48 ± 10.48 ±
− (Absorbance of blank). 0.47 0.43 0.17 0.26 0.08
FW (g)
2.6.6. Alternative complement pathway activity (ACH50) 4 32.07 ± 33.52 ± 33.90 ± 35.97 ± 31.98 ±
weeks 1.60b 1.15ab 0.26ab 0.78a 0.50b
ACH50 was determined using method of Yano (1992), with several
8 84.12 ± 94.82 ± 99.23 ± 108.67 ± 84.28 ±
changes. Briefly, rabbit red blood cells (R-RBC) were washed with PBS weeks 2.69c 3.12b 0.79b 1.48a 0.20c
by centrifugation at 3000 rpm, and in 0.01 M ethylene glycol tetra-acetic WG (g)
acid-magnesium-gelatin veronal buffer (0.01 M – EGTA-Mg-GVB) for 4 21.55 ± 23.05 ± 23.43 ± 25.48 ± 21.50 ±
twice. The R-RBC concentration was adjusted to 2 × 108 cells mL− 1 in weeks 1.13b 0.57ab 0.15ab 0.53a 0.46b
8 73.60 ± 84.36 ± 88.76 ± 98.19 ± 73.79 ±
0.01 M – EGTA-Mg-GVB buffer. Then 100 μL of the R-RBC suspension weeks 2.27c 2.87b 0.64b 1.23a 0.12c
was lysed with 3.4 mL of distilled water. The absorbance of hemolysate SGR (%)
was measured at 414 nm against distilled water as a blank and was 4 3.71 ± 3.88 ± 3.92 ± 4.11 ± 3.72 ±
brought to be close to 0.740. weeks 0.02c 0.03b 0.03b 0.03a 0.04c
8 3.47 ± 3.68 ± 3.75 ± 3.89 ± 3.48 ±
For the ACH50 test, 100 μL of serum was diluted with 400 μL of 0.01
weeks 0.03c 0.03b 0.02b 0.02a 0.01c
M-EGTA-Mg-GVB and serial two-fold dilution was conducted. The tubes FCR
were performed on ice to retard the reaction of complement until all 4 1.18 ± 1.12 ± 1.11 ± 1.06 ± 1.16 ±
tubes were prepared. Consequently, 100 μL of R-RBC suspension was weeks 0.02a 0.001b 0.007c 0.01b 0.01a
loaded into each tube and incubated at 20 ◦ C for 1.5 h with occasional 8 1.31 ± 1.21 ± 1.20 ± 1.14 ± 1.28 ±
weeks 0.01a 0.01b 0.02b 0.01c 0.02a
shaking. After incubation, 3.15 mL of cold normal saline solution was
SR (%)
placed into each tube to stop the reaction, and then the tube was 4 100 ± 100 ± 0.00 100 ± 0.00 100 ± 0.00 100 ±
centrifuged at 1600 g for 5 min. After centrifugation, 100 μL of super­ weeks 0.00 0.00
natant in each dilution was loaded into 96-well plate and read at 414 8 93.33 ± 96.67 ± 96.67 ± 98.33 ± 96.67 ±
weeks 1.44 1.44 1.44 1.44 1.77
nm. The degree of hemolysis was calculated by dividing the corrected
absorbance 414 value by the corrected absorbance 414 of the 100% Note: AQF1, Control; AQF2, 25 g kg− 1 diet, AQF3, 50 g kg− 1 diet, AQF4, 100 g
hemolysis control. The degree of hemolysis and the serum volume were kg− 1 diet, and AQF5, 200 g kg− 1 diet, IW, Initial weight; FW, Final weight; WG,
plotted on a log-log paper. The volume of serum that gave 50% hemo­ Weight gain; SGR, Specific growth rate; FCR, Feed conversion ratio; SR, Survival
lysis was used for calculating the ACH50 using the formula: ACH50 rate, Different letter in a row denotes significant difference (P < 0.05).
(units/mL) = 1/K x r x ½. Where K is the amount of serum giving 50%
hemolysis, r is the reciprocal of the serum dilution, and ½ is the with the best values, were detected in the AQF4 diet. However, there
correction factor. The assay was performed on a ½ scale of the original were no variations (P > 0.05) found in fish fed AQF2 and AQF3, as well
method. as AQF5 and the control (Table 3).

2.7. Statistical analysis 3.3. Analysis of serum immune response

SAS software was used to perform all statistical analyses (SAS,
Version, 2003). The Kolmogorov-Smirnov Test was used to determine
the normality of the data. One-way analysis of variance (ANOVA) and
Duncan’s multiple range test were used to evaluate significant differ­
ences in growth performance parameters and immunological activities
at level of 95% (P < 0.05). In the challenge experiment, the
Kaplan–Meier was used to assess survival rates, and the log-rank test was
used to examine treatment group differences.
3. Results
3.1. Analysis of growth performance
Fish gave administrated diets resulted in better (P < 0.05) specific
growth rate (SGR), weight gain (WG), final weight (FW) as opposed to
the control diet (Table 2). The highest SGR and WG values were
observed in the AQF4 supplemented diet. However, no significant dif­
ferences (P > 0.05) in these parameters were noticed in fish fed the
AQF2, AQF3, and AQF5 diets. The lowest FCR value was viewed (P <
0.05) in fish fed AQF4 diet as compared to other groups, while the
highest (P < 0.05) FCR values were observed in the AQF1 and AQF5
diets. Conversely, there were no differences in survival rates between
the control and administrated groups (P > 0.05).
3.2. Analysis of mucosal immune response
After 8 weeks of feeding, supplementations of AQF led to higher (P <
0.05) lysozyme values in mucus and serum versus the control (Table 3),
Effects of AQF on serum immunities are displayed in Table 4. The
best serum lysozyme (SL) level was noticed in fish fed AQF4 as opposed
to AQF1, AQF2, AQF3, and AQF5 diets. Likewise, significant differences
in serum peroxidase (SP), alternative complement activity (ACH50),
phagocytosis index (PI), and respiratory burst activity (RB) were noticed
in fish fed AQF (P < 0.05) versus the control (Table 4). The highest
values were observed in fish fed the AQF4 diet (Table 4). Nevertheless,
no significant differences in serum immunities (P > 0.05) were noticed
in fish fed AQF2, AQF3, and AQF5 diets (Table 4). (Table 5).
3.4. Analysis of fish survival rate
The fish mortality was occurred earlier in the control group (day 4)
and lasted until day 8 post-challenge, while the mortality in AQF sup­
plemented diets was found on day 5 and last until day 7 post-challenge.
The relative percentage of survival (RPS) showed that in contrast to the
control (26.67%), the AQF administrated diets led to significantly (P <
0.05) higher survival rate by 60% (AQF2), 66.67% (AQF3), 83.33%
(AQF4), and 53.33 for AQF5 (Fig. 1). The relative percent survival (RPS)
was 45.45%, 54.55%, 77.27%, and 36.36% in AQF2, AQF3, AQF4, and
AQF5, respectively. The highest survival rate and RPS against
S. agalactiae were noted in fish fed AQF4 diet.
4. Discussion
Moving from traditional to non-traditional aquaculture systems re­
quires considerable efforts to maintain high production using available
resources (Craig, 2019; Dawood and Koshio, 2020). Remarkably, the

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C. Lumsangkul et al. Aquaculture Reports 24 (2022) 101114

Table 3
Skin mucus lysozyme and peroxidase activities on Nile tilapia 4- and 8-week post-feeding.
AQF1 AQF2 AQF3 AQF4 AQF5
c b b a
4 weeks SMLA 1.65 ± 0.11 2.79 ± 0.11 2.81 ± 0.07 3.59 ± 0.23 1.64 ± 0.04c
SMPA 0.08 ± 0.005c 0.11 ± 0.005b 0.12 ± 0.003b 0.16 ± 0.008a 0.08 ± 0.005c
8 weeks SMLA 2.85 ± 0.13c 3.95 ± 0.05b 3.89 ± 0.06b 4.81 ± 0.12a 2.92 ± 0.12c
SMPA 0.12 ± 0.008c 0.18 ± 0.005b 0.18 ± 0.02b 0.21 ± 0.008a 0.12 ± 0.01c

Note: SMLA (µg mL− 1), Skin mucus lysozyme activity, SMPA (µg mL− 1), Skin mucus peroxidase activity, Different letter in a row denotes significant difference (P <
0.05).

Table 4
Serum immunity on Nile tilapia 4- and 8-week post-feeding.
AQF1 AQF2 AQF3 AQF4 AQF5

4 weeks SL 4.38 ± 0.16c 5.57 ± 0.25b 5.58 ± 0.24b 6.85 ± 0.14a 5.08 ± 0.11b
SP 0.10 ± 0.008c 0.13 ± 0.005b 0.13 ± 0.008b 0.17 ± 0.008a 0.12 ± 0.003b
ACH50 120.71 ± 4.77c 139.21 ± 1.34b 141.74 ± 1.56b 152.79 ± 2.37a 127.15 ± 3.30c
PI 1.80 ± 0.06c 2.40 ± 0.12b 2.59 ± 0.07b 3.05 ± 0.03a 1.98 ± 0.05c
RB 0.06 ± 0.006c 0.08 ± 0.006b 0.10 ± 0.005b 0.13 ± 0.005a 0.08 ± 0.005b
8 weeks SL 6.17 ± 0.23c 7.60 ± 0.28b 7.91 ± 0.19b 8.87 ± 0.10a 7.37 ± 0.32b
SP 0.14 ± 0.01c 0.19 ± 0.005b 0.21 ± 0.02b 0.25 ± 0.008a 0.19 ± 0.005b
ACH50 141.34 ± 1.94c 157.37 ± 3.85b 157.07 ± 2.91b 174.18 ± 3.41a 143.41 ± 2.58c
PI 2.26 ± 0.10c 2.74 ± 0.08b 2.78 ± 0.08b 3.36 ± 0.08a 2.37 ± 0.09c
RB 0.11 ± 0.005c 0.14 ± 0.003b 0.15 ± 0.005b 0.18 ± 0.005a 0.14 ± 0.003b

Note: SL, Serum lysozyme activity (µg mL− 1); SP, Serum peroxidase activity (µg mL− 1); ACH50, Alternative complement activity (unit mL− 1); PI, Phagocytosis activity
(bead cell− 1); RB, Respiratory burst activity (OD655). Different letter in a row denotes significant difference (P < 0.05).

Table 5
Differences between investigated groups using log-rank test.
Group Significance

AQF1 AQF2 AQF3 AQF4

AQF2 0.003*
AQF3 0.001* 0.552ns
AQF4 0.000* 0.039* 0.128ns
AQF5 0.068ns 0.348ns 0.141ns 0.006*

Note: “*” denotes a statistically significant difference (P < 0.05)

“ns” means not significant

purity and availability of the rearing water and the optimum dietary
formulations are the main factors affecting the performances of aquatic
organisms (Ciji and Akhtar, 2020; Enayat Gholampour et al., 2020;
Harikrishnan et al., 2021; Meade, 2012; Rashidian et al., 2021; Sinha
et al., 2021; Vazirzadeh et al., 2020). The current investigation exam­
ined the possibility of including aquatic fern (AQF) as a sustainable feed
supplement in Nile tilapia diets reared in biofloc system (BFT), focusing
exclusively on the growth, skin mucus and serum immunities, and
tolerance against S. agalactiae infection.
The present study indicated that Nile tilapia fed 100 g kg− 1 Azolla
caroliniana (AQF) diet resulted in improvement of FW, WG, SGR, and
FCR. These results were similar to previous studies reported in carp,
Labeo fimbriatus (Gangadhar et al., 2015); Thai silver barb, Barbonymus
gonionotus (Das et al., 2018), and Nile tilapia, Oreochromis niloticus
(Magouz et al., 2020; Mosha et al., 2020). The significant increase in Fig. 1. Kaplan–Meier analysis of cumulative survival in Nile tilapia (n = 30)
challenged with S. agalactiae at day 21 post-vaccination. AQF1, Control; AQF2,
growth performance in fish that fed AQF diets may be attributable to the
25 g kg− 1 diet, AQF3, 50 g kg− 1 diet, AQF4, 100 g kg− 1 diet, and AQF5,
high content of essential amino acids and protein in aquatic ferns (Azhar
200 g kg− 1 diet.
et al., 2018; Das et al., 2018; Rashad, 2021). Furthermore, it could be
due to the effects of Azolla on gut villi and bacterial ecosystem as noticed
with more than 25% Azolla pinnata drastically inhibited the growth rate
by its effect on short-chain fatty acid levels (Abdelatty et al., 2020). In
of Thai silver barbs. It has been reported that high aquatic Azolla levels
addition, Azolla supplement was found to affect blood glucose (Anhita
reduce feed intake and compromise digestive enzyme activities in fish
et al., 2016), implying a likely increase in hepatic gluconeogenesis,
intestines due to anti-nutritional factors (Kamali-Sanzighi et al., 2019; Li
which is primarily regulated by adenosine monophosphate-activated
et al., 2012). Additionally, aquatic ferns contain a high concentration of
protein kinase that plays an important role in controlling hepatic
indigestible fiber and carbohydrates, and are frequently deficient in
gluconeogenesis (Li et al., 2020). Interestingly, a higher dose level of
certain essential amino acids, methionine, and lysine, when compared to
AQF diet (200 g kg− 1) led to decrease growth performance and feed
animal protein, which has a detrimental effect on feed absorption,
utilization. Similarly, Das et al. (2018) observed that dietary inclusion
digestion, and utilization, and may result in decreased growth

5
C. Lumsangkul et al.
performance when used in large quantities (Das et al., 2018).
There is a strong association between the balanced feeding formu­
lations and the immune defense of aquatic organisms (Ahmadifar et al.,
2019; Hoseinifar et al., 2020; Rashidian et al., 2020). Intestinal immu­
nity is the first immune line affected by feed quality (Dawood, 2021),
whereas skin mucus is the external line associated with the surrounding
environmental conditions (Pérez-Sánchez et al., 2017). The current
study evaluated the immune response of Nile tilapia fed dietary AQF
reared in bio-floc system in both serum and skin mucus. The results
provide more evidence about the internal effect of AQF on intestinal
immunity, which is linked to the body’s immune system (serum and skin
mucus immunities). Concurrent with the growth performance results,
the serum and skin mucus immunities showed enhanced responses in
fish fed 100 g kg− 1 AQF under bio-floc conditions. After eight weeks of
AQF feeding, skin mucus samples showed higher lysozyme (SMLA) and
peroxidase (SMPA) activities than the control group. Serum SL, SP,
ACH50, PI, and RB were higher in fish fed AQF than the control with the
highest value in fish provided a 100 g kg− 1 AQF diet. Fish fed
200 g kg− 1 AQF diet had similar serum immune responses with the
control, except for the SL, SP, and RB activities, which were higher in
fish provided 200 g kg− 1 AQF diet than the control. Similar results were
reported in convict cichlid, Amatitlania nigrofasciata (Hoseinifar et al.,
2020); common carp, Cyprinus carpio (Hoseinifar et al., 2021; Hosseini
et al., 2020); barramundi, Lates calcarifer (Chaklader et al., 2021); Nile
tilapia, O. niloticus (Mosha et al., 2020; Van Doan et al., 2021). These
results can be attributed to the role of lysine, vitamins, and
phyto-contents (carotenoids, flavonoids, and tannins), which act as
natural antioxidants and immunostimulants (Brouwer et al., 2019;
Thavasi Alagan et al., 2020). Moreover, the high content of carotene,
phenolic, and flavonoid components in Azolla has been proved for its
immune-potentiating impacts (Dhumal et al., 2009; Kumar et al., 2014;
Nayak and Padhy, 2017; Noor Nawaz et al., 2014). Additionally, dietary
inclusion of Azolla meal could increase short-chain fatty acid pro­
ductions (e.g., propionate, acetate, and butyrate) (Brouwer et al., 2019),
which in turn enhance immune response (Kim, 2021; Schulthess et al.,
2019).
Interestingly, the results showed increased resistance against
S. agalactiae infection in Nile tilapia fed dietary AQF. The increased
resistance against bacterial infection is in line with enhanced respiratory
burst activity, which indicates the activated immunity of fish fed dietary
AQF. Similarly, Mosha, Felix, Manikandavelu, Felix, TLS, Menaga
(2020) reported that Nile tilapia fed aquatic Azolla had high tolerance
against bacterial infection with Aeromonas hydrophila. The improved
survival rate after infection with S. agalactiae is probably attributed to
the antimicrobial effect of Azolla associated with its content of proline
and fucoxanthin (Kösesakal, Yıldız, 2019; Mosha et al., 2020). These
phytoconstituents have enzymatic functions that reduce platelet acti­
vation and aggregation against bacterial diseases and anti-inflammatory
activity (Biller-Takahashi et al., 2013; Yilmaz, 2019).
The addition of feed additives into bio-floc system, such as pre­
biotics, probiotics, and medicinal plants, has recently attracted much
attention. Dietary inclusion of AQF in bio-floc system generated higher
growth, immune response, and disease prevention. The findings were in
line with earlier studies in gibel carp (Carassius auratus gibelio) (Qiao
et al., 2020); Pacific white shrimp (Penaeus vannamei) (Hussain et al.,
2021), and Nile tilapia (Laice et al., 2021; Van Doan et al., 2021; Van
Doan et al., 2021). Previous studies have demonstrated that bio-floc
provides several essential nutrients for tilapia (Ekasari et al., 2014;
Green et al., 2019; Haridas et al., 2017; Mohammadi et al., 2021; Oli­
veira et al., 2021; Zaki et al., 2020), which in turn improves fish growth
and well-being. Azolla spp. are not only a good nutrient source but are
also act as ammonium absorption substance (Bhuvaneshwari and Singh,
2015; Brouwer et al., 2018).
6
Aquaculture Reports 24 (2022) 101114
5. Conclusions
In this study, Nile tilapia fed 100 g kg− 1 AQF resulted in better
growth performance, feed intake, skin mucus, serum immunity, and
disease resistance of Nile tilapia against S. agalactiae. The results suggest
that dietary aquatic fern (AQF) can be used as a non-traditional strategy
to produce a feasible diet for sustainable aquaculture.
CRediT authorship contribution statement
Chompunut Lumsangkul: Investigation. Nguyen Vu Linh: Data
curation and Writing - review & editing. Fapailin Chaiwan: Aquatic
fern preparation. Mohsen Abdel-Tawwab: Data computation. Mah­
moud A.O. Dawood: Writing – original draft. Caterina Faggio: Final
approval. Sanchai Jaturasitha: Resources. Hien Van Doan: Visuali­
zation, Writing – review & editing.
Declaration of Competing Interest
The authors declare that there is no conflict of interest.
Data availability statement
The datasets generated and/or analyzed during this investigation are
accessible upon reasonable request from the corresponding author.
Acknowledgments
This work was supported by the National Research Council of
Thailand (NRCT2562) and Chiang Mai University, Chiang Mai, Thailand
(CoE 2564). This research work was partially supported by Chiang Mai
University.
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