Accepted Manuscript

Changes in the growth, humoral and mucosal immune responses

following β-glucan and vitamin C administration in red sea bream,
Pagrus major

Mahmoud A.O. Dawood, Shunsuke Koshio, Mabrouk El-Sabagh,

Md Masum Billah, Amr I. Zaineldin, Mohamed Mamdouh Zayed,
Amira Alaa El-Dein Omar

PII: S0044-8486(16)31290-X
DOI: doi: 10.1016/j.aquaculture.2016.12.036
Reference: AQUA 632475
To appear in: aquaculture
Received date: 29 August 2016
Revised date: 27 December 2016
Accepted date: 28 December 2016

Please cite this article as: Mahmoud A.O. Dawood, Shunsuke Koshio, Mabrouk El-
Sabagh, Md Masum Billah, Amr I. Zaineldin, Mohamed Mamdouh Zayed, Amira Alaa
El-Dein Omar , Changes in the growth, humoral and mucosal immune responses following
β-glucan and vitamin C administration in red sea bream, Pagrus major. The address
for the corresponding author was captured as affiliation for all authors. Please check if
appropriate. Aqua(2016), doi: 10.1016/j.aquaculture.2016.12.036

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 ACCEPTED MANUSCRIPT

Changes in the growth, humoral and mucosal immune responses following β-glucan and
vitamin C administration in red sea bream, Pagrus major

Mahmoud A.O. Dawood a, d,*, Shunsuke Koshio b, Mabrouk El-Sabagh f, Md. Masum Billah b,c,
Amr I. Zaineldin a,e, Mohamed Mamdouh Zayed g, Amira Alaa El-Dein Omar h

a
Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University, 4-50-20,

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Kagoshima 890-0056, Japan

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b
The United Graduate School of Agriculture Sciences, Kagoshima University, 1-21-24

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Korimoto, Kagoshima 890-0056, Japan
c
Education and Research center for Marine Resources and Environment, Faculty of Fisheries,

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Kagoshima University, Kagoshima, Japan
d
Department of Animal Production, Faculty of Agriculture, Kafrelsheikh University, 33516,
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Kafrelsheikh, Egypt
e
Animal Health Research Institute(AHRI-DOKI), Egypt
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Department of Nutrition and clinical nutrition, Faculty of Veterinary Medicine, Kafrelsheikh

University, 33516, Kafrelsheikh, Egypt
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g
Department of Aquaculture, Faculty of Aquatic and Fisheries Sciences, Kafrelsheikh
University, 33516, Kafrelsheikh, Egypt
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h
Department of Fish Diseases and Management, Faculty of Veterinary Medicine Kafrelsheikh
University, 33516, Kafrelsheikh, Egypt
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Abstract: In order to study the effects of dietary β-glucan (BG), vitamin C (VC) and their
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combination (BG/VC) on growth, humoral and mucosal immune responses of red sea bream
(Pagrus major) and its resistance to low water salinity stress, 240 healthy fish with initial body
weight of 1.98±0.01g were randomly divided into four groups: a control group fed with basal
diet and three treated groups fed with basal diets supplemented with 1000 mg kg-1 BG, 800 mg
kg-1 VC, and the combination of 500 mg kg-1 BG + 400 mg kg-1 VC, respectively. The obtained
results showed that, compared with the control, the final body weight, weight gain and specific
growth rate, feed and protein efficiency ratio, body protein content, nitro blue tetrazolium
(NBT), total serum protein and tolerance against low salinity stress increased in fish fed BG
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or/and VC supplementations with the highest being in fish fed both BG and VC supplements
while body moisture content, plasma glucose, triglyceride and malondialdehyde (MDA) contents
decreased (P < 0.05); body lipid content, hematocrit, serum and mucus lysozyme activities,
superoxide dismutase (SOD), alternative complement pathway and mucus secretion also
significantly increased in fish fed BG and VC supplements (P < 0.05). Moreover, the fish fed BG
diet had better improvement (P < 0.05) in mucus bactericidal activity than the VC group.
Furthermore, fish fed BG diet exhibited higher serum peroxidase activity (PA), however, fish fed

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VC diet showed higher mucus PA than the control group (P < 0.05). Thus, the basal diet

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supplemented with BG or/and VC could promote the growth, antioxidant capacity, humoral and

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mucosal immune responses of red sea bream, and enhance the resistance to low salinity stress.

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Keywords: Red sea bream; β-glucan; Vitamin C; Growth; Immune responses; Mucus secretion;
Stress resistance
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1. Introduction
Red sea bream (Pagrus major) has become a commercially important fish species in aquaculture
in Japan and eastern Asia (Dawood et al., 2016a; El Basuini et al., 2016), and research related to
red sea bream within the last decade has mainly concentrated on nutrition requirements of this
species. However, information on the red sea bream immune response has rarely been reported,
although high-density aquaculture has led to excessive stress and has contributed to outbreaks of
bacterial infection that have caused high mortality in red sea bream aquaculture (Dawood et al.,

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2015c). Excessive stress suppresses the immune system and results in physiological dysfunction,

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increased probability of infection, and even death (Thompson et al., 1993). Therefore, efforts

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have been made to relieve stress and enhance immunity in aquatic animals using various
immunostimulants in the diet, including probiotics (Dawood et al., 2016a), prebiotics (Dawood

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and Koshio, 2016a), emodin (Ming et al., 2012), fucoidan (Yang et al., 2014), lactoferrin
(Eslamloo et al., 2012; Kakuta et al., 1996), β-glucan (Brogden et al., 2014) and vitamin C
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(Liang et al., 2015; Roosta et al., 2014). β-glucan (BG), a homopolymer of glucose, has been
isolated from a wide range of natural sources, including cell walls of bacteria, fungi, yeast and
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cereal grains, and used as a feed additive (Dalmo and Bøgwald, 2008). Dietary
cosupplementation with vitamin C (VC), which is defined as immunostimulant, is a potentially
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useful approach for providing feed additives to aquatic animals (Dawood et al., 2016c; Pezzato et
al., 2014; Verlhac et al., 1996). Despite the potential for synergistic effects, research into the use
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of BG and VC as feed additives in fish is limited.

BG has been shown to improve growth, immune response and resistance against certain
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pathogens by increasing the concentration of lysozyme and the complement system components
that enhance the phagocytic activity of macrophages in various fish species (Dobšíková et al.,
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2013; Kumari J, Sahoo, 2006; Li et al., 2009; Misra et al., 2006). Besides its role in the growth
performance and feed utilization for fish, dietary VC has been shown to stimulate antioxidant
capacity and immune responses such as macrophage activities, cell proliferation, natural killer
cell activity, complement and lysozyme levels (Ai et al., 2004; Azad et al., 2007; Chen et al.,
2015; Eo et al., 2008; Gao et al., 2013; Liu et al., 2011; Shahkar et al., 2015; Tewary and Patra,
2008).
The present study analyzed BG and VC as immunostimulants that could provide protection to
red sea bream from different stressors and disease outbreaks. Further, these compounds could be
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used by the aquaculture industry as a nutritional strategy for minimizing economic losses due to
the stress conditions under intensive culture systems. Until now, there are no studies on BG
or/and VC as functional feed additives for potential growth and health benefit of red sea bream.
Therefore, the current research was conducted to evaluate the comparative effects of BG or/and
VC as functional feed additives on growth performance, immune responses, skin mucus and
stress resistance of juvenile red sea bream.

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2. Materials and methods

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2.1 Experimental diets

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The basal diet formulation for red sea bream contained approximately 49.6% protein and 11.4%
lipids (Dawood et al., 2015a), and the proximate composition is shown in Table 1. After

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reviewing the dosages used in previous reports (Dawood et al., 2015b,c) and Gao et al. (2013),
the identified dosages were 500 or 1000 mg kg-1 for β-glucan (BG, Daigon do, Tokyo, Japan)
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containing 85% BG and 400 or 800 mg kg-1 for vitamin C (VC, L-Ascrobil-2-Phosphate-Mg).
Four experimental diets were formulated containing [0 mg BG or/and VC (Ctrl.), 1000 mg BG
(BG), 800 mg VC (VC), or both 500 mg BG and 400 mg VC (BG/VC) kg-1 diet] (Table 1). To
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prepare the diets, the ingredients were mixed together for 15 min in a food mixer, then soybean
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lecithin and pollack liver oil were slowly added and the feed was mixed for a further 15 min.
Distilled water was then slowly added to the diets and mixed for another 15 min to form a soft
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dough. The diets were pelleted in a laboratory pellet mill, through a (1.6–2.1 mm) diameter die.
Pellets were dried in an oven at 50 °C for 2 h, and then stored in a freezer in airtight bags until
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use.
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2.2. Experimental design

The experiment was conducted at the Kamoike Marine Production Laboratory, Faculty of
Fisheries, Kagoshima University, Japan. Juveniles of red sea bream (1.98±0.01g), were obtained
from Tawaki farm, Kumamoto, Japan and acclimatized to tank conditions for one week. Fish
health status was verified by normal colouration, the absence of cysts, spots or patches over the
body and gills and normal behavioral signs (swimming and feeding reflexes). After
acclimatization, the homogenous sized 20 fish per tank (3 tanks per treatment) were randomly
distributed in previously prepared 12 tanks of 100-L supplied with a flow through sea water
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system (1.5-2 L min−1) where each tank was equipped with an inlet, outlet, and continuous
aeration. The mean water quality parameters were as follows: temperature 25.2±1.5°C, dissolved
oxygen 6.1±0.5 mg L-1 and pH 7.1. The light–dark cycle was fixed at 12 h light and 12 h dark.
For 56 days, the fish were fed by hand, twice daily, until apparent visual satiation level.

2.3. Growth performance, feed utilization and survival

Growth performance, feed utilization and survival parameters were calculated as described by

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Dawood et al. (2015d) and according to the following formulae:

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Weight gain (WG, %) = (W0 – Wf) ×100/W0; specific growth rate (SGR, % day−1) = {(Ln (Wf) –

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Ln (W0)) / t (56 days)} ×100; survival (SUR, %) =100× (Nf/ N0)
Where W0 and Wf were initial and final body weight (g) of fish, respectively; t was duration of

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experiment in days; Nf and N0 were initial and final number of fish, respectively.
Feed efficiency ratio (FER) =live weight gain (g) /dry feed intake (g); protein efficiency ratio
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(PER) =live weight gain (g)/dry protein intake (g)
Where dry feed intake (FI, g fish−1 56 days−1)=(dry diet given−dry remaining diet recovered) /
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no. of fish
Condition factor (CF, %) = weight of fish (g)/ (length of fish)3 (cm)3×100
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2.4. Sample preparation

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At the end of the feeding trial, three fish from each tank were randomly selected and stored at −
20 °C for whole body analysis. Furthermore, five fish from each replicate tank were randomly
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selected and their surfaces were washed with distilled water individually, and skin mucus was
collected from body surface by using a small piece of sterilized cotton and immediately
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suspended in 1 ml of phosphate-buffered saline (PBS, pH=7.4). Sample was then put into a set of
two centrifugal tubes, the upper tube had a small filter (0.45 μm size), with which the mucus in
the cotton will be collected in the lower tube while centrifuged. The sets of the double-tube (1.5
ml centrifugal tube) were centrifuged at 3000×g for 5 min under 4 °C (MX-160, Tomy Seiko Co.,
LTD, Tokyo, Japan), and the supernatant in the under tube was transferred into another
centrifugal tube (510-GRD, QSP, San Diego, CA, USA) and kept at -80°C until the analysis.
Viscera and liver were dissected out from the fish above, weight individually to get
viscerasomatic index (VSI) and hepatosomatic index (HSI) using the following equations:
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VSI (%) =weight of viscera/weight of fish×100

HSI (%) =weight of liver /weight of fish×100
The fish were bled from the caudal vein using 1.0 mL heparinized syringes (1600 UI ml -1,
Nacalai Tesque, Kyoto, Japan) to collect blood from 5 fish in each replicate tank and pooled.
Partial heparinized whole blood was used to analyze the hematocrit and nitro blue tetrazolium
(NBT) activity while plasma was obtained by centrifugation at 3000×g for 15min under 4 °C.
Additionally, blood was collected using non-heparinized disposable syringes in order to collect

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serum. For serum isolation, non-heparinized disposable syringes were used to collect blood, then

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samples were left for 4 h at 4 °C, centrifuged at 3000×g for 15min under 4°C and the supernatant

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retained as serum. Plasma and serum samples were stored at −80 °C until analysis.

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2.5. Biochemical analysis
The diets and fish whole body were analyzed for moisture, crude protein, crude lipid and ash, in
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triplicate, using standard methods (AOAC, 1998). VC contents of test diets were determined
based on the method of Gao et al. (2013). Total serum protein and plasma chemical parameters
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including: glucose, total bilirubin, blood urea nitrogen, glutamyl oxaloacetic transaminase,
glutamic-pyruvate transaminase, triglycerides and total cholesterol were measured
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spectrophotometrically with an automated analyzer (SPOTCHEM™ EZ model SP-4430, Arkray,

Inc. Kyoto, Japan) according to Tatsumi et al. (2000).
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2.6. Immunological and antioxidant enzymes analysis

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Lysozyme, bactericidal and peroxidase activities in serum or mucus were determined as

described by Dawood et al. (2016a,b). Serum alternative complement pathway (ACP) was
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measured as described by Dawood et al. (2016a). The nitro blue tetrazolium (NBT) assay as
described by Anderson and Siwicki (1995) with some modifications by Kumari and Sahoo
(2005). Briefly, Blood (0.1 ml) was placed in microtiter plate wells, to which an equal amount of
0.2% NBT solution (Sigma, USA) was added and incubated for 30 min at room temperature. A
sample of NBT blood cell suspension (0.05 ml) was added to a glass tube containing 1m l N, N-
dimethylformamide (Sigma, USA) and centrifuged for 5 min at 3000 rpm. Finally, the optical
density of supernatant was measured at 540 nm. Dimethylformamide was used as the blank.
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Serum superoxide dismutase (SOD) activity was measured by the percentage reaction inhibition
rate of enzyme with WST (Water Soluble Tetrazolium dye) substrate and xanthine oxidase as
described by Dawood et al. (2016a). The catalase (CAT) enzyme activity was performed using
spectrophotometric determination of hydrogen peroxide (H2O2) which form stable complex with
ammonium molybdate (Goth, 1991). The serum (50 μl) was added to the 1.0 ml substrate (65
μmol per ml H2O2 in 60 mmol l-1 phosphate buffer, pH 7.4) and incubated for 60 s at 37 °C. The
enzymatic reaction was stopped with 1.0 ml of 32.4 mmol l-1 ammonium molybdate

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((NH4)6Mo7O24·4H2O). A yellow complex of ammonium molybdate and hydrogen peroxide was

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formed. The absorbance of this yellow colour was measured at 405 nm with a spectrophotometer

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(HACH, DR/4000U) against a blank (serum was replaced with distilled water). CAT activities
are expressed as kilo unit per liter. The malondialdehyde (MDA) concentration was used as a

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marker of lipid peroxidation in fish serum and measured using Colorimetric TBARS Microplate
Assay Kit (Oxford Biomedical Research, Inc., USA) according to the manufacturer's
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instructions. The absorbance was measured at 532 nm. The MDA level was expressed as nmol
per ml serum.
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2.7 Mucus secretion

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Amounts of secreted mucus were quantitated based on the method described by Kakuta et al.
(1996). Bovine serum albumin (Sigma Aldrich) was used as standard and the soluble protein
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concentration of mucus samples was determined following the method of Lowry et al. (1951).
The amounts of secreted mucus were expressed as mg of total protein per ml collected mucus.
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2.8 Low salinity stress test

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Low salinity stress test was conducted to determine the lethal time of 50% mortality (LT 50) in
low salinity water according to Dawood et al. (2015a). Total 15 fish per treatment (5 fish per
each tank) were randomly selected and transferred into a 20-L rectangular glass aquarium
equipped with continuous aeration and kept under ambient temperature during the stress test. The
city water was de-chlorinated by strongly aerating for at least 24 h and mixed with seawater
(50%), and then used as low salinity water. The passing of time to reach 50% death (LT 50) was
calculated according to Moe et al. (2004).
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2.9. Statistical analysis

All data were subjected to statistical verification using one-way ANOVA (Package Super
ANOVA 1.11, Abacus Concepts, Berkeley, California, USA). Probabilities of P < 0.05 were
considered significant. Significance differences between means were evaluated using the Tukey
Kramer test.

3. Results

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3.1 Growth, nutrient utilization, and survival

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The effects of BG or/and VC on the growth, nutrient utilization, and survival of red sea bream

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are shown in Table 2. Administration of BG or/and VC diets for 56 days resulted in significantly
higher final weight, weight gain (WG), and specific growth rate compared to values for the

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control group (P < 0.05; Table 2). Furthermore, the highest value of WG was found in diet group
BG/VC when compared with other groups, however, the value did not significantly differ from
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those in BG group (P > 0.05). Moreover, fish fed BG or/and VC showed higher significant feed
efficiency ratio (FER) and protein efficiency ratio (PER) values than the control group (P < 0.05;
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Table 2). However, no significant difference was observed between BG or VC groups (P > 0.05).
The highest values of FER and PER were obtained in fish fed the diet containing both BG and
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VC (BG/VC) (P < 0.05). Also, survival was not significantly affected by the dietary
supplementation. Fish fed BG or/and VC free diet (Ctrl.) showed significantly lower growth and
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feed utilization performances in the present study.

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3.2 Whole body proximate analysis and biometric parameters

The whole body proximate compositions of red sea bream are shown in Table 3. In comparison
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with the control group, dietary treatments had no significant influence on the ash content at the
end of the feeding trial. Fish fed VC or BG/VC supplemented diets showed decreased moisture
level significantly than the fish fed the control diet (P < 0.05), but the values did not significantly
differ from those in BG group. Dietary BG or/and VC supplements affected on whole body crude
protein content, the level of crude protein content in fish fed BG, VC, and BG/VC diets were
significantly higher than those in the control group (P < 0.05). Additionally, total lipid content
increased significantly in fish fed both BG and VC diet (BG/VC) when compared with the
control group (P < 0.05), however, no significant difference was observed between VC and
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BG/VC groups (P > 0.05). Whole body biometric parameters (CF, HSI, and VSI) were not
significantly influenced by the dietary BG or/and VC supplementations.

3.3. Hematocrit and blood chemical parameters

Fish fed BG and VC diet (BG/VC) showed a significant improved hematocrit level when
compared with fish fed VC supplemented diet (P < 0.05; Table 4), but the values did not
significantly differ from those in Ctrl. or BG groups. Test diets had no significant effect on blood

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chemical parameters of fish among different treatments, except for those of plasma glucose

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(GLU) and triglycerides (TG) (Table 4). The obtained results showed that, fish fed VC or

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BG/VC diets showed significantly lower plasma GLU values compared to fish fed the control
diet (P < 0.05). However, GLU values showed no significant differences between Ctrl. and BG

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groups (P > 0.05). Additionally, fish fed Ctrl. and VC diets showed significantly higher TG
values when compared with the other groups (P < 0.05).
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3.4. Humoral and mucosal immune responses
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Table 5 shows the serum and mucus lysozyme (LZY), bactericidal (BA), and peroxidase (PA)
activities of red sea bream fed the test diets for 56 days. Significantly higher serum and mucus
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LZY was observed in fish fed BG/VC diet (P < 0.05), however, serum LZY showed no
significant differences in case of BG and BG/VC groups (P > 0.05). Mucus BA of fish fed BG
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supplemented diet was significantly higher than those of fish fed VC supplemented diet (P <
0.05), however, no significant differences were observed among the other groups (P > 0.05). On
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the other hand, serum BA was not significantly influenced by the dietary BG or/and VC
supplementations. Serum PA was significantly enhanced in VC group when compared with the
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Ctrl. group (P < 0.05), however, no significant differences were observed among the other
groups (P > 0.05). Moreover, mucus PA was significantly enhanced in BG group when
compared with the Ctrl. group (P < 0.05), however, no significant differences were observed
among the other groups (P > 0.05).
Alternative complement pathway was significantly enhanced in BG/VC group when compared
with the Ctrl. and BG groups (P < 0.05; Fig. 1), however, no significant differences were
observed among the other groups (P > 0.05). Furthermore, nitro blue tetrazolium activity was
significantly increased in all BG or/and VC supplemented groups (P < 0.05; Fig. 2). Also,
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supplementation of BG or/and VC tended to increase the total serum protein content significantly
when compared to the control group (P < 0.05; Fig. 3). Overall, relatively lower humoral and
mucosal immune responses were measured in fish fed control diet.

3.5. Amounts of secreted mucus

Supplementation of both BG and VC improved the mucus secretion on body surface of red sea
bream and significantly higher value was observed in fish fed diet group BG/VC when compared

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with the fish fed the other test diets (P < 0.05; Fig. 4).

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3.6. Antioxidant enzymes activities
Table 6 shows the detected specific activities of antioxidant enzymes including: superoxide

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dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) of red sea bream fed the test
diets for 56 days. SOD was significantly increased in case of fish fed BG/VC diet when
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compared with the fish fed the control diet (P < 0.05), however, no significant differences were
observed among the other groups (P > 0.05). On the other hand, MDA was significantly
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decreased in case of fish fed BG/VC diet when compared with the fish fed the control diet (P <
0.05), however, no significant differences were observed among the other groups (P > 0.05).
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Further, CAT was not significantly influenced by the dietary BG or/and VC supplementations.
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3.7. Low salinity stress challenge

The results of lethal stress test against low salinity water shock (LT50) obtained by regression
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analysis are shown in Fig. 5. Supplementation of either BG or/and VC improved the tolerance
against low salinity stress of red sea bream and significantly higher LT50 obtained in fish fed all
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supplemented diets with BG or/and VC than the control group (P < 0.05).

4. Discussion
Increased susceptibility of fish to disease may result from stress induced immunosuppression due
to increasing stocking density, management practices and unfavorable environmental conditions
(Pezzato et al., 2014). Supplementation of immunostimulants such as β-glucan (BG) or/and
vitamin C (VC) have been used to enhance the growth, innate immunity and improve resistance
to different stressors in aquatic animals (Dawood and Koshio, 2016a,b; Dawood et al., 2015b;
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Brogden et al., 2014; Roosta et al., 2014; Shahkar et al., 2015). Existing dietary supplementation
strategies developed for other aquacultured species, especially the use of immunostimulants, may
be applicable to nutrition research on red sea bream (P. major) and to the development of feeds
for that fish (Hossain et al., 2016).
In the current study, the effects of BG or/and VC supplementation were investigated to provide
useful data for both immunological research and improved red sea bream production.
Supplementation of BG or/and VC enhanced the growth performance (FNW, WG, and SGR) of

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red sea bream. Although improvement of growth rate has been observed in P. major (Dawood et

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al., 2015b,c), mirror carp (Kühlwein et al., 2014), koi carp (Lin et al., 2011) and cyprinid rohu

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(Misra et al., 2006) fed with BG, there is no explanation on how these additives work to enhance
growth rate. The mode of penetration of BG through the gastrointestinal tract of fish remains to

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be determined, however, Dalmo and Bøgwald (2008) observed that different fish species are well
equipped to digest BG. If BG can be digested by fish and is a source of energy, it can be
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suggested that growth rate and feed utilization (FER and PER) were enhanced by the energetic
benefits obtained through BG. In contrast, the use of BG supplementation did not improve
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growth performance in Nile tilapia (Oreochromis niloticus) (Shelby et al., 2009), channel catfish
(Ictalurus punctatus) (Welker et al., 2007), or hybrid striped bass (Morone chrysops X M.
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saxatilis) (Li et al., 2009). VC is an essential nutrient needed to maintain the normal
physiological functions of fish (Chen et al., 2015; Liu et al., 2011). Growth enhancing effects of
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VC on red sea bream have not been reported previously to the authors’ knowledge, but enhanced
growth performance and feed utilization were reported recently in Caspian roach (Rutilus rutilus
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caspicus) fry (Roosta et al., 2014), Jian carp (Cyprinus carpio var. Jian) (Liu et al., 2011), and
cobia (Rachycentron canadum) (Zhou et al., 2012). However, dietary administration of VC failed
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to improve the growth performance of gilthead sea bream (Sparus aurata) (Henrique et al.,
1998). The improved feed utilization, as shown by FER and PER results in fish fed diet
supplemented with BG or/and VC in this study might be a possible reason for the higher
performances of fish in these groups. Probably BG was degraded in the digestive gland by
glucanases to produce energy, permitting the use of more proteins for feed utilization and growth
(Dawood et al., 2014; López et al., 2003).
High protein and lipid contents of whole body was observed in fish fed BG or/and VC-
supplemented diets in the present study indicates highly efficient utilization of dietary protein
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and lipid in red sea bream. Dawood et al. (2015c) and Yousefi et al. (2013) reported a significant
increase in P. major and Barbus sharpeyi whole body protein content due to BG or VC
supplementations, respectively. Condition factor (CF), hepatosomatic index (HSI) and
viscerasomatic index (VSI) are rough measurements of the energy reserved nutritional state of
the fish (Dawood et al., 2015b). Somatic parameters (CF, HSI, and VSI) of red sea bream were
not significantly altered by BG or/and VC supplementation. However, the results reflect the
improvements in body weight together with body length of fish fed BG or/and VC-supplemented

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diets when compared with the fish fed the control diet.

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Dietary BG or/and VC supplementations had significant influence on the hematological values of

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fingerling P. major in this study. Fish fed both BG and VC supplements (BG/VC diet) showed a
significant improved hematocrit level (HCT) when compared with fish fed only VC

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supplemented diet, indicating that neither BG nor VC could enhance the HCT level of red sea
bream individually. Similar to the current results, red sea bream fed diets supplemented with BG
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at 500 mg kg-1 improved the hematocrit level (Dawood et al., 2015c). Generally, HCT has been
increased in mirror carp (Kühlwein et al., 2014) and channel catfish (I. punctatus) (Welker et al.,
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2007) fed diets supplemented with BG. These data indicate that the effects of BG on HCT of P.
major might be depend on BG level of supplementation. Except for plasma glucose (GLU) and
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triglycerides (TG), we found that the BG or/and VC diets had no effects on the levels of most
examined metabolites, including total bilirubin, blood urea nitrogen, glutamyl oxaloacetic
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transaminase, glutamic-pyruvate transaminase and total cholesterol. Our results showed that fish
fed dietary VC or BG/VC diets had the lowest plasma GLU level, which indicated that they
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digested the VC and BG. Plasma GLU is commonly considered to be one stress indicator in fish,
high GLU levels often indicate the higher stress status of fish (Eslamloo et al., 2012). The lower
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plasma GLU content in fish fed VC or BG/VC supplemented diets indicated that dietary BG and
VC supplementation induced an optimal physiological condition of the fish. It is well known that
plasma TG play an important role in body health, immune response and antioxidant capability
(Yang et al., 2014). In the present study, plasma TG was significantly affected by the dietary BG
or BG/VC supplementations. Fish fed the control or VC diets had significantly higher TG than
those fed the diets supplemented with BG. Overall, results showing better physiological
condition of red sea bream fed BG or/and VC supplemented diets compared with control diet
indicate that BG or/and VC may help to maintain low level of plasma GLU and TG.
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Additionally, they may affect the lipid metabolism, but more study will be needed to clarify the
details on this mechanism.
The innate immune system of fish is considered to be the first line of defence against a broad
spectrum of pathogens and different stressors and is more important for fish as compared with
mamals (Adel et al., 2016; Uribe and Folch, 2011). A number of studies reported that fish fed
BG or/and VC-supplemented diets can positively influence both humoral and mucosal
components of the immune system (Dawood et al., 2015c; Dobšíková et al., 2013; Kühlwein et

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al., 2014; Nayak et al., 2007; Pezzato et al., 2014; Roosta et al., 2014). Results of the current

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study revealed that the skin mucus and serum immune parameters [lysozyme (LZY), bactericidal

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(BA), and peroxidase (PA) activities) were altered by dietary supplementation of BG or/and VC
in P. major diets. Lysozyme, an important hydrolytic enzyme of the non-specific immune

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system, can be recognized as a general immunity index (Callewaert and Michiels, 2010). In this
study, the fish fed diets adding 500 mg kg-1 of BG and 400 mg kg-1 VC (BG/VC) exhibited a
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significantly higher serum and mucus LZY compared to the control group, indicating that both
dietary BG and VC can improve the immunity of P. major. However, the individual
supplementation of BG at 1000 mg kg-1 or VC at 800 mg kg-1 did not further improve the LZY in
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P. major significantly, indicating that such positive effect was dose-dependent. Similar result
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was also discovered by Dawood et al. (2015b,c) in which a significantly higher serum LZY was
observed in the fish fed the diet supplemented with 1000 mg kg-1 BG. Furthermore, Shahkar et
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al. (2015) reported that Japanese eel (Anguilla japonica) fed diets supplemented with VC also
enhanced LZY. The increased LZY in BG/VC supplemented group was due to
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immunostimulating properties of BG and VC which may act by either an increase in the number
of phagocytes secreting lysozyme or an increase in the amount of lysozyme synthesized per cell
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(Kumari and Sahoo, 2006).

Bactericidal activity is one of the main immune responses responsible for the direct ability of
killing pathogenic bacteria. This activity can be carried out by many different compounds, but
unfortunately most of them are unknown (Guardiola et al., 2014). Among them one of the most
characterized is the lysozyme, which level and activity in the skin mucus depends on the
environmental conditions and the fish species (Fast et al., 2002; Dawood et al., 2016e). In the
present study, there was a trend of increased mucus BA with the supplementation of BG
compared with control. Increased BA due to BG supplementation was also reported in P. major
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(Dawood et al., 2015c) and Labeo rohita (Misra et al., 2006). The increased LZY and BA in BG
supplemented groups in the present study would be due to the immunostimulatory effects of
dietary BG.
Since peroxidases are important microbicidal agents that effectively eliminate H2O2 and maintain
the redox balance of immune system, it is tempting to consider that peroxidase in serum and
mucus will be essential for humoral, mucosal immunity and skin defense (Guardiola et al.,
2014). In this study, serum PA in VC group was higher than in the control group, meanwhile,

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mucus PA in BG group was higher than in the control group, confirming other results obtained

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by Kumari and Sahoo (2006) and Dawood et al. (2015c). On the contrary, feeding with BG

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resulted in no increase in PA in gilthead seabream (S. aurata L.) (Guzmán-Villanueva et al.,
2014). This was probably because of different species, fish size, and environmental conditions.

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The role of VC to enhance the humoral and mucosal immune responses was emphasized by the
presence of intracellular VC in leukocytes at concentrations related to dietary levels (Levy et al.,
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1996; Roosta et al., 2014; Verlhac and Gabaudan, 1994). The information about the effects of
VC on the mucosal immunity of fish still need to be more clarified in future studies.
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The respiratory burst activity generated by macrophages/monocytes and granulocytes has

effector mechanisms like direct killing of microorganisms by the end products of reactive
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oxygen species, such as superoxide anion, hydrogen peroxide and hydroxyl radical (Dalmo et al.,
1997). In the present study, enhancement of LZY, ACP, NBT and PA, were confirmed in the
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serum of fish fed dietary BG or/and VC. The effect of VC, which is an abundant source in citrus
(Cano et al., 2008), was studied in the immune responses in several fish species. Many studies
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reported enhanced immunity and disease resistance by dietary supplementation of VC at

optimum or overdose concentrations (Azad et al., 2007; Dawood and Koshio, 2016b; Eo et al.,
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2008; Nayak et al., 2007; Ortuno et al., 2003). These studies demonstrated that both the acquired
and innate immunity could be fortified by enhanced activity of LZY, ACP and NBT in turbot,
Scophthalmus maximus (L.), catfish, seabream, seabass, milkfish, Chanos chanos (Forsskal),
carp and tiger puffer, Takifugu rubripes (Temminck & Schlegel), although some studies reported
no effects of dietary VC on the immune response (Thompson et al., 1993; Bell et al., 1984).
Thus, the use of BG provides a potential advantage in terms of VC supplementation in fish diets
(Dawood et al., 2016c; Pezzato et al., 2014; Verlhac et al., 1996). In the current study, total
serum proteins also showed similar increasing trend with BG and VC supplementation compared
 ACCEPTED MANUSCRIPT

to the control group. The increased total serum protein in BG or/and VC-supplemented groups in
the present study would be due to the immunostimulatory effects of dietary BG and VC.
Increased total blood protein due to BG supplementation was also reported in P. major (Dawood
et al., 2015c) and in common carp (C. carpio L.) (Dobšíková et al., 2013). Also, VC
supplementation enhanced the total serum protein in L. rohita (Tewary and Patra, 2008) and
Mekong giant catfish (Pangasianodon gigas) (Pimpimol et al., 2012).
The skin mucus, an important component of the innate immunity, is the first line of defence

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against microorganisms because it forms a physico-chemical barrier containing a diverse range

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of innate and adaptive immune factors that protects fish against infections (Guardiola et al.,

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2014; Koshio, 2016). The present results revealed remarkable elevation of skin mucus secretion
following administration of BG and VC supplemented diets; the highest elevation observed in

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fish fed both BG and VC supplementations. Likewise, administration of dietary BG significantly
elevated serum mucus in P. major (Dawood et al., 2015c). Furthermore, Roosta et al. (2014)
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reported supplementation of Caspian roach (R. rutilus caspicus) with VC increased skin mucus
immune parameters. The increased skin mucus enzyme activities (LZY, BA and PA) and protein
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secretion level, possibly hint at more pronounced immunomodulatory nature of BG and VC

compared to singular administration of BG or VC; as observed above in the case of the humoral
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and mucosal immune parameters. However, confirmation of this hypothesis merits further
research on skin mucus immune parameters and mucosal immune following administration of
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dietary immunostimulants.
The antioxidant status in fish can be accurately reflected by the activity of SOD, CAT and MDA
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enzymes (Chen et al., 2015; Liang et al., 2015). Serum SOD and CAT activities are common
antioxidant enzymes that can protect organism against damage by reactive oxygen species (ROS)
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which may lead to many disorders through attack macromolecules (Li et al., 2008; Stadtman and
Levine, 2003; Seifried et al., 2007). Additionally, MDA is formed as a normal product of lipid
peroxidation, but high levels of MDA can damage cell structure and function (Sies, 1997). In the
present study, the serum SOD and MDA enzymes were significantly influenced by dietary BG
or/and VC supplementation, where SOD was significantly increased, however, the MDA was
significantly decreased in BG/VC group when compared to the control group. These results
demonstrated that dietary BG and VC has a positive effect on the antioxidant capacity of red sea
 ACCEPTED MANUSCRIPT

bream. The compound increased the antioxidant capacity of the yellow catfish might by
increasing the antioxidant enzymes to decrease the ROS and reducing the MDA contents.
Low salinity stress challenge has often been used as a final indicator of fish quality after nutrition
trials (Dawood et al., 2015a,b); our results revealed that dietary BG or/and VC significantly
increased red sea bream resistance to low salinity stress. Increased LT50 of red sea bream fed BG
or/and VC-supplemented diets indicated healthy status of red sea bream. Similarly, red sea
bream fed BG showed greater survival after salinity stress challenge (Dawood et al., 2015b,c).

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Pezzato et al. (2014) reported that, BG and VC is recommended for Nile tilapia especially when

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fish are likely to encounter transport-induced stress. It has been suggested that greater resistance

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to salinity stress challenges might be due to improved feed utilization; as a result, energy and
other nutrients will be available to synthesize adrenal steroids to face the physical stressors

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(Dawood et al., 2016d); however, future studies are required to test this speculative hypothesis.
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5. Conclusion
In conclusion, the results of the current study demonstrated that supplementation of both BG
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or/and VC can have positive influences on growth performance, survival, feed utilization,
immune response, blood parameters, skin mucus and tolerance against low salinity stress of red
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sea bream. Further studies should be developed under intensive culture conditions to validate
laboratory findings. Based on fish growth, biochemical and immune responses, diet
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supplemented with 500 mg of BG and 400 mg of VC per kg diet is recommended for red sea
bream.
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 ACCEPTED MANUSCRIPT

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Verlhac, V., Gabaudan, J., Obach, A., Schüep, W., Hole, R., 1996. Influence of dietary glucan
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and vitamin C on non-specific and specific immune responses of rainbow trout (Oncorhynchus
mykiss). Aquaculture. 143(2),123-133.
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Verlhac, V., Gabaudan, J., 1994. Influence of vitamin C on the immune system of salmonids.
Aquacult. Res. 25, 21–36.
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Welker, T.L., Lim, C., Yildirim-Aksoy, M., Shelby, R., Klesius, P.H., 2007. Immune response
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punctatus, fed diets containing commercial whole-cell yeast or yeast subcomponents. J. World
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Yousefi, P., Yavari, V., Zakeri, M., Salati, A., Keyvanshokooh, S., 2013. Effect of Dietary
Supplementation of Vitamin C on Growth Performance Feed Utilization and Carcass
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Composition of Barbus sharpeyi Fingerlings. Journal of the Persian Gulf (Marine Science). 4,
23-31.
Zhou, Q., Wang, L., Wang, H., Xie, F., Wang, T., 2012. Effect of dietary vitamin C on the
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Shellfish Immunol. 32, 969–975.

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80
b
ACP (ACH 50% units ml-1)
70
ab
60 a a
50
40
30

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20

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Ctrl. BG VC BG/VC
Test diets

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Fig. 1. Alternative complement pathway (ACP) of red sea bream fed test diets for 56 days. Data
represent means± pooled SEM (n=9). Values with different letters are significantly different (P <
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0.05). Values with the same letter are not significantly different (P > 0.05).
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0.20
0.18 b
b
NBT (OD at 540 nm)

0.16 ab
0.14
0.12 a
0.10
0.08
0.06

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0.04
0.02

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Fig. 2. Nitro blue tetrazolium (NBT) activity of red sea bream fed test diets for 56 days. Data
represent means± pooled SEM (n=9). Values with different letters are significantly different (P <
0.05). Values with the same letter are not significantly different (P > 0.05).
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Total serum protein (g dl-1) 4 b b

3.5 ab
3 a
2.5
2
1.5
1

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0.5

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Fig. 3. Total serum protein of red sea bream fed test diets for 56 days. Data represent means±

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pooled SEM (n=9). Values with different letters are significantly different (P < 0.05). Values
with the same letter are not significantly different (P > 0.05).
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b
Mucus amount (mg ml-1)

5
a a
a
4

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1

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Fig. 4. Amounts of mucus secretion on body surface of red sea bream. Values are expressed as
mean relative means ± pooled SEM (n=9). Values with the same letter are not significantly
different (P > 0.05). Means with different alphabet are significantly different (P < 0.05).
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120
b b b
100

80 a
LT50 (min.)

60

40

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Fig. 5. Time to 50% mortality (LT50) of red sea bream exposed to low salinity water. Values are
expressed as mean ± SEM from triplicate groups (n=15). Values with the same letter are not
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significantly different (P > 0.05). Means with different alphabet are significantly different (P <
0.05).
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Table 1
Formulation and proximate composition of the experimental diets for red sea bream (%).

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Test diets (g kg-1 dry diet)
Ingredients
Ctrl. BG VC BG/VC
Brown fish meal 280 280 280 280
Casein 280 280 280 280

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Dextrin 70 70 70 70
α-starch

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60 60 60 60
Soybean lecithin 50 50 50 50

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Pollack liver oil 60 60 60 60
Vitamin mixture (VC free) 30 30 30 30

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Mineral mixture 30 30 30 30
Activated gluten 50 50 50 50
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α-Cellulose 90 89 89.2 89.1
β-glucan (BG)2 0 1 0 0.5
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Vitamin C (VC)3 0 0 0.8 0.4

Proximate composition
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Crude protein (%) 49.9 49.3 49.8 49.9

Total lipid (%) 11.8 11.9 10.9 11.9
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Moisture (%) 8.9 9.5 9.7 9.8

Ash (%) 11.9 11.3 11.2 11.9
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Gross energy (KJ g-1)4 19.55 19.3 19.6 19.4

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VC content (mg kg ) Trace Trace 818 403.2
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According to Dawood et al. (2015a).
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BG: preparation of β-glucan used is a commercial product containing 85%BG produced by
Daigon do (Tokyo, Japan) (Dawood et al., 2015b).
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VC: vitamin C source (L-Ascrobil-2-phosphate-Mg).
4
Calculated using combustion values for protein, lipid and carbohydrate of 23.6, 39.5 and 17.2 kJ
g−1, respectively. Carbohydrate was calculated by the difference:
100−(protein+lipid+ash+moisture).
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Table 2
Growth performance and feed utilization parameters of red sea bream fed diets supplemented
with BG or/and VC for 56 days.
Test diets
Parameters1
Ctrl. BG VC BG/VC
INW (g)2 1.99±0.002 1.97±0.01 1.97±0.01 1.98±0.01
FNW (g)3 18.43±0.72a 22.14±0.42b 21.87±0.25b 24.37±0.46c

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WG (%)4 826.31±35.15a 1035.69±21.8bc 1020.46±16.79b 1131.46±18.07c

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SGR (% day−1)5 3.97±0.07a 4.34±0.03b 4.31±0.03b 4.48±0.03b

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FER6 1.09±0.09a 1.54±0.02bc 1.52±0.02b 1.69±0.02c
PER7 2.17±0.17a 3.13±0.04bc 3.05±0.06b 3.39±0.02c

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SUR (%)8 96.67±3.33 100±0.00 96.67±0.00 100±0.00
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In the same line, means with different letters are significantly different (P < 0.05), means with
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the same letters are not significantly different (P > 0.05). Absence of letters indicates no
significant difference between treatments. Data represent means± pooled SEM (n=60).
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INW: initial weight, 3FNW: final weight, 4WG: percent weight gain, 5SGR: specific growth rate,
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FER: feed efficiency ratio, 7PER: protein efficiency ratio, 8SUR: survival.
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Table 3
Whole body proximate analysis (%) and somatic parameters in juvenile red sea bream fed diets
supplemented with BG or/and VC for 56 days.
Test diets
Parameters1
Ctrl. BG VC BG/VC
Moisture 71.7±0.8b 69.66±0.24ab 69.2±0.09a 68.58±0.37a
13.48±0.52a 15.67±0.51b 16.38±0.1b 17.06±0.31b

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Crude protein
5.92±0.38a 6.79±0.06a 7.36±0.52ab 8.57±0.05b

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Total lipid
Crude ash 5.45±0.33 4.55±0.22 4.51±0.35 4.82±0.34

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CF (%)2 1.85±0.27 1.75±0.33 1.85±0.38 1.77±0.1
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HSI (%) 1.08±0.34 1.74±0.46 1.32±0.21 1.6±0.33

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VSI (%)4 2.31±0.13 3.24±0.29 2.67±0.21 2.65±0.13
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In the same line, means with different letters are significantly different (P < 0.05), means with
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the same letters are not significantly different (P > 0.05). Absence of letters indicates no
significant difference between treatments. Data represent means± pooled SEM (n=9).
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2
CF: condition factor, 3HSI: hepatosomatic index, 4VSI: viscerasomatic index.
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Table 4
Hematocrit and blood chemistry parameters in juvenile red sea bream fed diets supplemented
with BG or/and VC for 56 days.
Test diets
Parameters1
Ctrl. BG VC BG/VC
HCT (%)2 36±1.16ab 36.67±1.76ab 33.33±0.67a 39±0.58b
GLU (mg dl-1)3 88.33±4.67c 80.33±44.26bc 68.33±4.26ab 60.67±3.18a

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T-BIL (mg dl-1)4

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0.37±0.07 0.43±0.09 0.4±0.06 0.57±0.15
BUN (mg dl-1)5 5.67±0.33 6.67±0.88 5.67±0.33 6.33±0.67

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GOT (IU l-1)6 69.67±10.99 443.33±6.17 53±8.72 47.67±8.67
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GPT (IU l ) <10 <10 <10 <10

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TG (mg dl-1)8 288.67±11.98c 255.67±8.95ab 285±5.13bc 244.67±11.35a
T-CHO (mg dl-1)9 220±7.77 213±21.55 219±6.08 188.67±9.56
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In the same line, means with different letters are significantly different (P < 0.05), means with
the same letters are not significantly different (P > 0.05). Absence of letters indicates no
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significant difference between treatments. Data represent means± pooled SEM (n=15).
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HCT: hematocrit, 3GLU: glucose, 4T-BIL: total bilirubin, 5BUN: blood urea nitrogen, 6GOT:
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glutamyl oxaloacetic transaminase, 7GPT: glutamic pyruvate transaminase, 8TG: triglyceride, 9T-
CHO: total cholesterol.
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Table 5
Humoral and mucosal immune responses of red sea bream fed diets supplemented with BG
or/and VC for 56 days.
Test diets
Parameters1
Ctrl. BG VC BG/VC
Serum LZY (unit ml-1)2 29.25±2.88a 35.83±0.87ab 33.08±1.81a 0.33±5.92b
Mucus LZY (unit ml-1) 28.42±0.42a 30.92±1.53a 29.5±0.66a 35.42±1.2b

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Serum BA (%)3

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49.82±2.24 71.88±2.69 61.06±13.04 76.72±10.37
Mucus BA (%) 57.4±6.13ab 71.88±2.69b 54.3±6.51a 63.21±1.46ab

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Serum PA (OD at 40 nm)4 1.46±0.1a 1.66±0.04ab 1.69±0.06b 1.67±0.04ab
Mucus PA (OD at 40 nm) 1.55±0.1a 1.91±0.08b 1.88±0.05ab 1.67±0.01ab

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In the same line, means with different letters are significantly different (P < 0.05), means with
the same letters are not significantly different (P > 0.05). Absence of letters indicates no
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significant difference between treatments. Data represent means± pooled SEM (n=15).
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LZY: lysozyme activity, 3BA: bactericidal activity, 4PA: peroxidase activity.
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Table 6
Specific activities of antioxidant enzymes and lipid peroxidation levels of red sea bream fed diets
supplemented with BG or/and VC for 56 days.
Test diets
Parameters1
Ctrl. BG VC BG/VC
SOD (U mg protein-1)2 52.48±1.02a 63.84±4.48ab 65.19±3.81ab 74.21±5.47b
CAT (kU L-1)3

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553.49±7.13 67.7±3.57 57.05±12.86 74.88±3.57
MDA (nmol ml-1)4 12.65±0.92b 10.03±1.59ab 11.77±1.36ab 6.7±0.26a

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In the same line, means with different letters are significantly different (P < 0.05), means with

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the same letters are not significantly different (P > 0.05). Absence of letters indicates no
significant difference between treatments. Data represent means± pooled SEM (n=9).

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2
SOD: superoxide dismutase, 3CAT: catalase, 4MDA: malondialdehyde concentration.
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Statement of relevance

A combination of β-glucan and vitamin C presented an additive effect on inducing the growth,
immune responses and oxidative status of red sea bream. We recommend that treatment of 500
mg of β-glucan and 400 mg of vitamin C per kg diet is the appropriate concentration of inducing
the red sea bream immune response.

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Highlights

 Red sea bream (Pagrus major) were fed β-glucan (BG) or vitamin C (VC) alone
or in combination.
 The mixture supplementation significantly improved growth performance and
feed utilization of fish.
 Fish fed BG or/and VC supplementations showed enhanced oxidative status and

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tolerance against stress.

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 Fish fed BG or/and VC supplementations showed enhanced humoral and mucosal

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immune responses.
 BG and VC synergistically enhance the performances of red sea bream.

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