Accepted Manuscript

Substituting fish meal with soybean meal in diets for Japanese

seabass (Lateolabrax japonicus): Effects on growth, digestive
enzymes activity, gut histology, and expression of gut
inflammatory and transporter genes

Chunxiao Zhang, Samad Rahimnejad, Ya-ru Wang, Kangle Lu,

Kai Song, Ling Wang, Kangsen Mai

PII: S0044-8486(17)31897-5
DOI: doi:10.1016/j.aquaculture.2017.10.029
Reference: AQUA 632883
To appear in: aquaculture
Received date: 22 September 2017
Revised date: 18 October 2017
Accepted date: 20 October 2017

Please cite this article as: Chunxiao Zhang, Samad Rahimnejad, Ya-ru Wang, Kangle Lu,
Kai Song, Ling Wang, Kangsen Mai , Substituting fish meal with soybean meal in diets for
Japanese seabass (Lateolabrax japonicus): Effects on growth, digestive enzymes activity,
gut histology, and expression of gut inflammatory and transporter genes. The address
for the corresponding author was captured as affiliation for all authors. Please check if
appropriate. Aqua(2017), doi:10.1016/j.aquaculture.2017.10.029

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 ACCEPTED MANUSCRIPT

Substituting fish meal with soybean meal in diets for Japanese seabass

(Lateolabrax japonicus): Effects on growth, digestive enzymes activity, gut

histology, and expression of gut inflammatory and transporter genes

Chunxiao Zhanga,*, Samad Rahimnejada, Ya-ru Wanga, Kangle Lua, Kai Songa, Ling

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Wanga, Kangsen Maib

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a
Xiamen Key Laboratory for Feed Quality Testing and Safety Evaluation, Fisheries

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College, Jimei University, Xiamen 361021, China
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b
The Key Laboratory of Mariculture (Education Ministry of China), Ocean University

of China, Qingdao 266003, China

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*
Corresponding Author: Dr Chun-Xiao Zhang, Tel.: +86 592 6181054; Fax: +86
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592 6181054; Fisheries College, Jimei University, No. 43 Yindou Road, Xiamen
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361021, China. E-mail: cxzhang@jmu.edu.cn

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Abstract

The aim of this study was to evaluate the effects of substituting fish meal (FM) with

soybean meal (SM) on growth performance, digestive enzymes activity, gut histology,

and intestinal pro-inflammatory and transporter genes expression in Japanese seabass

(Lateolabrax japonicus). Totally three test diets were prepared: a basal FM-based diet

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and two SM diets by substituting 50 or 75% of FM with SM (FM, SM50 and SM75

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diets). Each diet was fed to triplicate groups of fish (6.67±0.03 g) to apparent satiation

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twice a day for eight weeks. The results showed no significant (P > 0.05) differences

in growth performance between FM and SM50 groups while further increment of

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replacement level to 75% led to a significantly (P < 0.05) reduced growth rate.
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However, both SM50 and SM75 groups showed significantly lower feed efficiency

and protein efficiency ratio than FM group. Significantly lower digestibility

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coefficients of dry matter and protein were achieved in the group received SM75 diet
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and digestibility coefficient of gross energy decreased in both SM50 and SM75
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groups. Also, SM75 fed fish exhibited remarkably lower survival rate than the other
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treatments. SM75 group had lower whole-body protein and lipid contents than FM fed
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fish. Drastic decreases in protease, amylase and lipase activities were found in foregut

of SM groups compared to FM group. Offering SM75 diet resulted in significant

reduction of villus height, villus thickness, and muscular thickness in foregut and

midgut. A remarkable increase in serum D-lactate concentration was detected in SM

groups, and serum diamine oxidase activity elevated in SM75 group. Replacement of

FM resulted in elevated expression of gut pro-inflammatory genes such as TNF-α,

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IL-1β, IL-2, IL-8 while an opposed trend was observed for the anti-inflammatory

gene IL-4. Expression of intestinal transporter genes including PepT1, LAT1 and

SLC1A5 were significantly up-regulated by SM replacement. To conclude, replacing

50% of FM with SM did not significantly influence growth performance, but adverse

effects were found on feed utilization, digestive enzymes activity and gut health being

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more evident at the higher replacement level.

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Keywords: Lateolabrax japonicus; Fish meal replacement; Soybean meal; Digestive

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enzymes activity; Gut health; Gene expression
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1. Introduction

Fish meal (FM) has long been utilized as the best dietary protein source for

aquafeeds production because apart from being a balanced source of indispensible

amino acids, is a rich source of long-chain omega-3 fatty acids, vitamins and minerals

essential for normal animal growth (Olsen and Hasan, 2012). However, its stagnant

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supply as well as increasing demand has resulted in higher prices for FM during the

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recent decades. Furthermore, roughly two-third of the total produced FM is used for

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aquafeeds production (FAO, 2012); thus considering the rapid expansion of

aquaculture industry there are concerns over sufficient FM supply to meet the
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industry’s demand in the future regardless of its price (Faggio et al., 2014a,b; Fazio et
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al., 2013). The requisite for sustainable protein sources for aquafeeds production has

urged aquaculture nutritionists to explore alternative protein sources to replace for FM

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(Aragona et al., 2017; Carbone and Faggio., 2016; Faggio et al., 2015; Guardiola et al.,
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2016; Gatlin et al., 2007; Glencross, 2009).

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Terrestrial animal and plant proteins have been recognized as sustainable protein
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sources to substitute FM (Bowyer et al., 2013; Burgos-Aceves et al., 2016; Naylor et

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al., 2009; Tacon and Metian, 2008). Soybean meal (SM) has been identified as one of

the most promising candidates for FM replacement mainly due to its compatible

nutritional composition, relatively well-balanced amino acid profile, widespread

availability, and low cost (Gatlin et al., 2007; Storebakken et al., 2000; Trushenski et

al., 2006). However, digestibility, palatability and utilization of feeds (particularly in

carnivorous fish) could be negatively influenced when SM is used as the primary

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protein source (Brown et al., 1997; Davis et al., 1995; Refstie et al., 1998;

Sitjá-Bobadilla et al., 2005; Watanabe and Pongmaneerat, 1993; Zhou et al., 2005).

Standard SM can be incorporated only at relatively low levels in carnivorous fish feed

due to its adverse effects on gut health (Krogdahl et al., 2010; Merrifield et al., 2011).

The negative impacts of high dietary inclusion levels of SM are associated with its

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various antinutritional factors (ANFs) such as protease inhibitors, phytate, saponins,

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lectins, and oligosaccharides (Francis et al., 2001). There are several reports

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indicating that ANFs activate inflammatory signals in digestive tract of several fish

species (Bakke-McKellep et al., 2000; Krogdahl et al., 2010). Also, several

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researchers have reported the induction of inflammatory responses in the distal
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intestinal mucosa of cultured fish species following SM administration (Baeverfjord

and Krogdahl, 1996; Hedrera et al., 2013; Rumsey et al., 1994; Urán et al., 2008; van
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den Ingh et al., 1991; Yamamoto et al., 2008). The severity of histopathological
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changes observed by SM application depends on the content of the soybean protein

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and level of inclusion (Barrows et al., 2007; Francis et al., 2001), and is characterized
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by a shortening of the mucosal folds, a swelling of the lamina propria and

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subepithelial mucosa, loss of supranuclear vacuolization in the absorptive cells of the

intestinal epithelium, infiltration of a mixed leukocyte population in the lamina

propria and submucosa, and decreased numbers of absorptive vacuoles in the

enterocytes (Baeverfjord and Krogdahl, 1996; Bakke-McKellep et al., 2000;

Bakke-McKellep et al., 2007; van den Ingh et al., 1991). However, it has been

demonstrated that sensitivity to soy ANFs varies among different fish species, e.g.,
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Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) showed

distinct morphological alternations in the intestine (Krogdahl and Bakke-McKellep,

2001) while no apparent adverse effects could be found in red drum (Sciaenops

ocellatus) (Reigh and Ellis, 1992) and Japanese flounder (Paralichthys olivaceus)

(Kikuchi, 1999) offered diets containing almost equal amounts of FM and SM.

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Japanese seabass (Lateolabrax japonicus) is a carnivorous species that has been

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largely cultured in East Asia, particularly in China (Islam et al., 2015; Li et al., 2012).

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Previous studies showed that FM can be replaced up to 30% in seabass diet without

suppressing growth or negative effects on gut integrity (Hu et al., 2013; Laporte and
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Trushenski, 2012; Li et al., 2012). Wang et al. (2017) reported the incidence of
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enteritis in Japanese seabass fed a diet in which 50% of FM was replaced with SM.

The aim of the present study was to examine the impacts of replacing 50 and 75% of
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FM with SM on growth, digestive enzymes activity, and gut health of Japanese

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seabass. In order to further elucidate the molecular mechanisms associated with

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intestine inflammation, expression of inflammatory genes was also examined which

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has not been explored in previous studies.

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2. Materials and methods

2.1. Experimental diets

Formulation and proximate composition of the experimental diets are presented

in Table 1. Three experimental diets were formulated to be isonitrogenous (42%

protein) and isolipidic (12% lipid). A basal diet was formulated to contain 42% FM
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(FM diet), and two other diets were prepared by substituting 50 or 75% of FM by SM

(SM50 and SM75 diets). The coarse dry ingredients were finely ground using a

hammer mill and passed through a 60-μM mesh. All the dry ingredients were

thoroughly mixed and a mash was produced after adding fish oil, soybean oil and

deionized water. The resultant dough was conveyed into multifunctional spiral

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extrusion machinery (CD4XITS, South China University of Technology, Guangzhou,

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China) with 1.5 and 2.5-mm diameter. The pellets were dried overnight in a dry oven

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at 35 °C, then sealed in bags and stored at −20 °C until used.
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2.2. Experimental fish and feeding trial
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Japanese seabass juveniles were purchased from a private farm (Zhangpu Hui

Feng farm, Xiamen, China) and transported to the aquaculture laboratory of Jimei
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University. The fish were stocked into two 1000-L tanks supplied with filtered
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seawater in a recirculating system and fed the basal diet twice daily for one month to
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adapt them to the experimental facilities and conditions. At the termination of the
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acclimation period, 180 healthy fish averaging 6.67±0.03 g were stocked into nine
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150-L tanks (containing 120 L water) in a recirculating system at a density of 20 fish/

tank. The recirculating system consisted of a reservoir with a biological filter, a

circulation pump and an automatic temperature control device. The experiment was

conducted in triplicates and fish were fed their respective diets to apparent satiation

two times a day (8:30 and 17:30) for eight weeks. The fish were fed feed of 1.5-mm

diameter for the first 2 weeks and 2.5-mm diameter feed for the following 6 weeks.
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Uneaten feed, if any, was siphoned out 30 min after feeding, dried and weighted for

subsequent calculation of feed intake, and then approximately two-third of the water

was replenished. The fish were fasted for 24 h prior to weighing or sampling to

minimize handling stress on fish. The average temperature during the feeding trial

was 28±2 °C and the photoperiod was maintained on a 12:12 light:dark schedule.

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2.3. Sample collection

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At the termination of the feeding period, all the fish in each tank were counted

and bulk weighted for calculation of growth parameters and survival. Fish were
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anesthetized in eugenol (1:10000) prior to dissection or blood sampling. Three fish
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per tank were randomly captured for histological analyses. Prior to dissection, the fish

were first disinfected with 75% alcohol. Foregut and midgut were collected, flushed
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with ice-cold phosphate-buffered saline (PBS saline, pH 7.4), and then fixed in 10%
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formalin for morphological measurements. Also, another set of foregut and midgut
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samples were dissected from another set of three fish/tank and immediately immersed
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in RNA keeper (Vazyme Biotech Co., Ltd, NanJing) for 12 h at 4 °C and then
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preserved at −80 °C for analysis of Peptide Transporter 1 (PepT1), L-amino acid

transporter 1 (LAT1), solute carrier family 1 member A5 (SLC1A5), TNF-α, IL-1β,

IL-2, IL-8 and IL-4 genes expression. For analysis of digestive enzymes activity, new

sets of foregut and midgut samples were collected from three fish per tank,

immediately flash frozen in liquid nitrogen and subsequently kept in −80 °C. Three

fish from each tank (nine fish per dietary treatment) were randomly captured, and
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blood samples were collected from caudal vein with non-heparinized syringes and

allowed to clot at 4 °C for 24 h, then serum samples were separated by centrifuging at

3000×g for 10 min at 4 °C. Serum samples were used for determination of D-lactate

concentration and diamine oxidase (DAO) activity. The same fish were used for

determination of whole-body proximate composition. After six weeks of feeding, in

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order to determine the digestibility coefficients of protein, dry matter and gross energy,

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feces were collected from bottom of the tanks, centrifuged for 30 min at 2100×g and

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the supernatant was discarded, and feces were frozen at −20°C until analysis.
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2.4. Analytical methods
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2.4.1. Chemical composition and diet digestibility

Analyses of moisture, crude protein, crude lipid and ash contents of the
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experimental diets, feces and whole-body samples were performed by the standard
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procedures (AOAC, 2002). Moisture was determined by drying the samples in an

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oven at 105 °C to constant weight; crude protein was analyzed by the Kjeldahl
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method (N×6.25) with a FOSS Kjeltec 8400 analyzer (Tecator, Höganäs, Sweden)
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after acid digestion in an auto-digester (FOSS; Tecator); crude lipid was determined

by Soxhlet extraction in ether; ash content was measured by the combustion method

in a muffle furnace at 550 °C for 8 h. Gross energy content of diets was determined

using an adiabatic bomb calorimeter (Parr 6300; Parr Instruments Inc., Moline, IL,

USA). Yttrium oxide in the diets and feces samples were determined by inductively

coupled plasma-atomic emission spectrophotometer (ICP-OES, Prodigy7, Leeman

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Labs, USA). The apparent digestibility coefficients (ADCs) of dry matter, protein and

energy for the test diets were calculated using the following formula (NRC, 2011):

ADC of dry matter (%) = (1 − Y2O3 in diet/ Y2O3 in feces) ×100%

ADC of nutrient (%) = [1 − (Y2O3 in diet/ Y2O3 in feces) × (nutrient in feces/ nutrient

in diet)] ×100%

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ADC of gross energy was calculated using gross energy data (kJ g-1) instead of

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nutrient data.

2.4.2. Gut morphology

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The fixed foregut and midgut samples were dehydrated in a graded series of
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ethyl alcohol and embedded in paraffin. Three sections (7 μm thick) were cut from

each sample and then stained with hematoxylin/eosin. The villus height, villus
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thickness and muscular thickness of each slice were measured using the image
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analysis software Image-Pro Plus 6.0 (Media Cybernetics, Inc.). Histological

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alterations in intestinal epithelia were evaluated based on the degree of changes in

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villi and the absorptive epithelial cell area was examined using a light microscope
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Olympus BX53 (Olympus, Japan).

2.4.3. Digestive enzymes activity

Amylase and lipase activities were analyzed using commercial assay kits

(Nanjing Jiancheng Institute, Nanjing, China) according to the manufacturer’s

instructions. The protein content of the homogenates was measured using

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Folin-phenol reagent (Lowry et al., 1951). Protease activity was measured using

casein as substrate as described by Pan and Wang (1997). A typical assay was

performed as follows: 2 ml of 0.5% casein, 0.1 ml of 0.04 mol L-1 EDTA-Na2, 0.4 ml

of appropriate buffer, 0.2 ml of enzymatic extract and 0.8 ml distilled water were

mixed and incubated for 15 min in specific conditions of pH, temperature and NaCI

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concentration. The reaction was terminated by adding 1 ml of 30% chilled

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trichloroacetic acid (TCA). The mixture was centrifuged at 2000 g for 15-20 min. One

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milliliter of clear supernatant, 5 ml of 0.55 mol L-1 Na2CO3 and 1 ml of Folin reagent

were mixed and stayed for 15 min, then OD was measured in a spectrophotometer at
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680 nm against blanks in distilled water. Controls were made in which the enzymatic
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extracts were added at the end of the incubation period and just before the

centrifugation. Enzymatic extracts were diluted if required. One unit of the activity
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was defined as the amount of the hydrolysis of casein that liberated 1μg of tyrosine
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per min.
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2.4.4. Serum D-lactate concentration and DAO activity

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Serum D-lactate concentration and DAO activity were determined using

Beckman CX4 Chemistry Analyser (Beckman Coulter, Brea, CA) with commercial

assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). The

quantification of D-lactate requires two enzyme reactions. In the first reaction

catalysed by D-lactate dehydrogenase, D-lactate is oxidised to pyruvate and nicotinic

acid dehydrogenase (NADH) in the presence of nicotinamide-adenine dinucleotide

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(NAD+). However, since the equilibrium of reaction lies firmly in the favour of

D-lactate and NAD+, a further reaction is required to trap the pyruvate product. This is

achieved by the conversion of pyruvate to D-alanine and 2-oxoglutarate, with the

enzyme D-glutamate-pyruvate transaminase in the presence of a large excess of

D-glutamate. The amount of NADH formed in the above coupled reaction is

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stoichiometric with the amount of D-lactate. It is the NADH which is measured by the

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increase in absorbance at 340 nm. Activity of DAO was measured by using histamine

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as the substrate and by monitoring the rate of ammonia formation following the

cleavage of histamine. The rate of decrease in absorbance at 340 nm is proportional to

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activity of DAO. The effect of endogenous ammonia present in the serum sample was
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eliminated by performing a 10 minute incubation of the sample with 2-oxoglutarate.

One unit of activity was defined as one mmol of ammonia formed per minute per mL
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of serum at 37°C.
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2.4.5. RNA extraction and real-time quantitative PCR (qPCR)

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RNA was isolated from foregut and midgut samples (approximately 80 mg)
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using TRIzol Reagent (Invitrogen, USA). The purity and concentration of RNA were

measured using a ND-2000 spectrophotometer (NanoDrop 2000, Wilmington, DE,

USA). RNA integrity was confirmed by running 1 μg RNA on an ethidium-bromide

stained 1.5% agarose gel with 1X Tris Acetate EDTA (TAE) buffer. Gels were then

observed under ultraviolet light and photographed in a GS-800 Ultraviolet

Transilluminator (UVP, Upland, CA, USA).

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For each sample, 3-μg (0.15 μg/μl) RNA was reverse-transcribed into cDNA

using a Revert Aid First-Strand Synthesis System (Thermo Scientific, Waltham, MA,

USA) for quantitative reverse transcription PCR (RT-qPCR) with Oligo (dT) 18

primers according to the manufacturer’s protocol. The reaction was incubated using a

Peltier Thermal Cycler 200 (MJ Research, Watertown, MA, USA). cDNA integrity

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was confirmed by running 1-μg cDNA on an ethidium-bromide stained 1.5% agarose

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gel with 1X TAE buffer. Gels were treated as reported before.

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The PCR was performed in a total volume of 20 μL, containing 1 μL of each

primer (10 μM), 9 μL of the diluted single strand cDNA product and 10 μL of AceQ®
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qPCR SYBR® Master Mix (Nanjing, China). The primers' sequence is presented in
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Table 2. The RT-qPCR program was 95 °C for 10 min, followed by 40 cycles of 95 °C

for 15 s and 60 °C for 15 s and an extension at °C for 60 s. At the end of each PCR
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reaction, melting curve analysis was performed to confirm that only one PCR product
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was present in these reactions. Expression levels of the PepT1, LAT1, SLC1A5,
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TNF-α, IL-1β, IL-2, IL-8 and IL-4 genes were normalized to β-actin using the 2−ΔΔCT
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method (Schmittgen and Livak, 2008). Each sample was analyzed via RT-qPCR in
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triplicate. PepT1, LAT1, SLC1A5, TNF-α, IL-1β, IL-2, IL-8, IL-4 and β-actin genes

were retrieved from NCBI (http://www.ncbi.nlm.nih.gov/) and primers were designed

using Primer 5.0 (Table 2).

2.5. Statistical analysis

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All the data were analyzed by one-way analysis of variance (ANOVA) using

SPSS version 17.0 software (SPSS Inc., Chicago, IL, USA). When ANOVA detected

a difference among groups, Duncan’s multiple range test was used to identify the

difference in the means. Data are presented as mean ± standard error of the mean (SE).

Statistical significance was determined at P < 0.05.

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3. Results

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No significant (P > 0.05) differences were found for final body weight, weight

gain (WG) and specific growth rate (SGR) between fish fed FM and SM50 diets
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(Table 3), but increasing FM replacement level to 75% resulted in significantly (P <
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0.05) reduced growth performance. The highest feed intake was observed in the group

received SM50 diet which significantly differed from that of the FM group. However,
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both SM50 and SM75 groups had significantly lower feed efficiency (FE) and protein
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efficiency ratio (PER) than FM group. The fish fed SM75 diet exhibited significantly
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lower apparent digestibility coefficients (ADC) of dry matter and protein than the FM
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fed fish and ADC of gross energy significantly decreased at both replacement levels.
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SM75 group showed the lowest survival rate (78.8%) which significantly differed

from those of the SM50 (98.3%) and FM (100%) groups.

The results of whole-body composition analysis revealed the significant reduction

of protein and lipid contents in SM75 group compared to the other treatments, and

their values were inversely correlated with whole-body moisture and ash contents

(Table 4).
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Activity of digestive enzymes including protease, amylase and lipase in foregut

and midgut are presented in Table 5. The results revealed the drastic reduction in

activity of all the three enzymes in foregut of SM fed groups compared to the FM

group. In midgut, the highest lipase activity was observed in SM50 group which was

significantly different from that of the SM75 group.

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Replacing 75% of FM with SM resulted in significant reduction of villus height,

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villus thickness, and muscular thickness in foregut and midgut compared to the FM

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group (Table 6 and Fig. 1). Significant decreases in villus and muscular thicknesses

were observed in foregut of SM50 group, also muscular thickness was significantly
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decreased in midgut of the same group.
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The results showed that replacement of FM at both levels leads to significant

increase of serum D-lactate concentration (Fig. 2a). Also, SM75 fed fish exhibited
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significantly higher serum DAO activity than the FM fed fish (Fig. 2b).
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A significant enhancement in expression of PepT1 gene was observed in foregut

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of SM50 and SM75 groups compared to FM fed fish (Fig. 3a). Expression of LAT1
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gene was enhanced in foregut by SM replacement and a similar trend was observed
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for midgut (Fig. 3b). Also, the results showed significant enhancement of SLC1A5

expression in foregut of SM50 group and midgut of SM50 and SM75 groups (Fig.

3c).

The results of pro-inflammatory genes expression showed significant

enhancements in expression of TNF-α, IL-1β and IL-8 in foregut of SM50 and SM75

(Fig. 4a,b,d). Also, expression of IL-2 was enhanced in foregut of SM75 and midgut
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of SM50 and SM75 groups (Fig. 4c). An opposite trend to those of pro-inflammatory

genes expression was observed for the expression of the anti-inflammatory gene IL-4;

in this case SM groups had significantly lower expression of this gene (Fig. 4e).

4. Discussion

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To date, several studies have been conducted on replacement of FM with

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different types of SM in formulated feeds for Japanese seabass. Li et al. (2012)

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evaluated two types of SM including a commercial SM and a high-value SM as

alternatives to FM in diets for Japanese seabass. Their results showed that FM can be
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replaced up to 30% with commercial SM while the high-value SM could successfully
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replace 45% of FM. Later, Zhang et al. (2014) examined the effects of replacing 25,

50 and 75% of FM with untreated, gamma-irradiated or fermented SM in Japanese

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seabass feed. Their findings showed that dietary FM can be only replaced up to 25%
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when untreated SM was used, while the replacement level could be increased up to 50%
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when the gamma-irradiated SM was used. The results of a recent study by Zhang et al.
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(2016) showed that SM can substitute 60% of FM in Japanese seabass feed. Similarly,
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in the current study substituting 50% of FM with SM did not significantly impair

growth performance of Japanese seabass. The observed variations in the optimum

inclusion level of SM in diets for Japanese seabass among the above studies could be

associated with the differences in age, feeding strategy, rearing conditions, dietary

composition, and varied amount of ANFs among different studies (Lin and Luo,

2011).
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It is still unclear how dietary inclusion of SM affects feed palatability. In this

study replacing 50% of FM with SM significantly enhanced feed intake (as shown by

feeding rate) while leading to significantly lower FE and PER. In agreement to our

results, Zhang et al. (2016) reported the significant increase of feed intake in Japanese

seabass by increasing dietary SM level and this was associated with elevated feed

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conversion ratio. However, in general the effects of FM substitution with SM on fish

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feed intake have been contradictory, e.g. significantly reduced feed intake has been

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reported in Asian seabass (Tantikitti et al., 2005), spotted rose snapper (Lutjanus

guttatus) (Silva-Carrillo et al., 2012) and silvery-black porgy (Sparidentex hasta)

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(Yaghoubi et al., 2016), while significant enhancements in feed intake have been
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reported in cuneate drum (Nibea miichthioides) (Wang et al., 2006), sharpsnout

seabream (Diplodus puntazzo) (Hernández et al., 2007) and hybrid tilapia

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(Oreochromis niloticus × O. aureus) (Lin and Luo, 2011). The lower utilization of SM
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diets has been attributed to several factors such as reduced bioavailability or

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deficiencies of some minerals and essential amino acids, low digestibility of protein
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and energy, and presence of ANFs (Silva-Carrillo et al., 2012; Song et al., 2014;
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Tantikitti et al., 2005; Trushenski et al., 2014; Yaghoubi et al., 2016; Ye et al., 2011;

Zhou et al., 2011). It has been earlier noted that increment of feed intake in response

to increased dietary SM inclusion enhances feed conversion ratio probably resulting

from reduced total diet digestibility (Hernández et al., 2007). In the present study,

significantly lower ADC of dry matter and protein were observed in SM75 group, and

ADC of gross energy was significantly decreased in both inclusion levels of SM.
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Additionally, significantly decreased digestive enzymes activity were observed in

foregut of SM groups. Theses alterations could be the reason for reduced FE and PER

in SM fed groups in the current study.

Replacement of FM with SM resulted in significantly reduced protease, amylase

and lipase activity in foregut of Japanese seabass and this was more evident in SM75

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group. In agreement to our results, Murashita et al. (2015) reported significantly

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reduced trypsin, chymotrypsin, lipase and amylase activity in intestinal content of red

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seabream (Pagrus major) fed SM diet compared to FM fed fish. Similar findings have

also been shown in Atlantic cod (Gadus morhua) (Lemieux et al., 1999), Atlantic
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salmon (Salmo salar L.) (Krogdahl et al., 2003), and hybrid tilapia (Lin and Luo,
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2011). In the current study, similar trends were observed for digestive enzymes

activity, feed utilization and growth performance suggesting that alterations in

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digestive enzymes activity affect digestion (as highlighted by lower ADCs) and
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nutrients absorption (Lemieux et al., 1999). There are an array of factors that impact
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digestive enzymes secretion in fish including feeding habits, feed preferences, diet
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formulation, and ANFs (Escaffre et al., 1997; Hidalgo et al., 1999; Pavasovic et al.,
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2007). Murashita et al. (2015) reported lower gene expression levels of the digestive

enzymes in the hepatopancreas of red seabream fed SM diet compared with the FM

fed fish. They demonstrated that FM diet stimulates secretion/synthesis of pancreatic

digestive enzymes to a larger extent than the SM diet. Also, they suggested that a

water-soluble fraction of FM is responsible for increased gene expression of trypsin,

lipase, cholecystokinin and cholecystokinin receptor (cck-1r) in FM fed fish. On the

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other hand, SM contains different ANFs such as protease inhibitors that may reduce

the activity of digestive enzymes in fish (Hendricks and Bailey, 1989; Huisman and

Tolman, 1992; Liener, 1989).

In the present study, the group received SM75 diet exhibited significantly lower

whole-body protein and lipid contents than the other treatments, while an opposite

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trend was observed for moisture and ash contents. Similarly, Zhang et al. (2014, 2016)

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found a decreasing tendency in body lipid content of Japanese seabass by increasing

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replacement level of FM with SM. However, Li et al. (2012) could not find any

specific changes in body composition of Japanese seabass when up to 60% of FM was

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replaced with SM. Reduced whole-body protein and/ lipid contents by replacement of
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FM with SM have also been reported in Asian seabass (Tantikitti et al., 2005), cuneate

drum (Wang et al., 2006), spotted rose snapper (Silva-Carrillo et al., 2012), and
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marbled spinefoot (Siganus rivulatus) (Monzer et al., 2017). The observed trend for
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whole-body protein and lipid was consistent with the results of growth performance,
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diet digestibility and digestive enzymes activity, indicating that replacing FM with
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SM results in lower digestibility of nutrients leading to reduced growth performance

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(Zhang et al., 2014).

Epithelium of the intestinal mucosa acts as a physical barrier that allows

nutrients to be absorbed while blocking the intrusion of pathogens (Faggio et al., 2011;

Lauriano et al., 2016; Sun et al., 1998); therefore an intact intestinal mucosal barrier is

important for proper function of intestine. Intestinal mucosal barrier function is

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predominantly evaluated through indirect measures based on determinations of

intestinal permeability, serum D-lactate concentration and DAO activity. D-Lactate

and DAO are considered as the two key indicators of intestinal mucosal integrity.

Their values in serum are highly elevated in cases of intestinal mucosal barrier

damage (Luk et al., 1980; Vella and Farrugia, 1998). DAO is the marker enzyme of

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intestinal mucosal cells that exists in low levels in serum under normal conditions, but

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it is released into the blood stream when these cells are damaged. In the current study,

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serum D-lactate concentration increased in SM50 group, and both D-Lactate

concentration and DAO activity were enhanced in SM75 group indicating increased
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intestinal permeability and impaired intestinal mucosal barrier function. To our
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knowledge, this is the first report on the effects of plant proteins on intestinal barrier

function in fish and the underlying mechanism remains to be elucidated in the future
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studies.
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To date, research on farmed fish species has mainly focused on the performance
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and the activity of the brush border enzymes and less attention has been paid to the
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molecular aspects. The end products of protein digestion in fish constitute a mixture
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of free amino acids and small peptides that are efficiently absorbed across the small

intestinal epithelium (Clements and Raubenheimer, 2006). PepT1 has been identified

as the key transporter of di- and tripeptides from the intestinal lumen into the

enterocytes (Daniel, 2004). There are increasing evidences showing that it plays a

major role in fish nutrition (Dabrowski et al., 2005). A large amount of amino acids is

transported by PepT1 in peptide form providing essential nutrients for fish growth. It
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has been demonstrated that expression of genes encoding enzymes and transporters of

the brush-border membrane is crucial for the final stages of digestion and absorption

of nutrients through the epithelial luminal membrane of the intestine (Hakim et al.,

2009). There are some reports indicating that activity and expression of PepT1 can be

modulated by diet particularly the dietary protein source (Ostaszewska et al., 2010a;

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Ostaszewska et al., 2010b). In the present study, replacing FM with SM led to

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enhanced expression of transporters genes such as PepT1, LAT1 and SLC1A5. Little

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information is available about the effects of diet composition on the intestinal gene

expression of PepT1 in fish. Hakim et al. (2009) showed that feed deprivation
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enhances expression of PepT1 mRNA in European sea bass (Dicentrarchus labrax). It
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has been demonstrated that animals are able to adapt themselves to the changes in

feed composition and nutrients availability through altering the types and amounts of
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nutrient transporters (Humphrey et al., 2004). It seems that there is a homeostatic

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control at the gastrointestinal level enabling the fish to maximize gastrointestinal

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function for maximum uptake of protein products. This includes the alterations in
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spatial expression of transporters along the post-gastric canal in order to maximize

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absorption of di- and tripeptide and reduce protein loss (Bakke et al., 2010). Taking

these into account, the increased expression of transporters genes in SM groups in the

current study could be attributed to the lower availability of amino acids from SM

compared to FM.

In the present study, replacing FM with SM resulted in shortening of villus

height, and reduced villus and muscular thickness both in foregut and midgut of
 ACCEPTED MANUSCRIPT

Japanese seabass (as shown in Fig. 1), and increasing SM inclusion level aggravated

the negative effects indicating likely occurrence of SM-induced enteritis. A recent

study by J. Wang et al. (2017) showed the incidence of enteritis in Japanese seabass

fed a diet in which 50% of FM was substituted with SM. These authors reported

reduced microvillus height and muscular thickness in distal intestine of Japanese

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seabass following SM administration. These results are also consistent with previous

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findings in rainbow trout (Romarheim et al., 2008), giant grouper (Epinephelus

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lanceolatusand) (Garcia-Ortega et al., 2016), and orange-spotted grouper

(Epinephelus coioides) (Y. Wang et al., 2017). It has been pointed out that
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up-regulated expression of inflammatory genes is a sign of dysfunction caused by the
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SM-induced enteritis (Gu et al., 2016). In the current study, expression of

pro-inflammatory genes such as TNF-α, IL-1β and IL-8 and IL-2 was enhanced by
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SM inclusion providing further evidence for incidence of SM-induced enteritis in

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Japanese seabass. The negative impacts of high dietary SM on intestinal structure

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have been ascribed to its ANFs content such as saponins (Chen et al., 2011), raffinose,
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stachyose and lectins (Peng et al., 2013; Venou et al., 2006). Buttle et al. (2001)
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reported that lectin combining with polysaccharides on gut epithelial cell surface

could destruct gut microvillus leading to reduced absorption and digestion of nutrients.

Recently, Krogdahl et al. (2015) declared that soya saponins alone are able to cause

SM-induced enteritis in Atlantic salmon. Also, there are several reports indicating that

other ANFs in SM may aggravate the saponins effects (Bureau et al., 1998; Knudsen

et al., 2008).
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Cytokines are categorized to pro-inflammatory or anti-inflammatory depending

on their properties (Polińska et al., 2009; Sartor, 1995). It has been noted that SM can

cause intestinal inflammation by increasing pro-inflammatory cytokines and

decreasing anti-inflammatory cytokines level (Y. Wang et al., 2017). Up-regulated

expression of pro-inflammatory genes by SM administration has been reported in

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several fish species including Atlantic salmon (Marjara et al., 2012), common carp

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(Cyprinus carpio L.) (Urán et al., 2008), zebra fish (Hedrera et al., 2013), turbot

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(Scophthalmus maximus) (Gu et al., 2016) and orange-spotted grouper (Y. Wang et al.,

2017). Similarly, in the present study expression of pro-inflammatory genes including

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TNF-α, IL-1β, IL-8 and IL-2 was significantly enhanced by replacing FM with SM.
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IL-4 is considered as an anti-inflammatory cytokine and its expression level changes

in response to gut inflammation (Kołodziejska-Sawerska et al., 2013). In the current

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study, expression of IL-4 was down-regulated by SM inclusion; this is in agreement

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with Urán et al. (2008), who reported the up-regulation of pro-inflammatory IL-1β
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and TNF-α1 genes, and down-regulation of the anti-inflammatory IL-10 in common

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carp fed a diet in which 20% of FM was replaced with SM. They suggested that IL-1β
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and TNF-α1 are both involved in the enteritis process as their expression level in

SM-fed fish was higher than FM fed fish after 1, 3 and 5 weeks of feeding on the SM

diet.

In conclusion, the findings in this study showed that although replacing 50% of

FM with SM did not significantly influence growth performance of Japanese seabass,

it had adverse effects on digestive enzymes activity, and gut health. Further studies
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with lower replacement levels than in this study are required for determination of

optimum replacement level of FM with SM in diets for Japanese seabass.

Acknowledgements

This work was supported by the National Natural Science Foundation of China

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(Grant no. 31572625) and China Agriculture Research System (CARS-47).

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Fig.1. Foregut (A: FM, B: SM50, C: SM75) and midgut (D: FM, E: SM50, F: SM75)
structure of Japanese seabass fed the experimental diets for 8 weeks.
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a: D-lactate b: DAO
1.0
Serum D-lactate concentration (nmol/ml)
b b 35
b

Serum diamin oxidase activity (U/L)

0.8 30

25
0.6 a a
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0.4 15

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0.2 a 5

0.0 0

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FM SM50 SM75 FM SM50 SM75

Fig. 2. Effects of substituting fishmeal with soybean meal on serum D-lactate

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concentration (a) and diamine oxidase (DAO) activity (b) of Japanese seabass fed the
experimental diets for 8 weeks.

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Fig. 3. Relative expression of PepT1 (a), LAT1 (b) and SLC1A5 (c) in foregut and
midgut of Japanese seabass fed the experimental diets for 8 weeks.
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Fig. 4. Relative expression of TNF-α (a), IL-1β (b), IL-2 (c), IL-8 (d), and IL-4 (e) in
foregut and midgut of Japanese seabass fed the experimental diets for 8 weeks.
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Table 1. Formulation and proximate composition of the experiment diets (% dry

matter)

Ingredients FM SM50 SM75

a 42 21 10.5
Fish meal
Soybean mealb 0 30 45
Wheat flour 41.4 24.92 15.13
Wheat gluten 5.4 9.6 11.5

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Squid liver meal 1.5 1.5 1.5
Fish oil 2.3 4 5.7

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Soybean oil 3.5 3.1 2.7
Lecithin 2 2 2

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L-ascorbyl polyphosphate 0.1 0.1 0.1
Vitamin premixc 0.3 0.3 0.3
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Mineral premixd 0.5 0.5 0.5
Choline chloride 0.5 0.5 0.5
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Calcium dihydrogen phosphate 0 1.54 3.03

Yttrium oxide 0.5 0.5 0.5
Methionine 0 0.19 0.41
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Lysine 0 0.25 0.63

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Proximate composition (%)

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Dry matter (DM) 86.4 86.3 89.8

In DM:
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Protein 42.63 43.24 43.04

Lipid 12.88 13.19 13.12
Ash 8.43 7.98 7.53
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a
Xiamen ITG group Corp., Ltd., Xiamen, China, imported from Peru. (crude protein:
69.4%, crude lipid:7.8%).
b
Soybean meal, obtained from Quanzhou Fuhai cereals and oils industry Co., Ltd.
(crude protein: 46.5%, crude lipid: 1.6%).
c
Vitamin premix (mg or g kg-1 diet): thiamin, 10 mg; riboflavin, 8 mg; pyridoxine HCl,
10 mg; vitamin B12, 0.2 mg, vitamin K3, 10 mg; inositol, 100 mg; pantothenic acid,
20 mg; niacin acid, 50 mg; folic acid, 2 mg; biotin, 2 mg; retinol acetate, 400 mg;
cholecalciferol, 5 mg; alpha-tocopherol,100 mg; ethoxyquin, 150 mg; wheat middling,
1.1328 g.
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d
Mineral premix (mg or g kg-1 diet): Na F, 2 mg; KI,0.8 mg; Co Cl2·6H2O (1%), 50
mg; Cu SO4· 5H2O, 10mg; FeSO4· H2O, 80 mg; ZnSO4·H2O, 50 mg; Mn
SO4·H2O,25 mg; MgSO4· 7H2O, 200 mg; Zoelite, 4.582g.

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Table 2. Sequence of the primers used for real-time PCR

Target genes Forward primer (5'-3') Reverse primer (5'-3') Annealing temperature (°C)

β-actin F: AACTGGGATGACATGGAGAAG R:TTGGCTTTGGGGTTCAGG

P T 60
SLC1A5 F:GACGTGCGGTCCACAAAATG R:TCATCCTGGCTGTTGACTGG
R I 60
LAT1
PEPT1
F:TGGCCTACTTCACCACCATT
F:GACTTCTGCAGCTGACTTCG
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R:CGGAGTGAAGAGGTCTGTGT
R:TCGGCCCAAAGTCAAAAGTG
60
60
TNF-α F:GATCGTCATCCCACAAACCG
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R:GCTTTGCTGCCTATGGAGTC
N
60
IL-1β
IL-2
F:GTCAACTTACGTGCACCCTG
F:GGGAAAGTGTCACATGTCCG
M A
R:AAATCGTACCATGTCGCTGC
R:ACGGCCTGGTTTAAAGATGC
60
60
IL-4 F:ACCATGCATTACTACAGCACTG R:CACATTCAGGGGCGTTTGTC 60
IL-8 F:GGATCAGTTTCTTCACCCAGG
E D R:CAGGTGGAGTCGAGGATCAT 60

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Table 3. Growth performance, feed utilization and apparent digestibility coefficients

(ADC) of Japanese seabass fed the experimental diets for 8 weeks

Item FM SM50 SM75

a a
Final body weight (g) 59.10±1.26 55.03±2.06 38.83±1.16b
WGa (%） 1078±24.9a 1018±32.9a 799±46.4b
SGRb (%/d） 4.40±0.04a 4.32±0.04a 3.74±0.05b
Feeding ratec (%/d) 3.26±0.07b 3.47±0.04a 3.39±0.03ab

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FEd 0.92±0.02a 0.86±0.01b 0.82±0.01b
PERe 2.17±0.05a 1.98±0.03b 1.91±0.03b

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ADCd (%)f 81.8±0.21a 80.2±1.54a 75.2±0.95b
ADCp (%)g 95.7±0.25a 95.4±0.66ab 94.4±0.15b

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ADCge (%)h 87.3±0.07a 85.9±0.21b 84.2±0.08c
Survival (%) 100±0.00a 98.3±1.67a 78.8±4.73b
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Values are presented as mean ± SE. Values in the same raw having different
superscript letters are significantly different (P < 0.05).
a
Weight gain = [(final body weight − initial body weight) / initial body weight] × 100.
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b
Specific growth rate = [(ln final body weight − ln initial body weight) / days] × 100.
c
Feeding rate =100 × feed intake /(Final weight(g)/2+initial weight(g)/2)/t.
d
Feed efficiency = weight gain/dry feed fed.
e
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Protein efficiency ratio = (total final weight(g)- total initial weight(g))/total dry
protein consumed (g).
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f
Apparent digestibility coefficient of dry matter.
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g
Apparent digestibility coefficient of protein.
h
Apparent digestibility coefficient of gross energy.
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Table 4. Whole-body composition of Japanese seabass fed the experimental diets for
8 weeks (% wet weight)

Diets Moisture Protein Lipid Ash

b a a
FM 67.85±0.22 17.62±0.04 10.28±0.18 4.71±0.06b
SM50 67.13±0.11b 17.77±0.04a 10.43±0.19a 4.94±0.03a
SM75 69.16±0.34a 16.86±0.28b 8.31±0.06b 5.02±0.04a
Values are presented as mean ± SE. Values in the same column having different

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superscript letters are significantly different (P < 0.05).

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Table 5. Digestive enzymes activity (U/mg prot) in foregut and midgut of Japanese seabass fed the experimental diets for 8 weeks

Foregut

P T Midgut

Diets Protease Amylase Lipase Protease

R IAmylase Lipase

FM 82.94±1.31a 1.13±0.17a 492.25±31.45a

S C
26.60±0.91a 0.51±0.02a 118.26±32.42ab

SM50 35.00±6.65b 0.47±0.01b 345.45±45.26b

N U
24.17±3.52a 0.30±0.06a 178.58±24.89a
SM75 22.39±0.92b 0.44±0.01b 307.04±32.19b

M A 21.80±1.08a 0.36±0.12a 65.99±6.75b

Values are presented as mean ± SE. Values in the same column having different superscript letters are significantly different (P < 0.05).

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Table 6. Foregut and midgut morphology of Japanese seabass fed the experimental
diets for 8 weeks

Villus height Villus thickness Muscular

Diets
(μm) (μm) thickness (μm)
FM 443.22±8.75a 49.22±1.27a 152.72±0.79a
Foregut SM50 430.42±3.68a 36.25±2.31b 80.19±3.89b
SM75 287.83±6.83b 29.63±1.61c 77.95±2.72b

FM 368.95±3.86a 52.26±2.09a 119.56±4.45a

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Midgut SM50 352.83±20.00a 47.50±3.37ab 89.39±2.73b
SM75 285.67±4.56b 41.71±2.86b 93.05±5.80b

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superscript letters are significantly different (P < 0.05).

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Highlights
►Effects of high soybean meal (SM) diets were evaluated in Japanese seabass.

►Growth performance was not influenced by replacing 50% of fish meal with SM.

► SM fed fish exhibited reduced feed utilization and digestive enzymes activity. ►

SM adversely affected morphological and molecular characteristics of fish gut.

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