Dietary effects of adenosine monophosphate to enhance growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream, Pagrus major

Accepted Manuscript Dietary effects of adenosine monophosphate to enhance growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream, Pagrus major Md. Sakhawat Hossain, Shunsuke Koshio, Manabu Ishikawa, Saichiro Yokoyama, Nadia Mahjabin Sony PII:

S1050-4648(16)30484-3

DOI:

10.1016/j.fsi.2016.08.009

Reference:

YFSIM 4109

To appear in:

Fish and Shellfish Immunology

Received Date: 20 April 2016 Revised Date:

18 July 2016

Accepted Date: 7 August 2016

Please cite this article as: Hossain MS, Koshio S, Ishikawa M, Yokoyama S, Sony NM, Dietary effects of adenosine monophosphate to enhance growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream, Pagrus major, Fish and Shellfish Immunology (2016), doi: 10.1016/ j.fsi.2016.08.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Dietary effects of adenosine monophosphate to enhance growth,

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digestibility, innate immune responses and stress resistance of juvenile red

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sea bream, Pagrus major

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Md. Sakhawat Hossain a, b,c*, Shunsuke Koshio a, b, Manabu Ishikawa a,b, Saichiro Yokoyamaa,

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Nadia Mahjabin Sonya

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a

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Shimoarata 4-50-20, Kagoshima 890-0056, Japan

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b

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21-24, Kagoshima 890-0065, Japan

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c

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3100, Bangladesh

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Laboratory of Aquatic Animal Nutrition, Faculty of Fisheries, Kagoshima University,

The United Graduate School of Agricultural Sciences, Kagoshima University, Korimoto 1-

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Department of Aquaculture, Faculty of Fisheries, Sylhet Agricultural University, Sylhet-

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*

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Md. Sakhawat Hossain

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Laboratory of Aquatic Animal Nutrition

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Faculty of Fisheries, Kagoshima University

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Shimoarata 4–50–20; Kagoshima City; Kagoshima 890–0056, Japan

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Phone: +81 99 286 4182; Fax: +81 99 286 4184

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Email: [email protected]

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Corresponding Author

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ACCEPTED MANUSCRIPT ABSTRACT

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Our study explored the dietary effects of adenosine monophosphate (AMP) to enhance

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growth, digestibility, innate immune responses and stress resistance of juvenile red sea bream.

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A semi-purified basal diet supplemented with 0% (Control), 0.1% (AMP-0.1), 0.2% (AMP-

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0.2), 0.4% (AMP-0.4) and 0.8% (AMP-0.8) purified AMP to formulate five experimental

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diets. Each diet was randomly allocated to triplicate groups of fish (mean initial weight 3.4g)

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for 56 days. The results indicated that dietary AMP supplements tended to improve growth

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performances.One of the best ones was found in diet group AMP-0.2, followed by diet

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groups AMP-0.1, AMP-0.4 and AMP-0.8. The Apparent digestibility coefficients (dry matter,

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protein and lipid) also improved by AMP supplementation and the significantly highest dry

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matter digestibility was observed in diet group AMP-0.2. Fish fed diet groups AMP-0.2 and

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AMP-0.4 had significantly higher peroxidase and bactericidal activities than fish fed the

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control diet. Nitro-blue-tetrazolium (NBT) activity was found to be significantly (P < 0.05)

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greater in fish fed diet groups AMP-0.4 and AMP-0.8. Total serum protein, lysozyme activity

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and agglutination antibody titer were also increased (P>0.05) by dietary supplementation. In

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contrast, catalase activity decreased with AMP supplementation. Moreover, the fish fed AMP

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supplemented diets had better improvement (P<0.05) in body lipid contents, condition factor,

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hematocrit content and glutamyl oxaloacetic transaminase (GOT) level than the control group.

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Supplementation also improved both freshwater and oxidative stress resistances. Interestingly,

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the fish fed diet group AMP-0.2 and AMP-0.4 showed the least oxidative stress condition.

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Finally it is concluded that, dietary AMP supplementation enhanced the growth, digestibility,

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immune response and stress resistance of red sea bream. The regression analysis revealed that

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a dietary AMP supplementation between 0.2 - 0.4 % supported weight gain and lysozyme

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activity as a marker of immune functions for red sea bream, which is also inline with the

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most of the growth and health performance parameters of fish under present experimental

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conditions.

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Keywords: Adenosine monophosphate, Growth, Digestibility, Innate immune responses,

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Oxidative stress, Pagrus major

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1. Introduction

Red sea bream (Pagrus major) is one of the most cultured marine fish species in East

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Asian countries, particularly in Japan and Korea, due to its desirable taste, high market

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demand and its traditional consumption. However, intensive cultures of this species are

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particularly prone to bacterial, viral, parasitic and other environmental diseases due to the

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deterioration of water quality and elevation of stress in culture environments. In aquaculture

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antibiotic, vaccination and other chemicals are currently used to varying degrees to control

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the aforementioned diseases. However, each of these treatment methods has its drawbacks,

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including suppression of aquatic animals’ immune systems, environmental hazards and food

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safety problems. Moreover, the development of antibiotic resistance in humans has led to a

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growing interest in antibiotic-free animal production worldwide [1]. In such circumstances,

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dietary supplementation of health promoting functional compounds has turned out to be

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increasingly of interest as an effective alternative for prophylactic treatment against disease

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outbreaks in intensive aquaculture. Recently aquacultural research has paid increasing

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attention to nucleotides and their related products has due to their promise as potential

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immunomodulators as well as functional nutrients [2, 3].

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Nucleotides are the base units for deoxyribonucleic acid (DNA) and ribonucleic acid

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(RNA) synthesis during cell construction and provide energy for normal cellular process and

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are therefore essential to growth and development [4]. Dietary supplementation of

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nucleotides has been shown to benefit many mammalian physiological and nutritional

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ACCEPTED MANUSCRIPT functions [5-8]. In aquatic animals both nucleotides and nucleosides have long been

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implicated as feed attractants in both vertebrate and invertebrate species [9-12]. However,

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research into potential growth and health benefits of dietary nucleotides in aquaculture

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species did not begin until the early 2000s [13]. To date, research pertaining to nucleotide

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nutrition in fishes has shown rather consistent and encouraging beneficial results in fish

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health management [2, 3]. Research to date on dietary nucleotides has focused on a mixture

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of nucleotides, rather than specific types of nucleotides except inosine and inosine

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monophosphate (IMP) in research on feed stimulants [14].

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(AMP) is a purine nucleotide which consists of a phosphate group, the sugar ribose, and the

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nucleobase adenine. It is an ester of phosphoric acid and the nucleoside adenosine and used

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as a monomer of RNA. In aquaculture AMP has been studied most extensively as a part of

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mixed nucleotide rather than considered as individual nucleotide for promoting growth, feed

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utilization and potential heath benefit. Mackie [15] and Kiyohara et al. [16] initially reported

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the chemo-attractant properties of AMP for lobster and puffer fish respectively. Lin et al. [17]

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reported increased growth and non specific immune performance of red drum (Epinephelus

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malabaricus). However, studies on nucleotide nutrition of red sea bream are very scarce.

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Recently, we evaluated mixed nucleotide effects on this species [2]. However, to the best of

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our knowledge there is no research on individual nucleotide AMP as feeding stimulant as

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well as a functional nutrient for potential growth and health benefit of red sea bream.

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Therefore, the present study was conducted to assess the dietary effects of AMP on growth

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performance, feed utilization, digestibility, innate immune response and stress resistance of

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red sea bream juveniles.

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Adenosine monophosphate

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2. Materials and Methods

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2.1. Test fish and experimental system

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ACCEPTED MANUSCRIPT Juvenile red sea bream were obtained from a private hatchery (Ogata Suisan), in

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Kumamoto prefecture, Japan, and transported to the Kamoike Marine Production Laboratory,

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Faculty of Fisheries, Kagoshima University, Japan. The fish were maintained in 500 L tank

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with continuous aeration and flow through sea water and fed a commercial formulated diet

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(54% crude protein and 10% crude lipid, Higashimaru Foods, Kagoshima Japan) for one

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week to acclimatize to the experimental facilities and water condition. The feeding trial was

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carried out in 100 L polycarbonate tanks (filled with 80 L of water) in a flow through sea

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water system where each tank was equipped with an inlet, outlet, and continuous aeration.

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The tanks were maintained with a natural light/dark regime. The seawater was pumped from

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the deep basin of Kagoshima Bay, Japan and gravel filtered and supplied to the system. A

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flow rate of 1.5 L min-1 was maintained throughout the experimental period.

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2.2. Ingredients and test diets

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Tables 1and 2 summarize the basal diet formulation and chemical composition of the

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experimental diets, respectively. Five fishmeal and casein based semi-purified diets were

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formulated to be nearly isonitrogenous (54% crude protein), isolipidic (11% crude lipid) and

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isocaloric (22 KJ g−1 gross energy). The experiential diets were prepared by supplementing

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purified AMP nucleotide as disodium salts (Sigma Aldrich Co., St. Louis, MO, USA) to the

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basal diet at concentrations of 0.1, 0.2, 0.4 and 0.8 % for the diet groups AMP-0.1, AMP-0.2,

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AMP-0.4 and AMP-0.8 respectively. Basal diet without AMP supplementation was used as

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control. For proper mixing of AMP with other ingredients, initially AMP and weighted

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arginine of the respective diets, according to basal diet formulation were mixed properly in a

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glass beaker by hand with the help of a stainless laboratory spatula. This mixture was then

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thoroughly mixed with other dry ingredients in a food mixer for 10 min. Pollack liver oil and

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soybean lecithin were premixed with a sonicator (CA-4488Z, Kaijo Corporation, Tokyo,

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ACCEPTED MANUSCRIPT Japan), added to the dry ingredients and mixed for another 10 min. Water was added

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gradually (35–40% of the dry ingredients), to the premixed ingredients and mixed for another

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10 min. The pH of the diets was adjusted to the range of 7.0–7.5 with 4 N sodium hydroxide.

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The mixture was then passed through a meat grinder with an appropriate diameter (1.2–2.2

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mm) to prepare pellets, which were then dried in a dry-air mechanical convection oven (DK

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400, Yamato Scientific, Tokyo, Japan) at 55 °C for about 150 min. The test diets were stored

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at −28 °C in a freezer until use.

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2.3. Feeding protocol

After being acclimatized to the laboratory environment, homogenous sized juveniles

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were sorted. Triplicate groups of fish were assigned to each dietary treatment. Twenty fish,

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having a mean initial body weight of 3.4±0.01 g were randomly allocated to fifteen

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previously prepared tanks. Fish were fed the experimental diets for 56 days by hand twice a

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day to visual satiation at 9.00 and 16.00 h. At the end of each day, daily feed supplied was

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obtained through the subtraction of the remaining feed from the previous day’s feeding and

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the amount was recorded. The uneaten feed was also collected 1 hour after feeding and dried,

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weighed and finally subtracted from the total amount of supplied diets to calculate the actual

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feed intake. All fish were weighed in bulk fortnightly to determine growth and to visually

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check their health condition. The monitored water quality parameters were: water

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temperature 22.6±1.9 °C; pH 8.1±0.7 and salinity 34.5±0.5 during the feeding trial. These

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ranges are considered within optimal values for juvenile red sea bream.

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2.4. Evaluation of performance parameters The following variables were evaluated: Weight gain (%) = (final weight – initial weight) × 100 / initial weight

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ACCEPTED MANUSCRIPT Specific growth rate (SGR, % day-1) = {Ln (final weight) – Ln (initial weight) / duration} ×

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100

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Survival (%) = 100 × (final no. of fish / initial no. of fish)

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Feed intake (g fish-1 56 days-1) = (dry diet given – dry remaining diet recovered) / no of fish

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Feed conversion efficiency (FCE) = live weight gain (g) / dry feed intake (g)

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Protein efficiency ratio (PER) = live weight gain (g) / dry protein intake (g)

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Condition factor (CF, %) = weight of fish / (length of fish)3 × 100

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2.5. Digestibility assessment

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A digestibility trial was conducted at the end of the growth trial; the remaining fish from the

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same treatments were distributed randomly into triplicate tanks. The fish were fed a diet

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containing chromium oxide (Wako Pure Chemical Industries, Ltd) as the inert marker at a

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level of 0.5% (Cr2O3, 5 g kg-1) to the previous formulation and fed to the fish under the same

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condition as the growth experiment apart from the difference in stocking density. After 5-day

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adaptation of the diets, feces were collected at 3-h intervals between the morning and

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afternoon feeds by using a siphon for two weeks. Feces of red sea bream very rapidly settled

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to the bottom of the tank and did not easily break up in the water so that nutrient and marker

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losses were minimized. Feces collection continued for two weeks until a sufficient amount of

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feces had been collected for analysis. Feces were freeze-dried freeze-dried immediately kept

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at - 20oC until analysis. The concentration of chromium oxide in diets and feces was

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determined according to Furukawa and Tsukahara [18].

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2.6. Sample collection

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At the end of the feeding trial, all experimental fish were fasted for 24 h. All the fish

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were anesthetized with Eugenol (4-allylmethoxyphenol, Wako Pure Chemical Ind., Osaka,

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Japan) at 50 mg L−1. Then individual body weight of fish was measured, and the growth

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ACCEPTED MANUSCRIPT parameters were calculated accordingly. Nine fish per treatment (three fish per each tank)

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were randomly collected, washed with distilled water and then stored at −80 °C (MDF-U383,

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SANYO, Tokyo, Japan) for the whole body proximate analysis. Six fish were randomly

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sampled from each dietary replicate tank and their blood were collected by puncture of the

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caudal vain using heparinized (1600 IU/ml, Nacalai Tesque, Kyoto, Japan) disposable

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syringes and pooled. In addition non-heparinized disposable syringes were used to collect

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blood for serum analysis. Partial heparinized whole blood was used to analyze the hematocrit

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levels while plasma and serum were obtained by centrifugation at 3000 ×g for 15 min under

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4 °C, and then stored at −80 °C until the analysis. Liver and viscera were dissected out from

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the fish identified above and were weighed individually to calculate the hepatosomatic index

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(HSI) and viscerasomatic index (VSI) using the following formula:

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Hepatosomatic index (HSI, %) = weight of liver / weight of fish × 100

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Viscerasomatic index (VSI, %)= viscera weight/fish weight × 100

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2.7. Biochemical analysis

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The ingredients, diets and fish whole body were analyzed in triplicate, using standard

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methods [19]. The moisture was determined by drying the sample at 105 °C to constant

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weight. The ash was analyzed by combustion at 550 °C for 12 h. The crude protein content

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was determined by measuring the nitrogen content (N × 6.25) using the Kjeldahl method with

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a Tecator Kjeltec System (1007 Digestion System, 1002 Distilling unit, and Titration unit;

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FOSS Tecator AB, Högendäs, Sweden). Crude lipid content was estimated using gravimetric

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method (954.02). Total serum protein (TSP) and plasma chemical parameters such as total

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albumin, total bilirubin, glucose, glutamyl oxaloacetic transaminase (GOT), glutamic-

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pyruvate transaminase (GPT), blood urea nitrogen (BUN), triglycerides and total cholesterol

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(T-Cho) were measured spectrophotometrically with an automated analyzer (SPOTCHEM™

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ACCEPTED MANUSCRIPT EZ model SP-4430, Arkray, Inc. Kyoto, Japan). Biological antioxidant potential (BAP) and

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reactive oxygen metabolites (d-ROMs) were also measured spectrophotometrically from

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blood plasma using commercial BAP (D1-002c) and d-ROM (D1-002b) test kits (Uisuma

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corporation, Tokyo, Japan) with an automated analyzer FRAS4, Diacron International s.r.l.,

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Grosseto, Italy by following the method described previously [20].

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2.8. Evaluation of immune parameters

Serum lysozyme activity (LA) was measured with turbidimetric assays [21]. Ten micro-

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liters of samples was put into well of microplate, then added 190 micro-liters of substrate (0.2

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mg Micrococcus lysodeikticus, lyophilized cell, Sigma, USA)/ml PBS, pH 7.4,their

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absorbance were measured after 1 and 5 min, which were incubated with gentle shaking at

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constant room temperature, at 450 nm with ImmunoMini NJ-2300 (System Instruments,

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Tokyo, Japan). A unit of enzyme activity was defined as the amount of enzyme that causes a

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decrease in absorbance of 0.001/min.

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Oxidative radical production by phagocytes during respiratory burst was measured

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through the NBT (nitro-blue-tetrazolium) assay described by Anderson and Siwicki [22] with

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some modifications. Briefly, blood and 0.2% NBT (Nacalai Tesque, Kyoto, Japan) were

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mixed in equal proportion (1:1), incubated for 30 min at room temperature. Then 50 mL was

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taken out and dispensed into glass tubes. One milliliter of dimethylformamide (Sigma) was

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added and centrifuged at 2000 × g for 5 min. Finally, the optical density of supernatant was

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measured at 540 nm using a spectrophotometer (Hitachi U-1000, Japan). Dimethylformamide

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was used as the blank.

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Serum bactericidal activity (BA) was assessed according to Yamamoto and Iida [23].

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Samples were diluted 3, 4 and 5 times with a Tris buffer (pH 7.5). The dilutions were mixed

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with a bacterial suspension (0.001 g/mL, Escherichia coli, IAM1239 cell line, Kagoshima,

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ACCEPTED MANUSCRIPT Japan) (Promega, Germany) and incubated at 25 °C for 24 h in micro tube rotator (MTR-103,

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AS ONE, Osaka, Japan). 50 microliter of the reaction solutions were incubated on TSA

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(Trypto-Soya agar, Nissui Pharmaceutical Co. Ltd., Japan) at 25 °C for 24 h (on X-plate).

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Colony forming unit (CFU) was counted by the plate counting method as described by Ren et

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al. [24]. The bactericidal activity was defined as follows, (CFU of blank group-CFU of each

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group)/CFU of blank group × 100.

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The total peroxidase activity (PA) in serum was measured according to Salinas et al. [25],

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with some modifications. Briefly, 15 µL of serum were diluted with 35 µL of Hank's

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Buffered salt solution (HBSS) without Ca+2 or Mg+2 in flat-bottomed 96-well plates. Then, 50

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µL of peroxidase substrate (3, 3', 5, 5'-tetramethylbenzidine hydrochloride) (TMB; Thermo

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Scientific Inc., USA) was added. The serum mixture was incubated for 15 min. The colour-

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developing reaction in serum samples was stopped by adding 50 µL of 2M sulphuric acid and

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the OD (450 nm) was measured in a plate reader. PBS was used as a blank instead of serum.

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The catalase activity (CAT) assay was performed using spectrophotometric determination

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of hydrogen peroxide (H2O2) which forms a stable complex with ammonium molybdate that

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absorbs at 405 nm [26]. The serum (50 µL) was added to the 1.0 ml substrate (65 µmol per

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ml H2O2 in 60 mmol/L phosphate buffer, pH 7.4) and incubated for 60 s at 37 °C. The

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enzymatic reaction was stopped with 1.0 ml of 32.4 mmol/L ammonium molybdate ((NH4)6

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Mo7O24 .4 H2O) and the yellow complex of molybdate and hydrogen peroxide is measured at

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405 nm against reagent blank. One unit of CAT decomposes 1 µmole of hydrogen peroxide/l

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minute under assay conditions. CAT activities are expressed as kilo unit per liter (kU/L)

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The agglutination antibody titer was conducted in a round bottomed ‘U’ shaped micro titer

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plates after Swain et al. [27] with slight modifications. 50 µL of serum was serially diluted in

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PBS (1/2, 1/4, 1/8, 1/16, 1/32and 1/64) and then equal volumes of Vibrio anguillarum (1×108

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cfu mL-1) was added to wells and kept for 24 h at 4 0C. The reciprocal of the highest dilution

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that gave agglutination was taken as the agglutination antibody titer which is expressed as

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log10.

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2.9. Fresh water stress test A fresh water stress test was conducted to determine the lethal time of 50% mortality

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(LT50) in fresh water. After the feeding trial, five fish from each rearing tank (total 15 fish per

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treatment) were randomly selected and transferred in to a blue colored 20 L rectangular glass

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aquarium with 15 L tap water which was aerated for 24 h. The glass aquaria for the stress test

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were continuously aerated and kept under ambient temperature during the stress test. The

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duration for 50% mortality of red sea bream juvenile was calculated according to Moe et al.

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[28] as follows: time to death (min) of a juvenile was converted to log10 values. When the

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juveniles were transferred to the low salinity water at first time, they were still alive so that

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the survival rate was assumed to be 100% (log value was log10 100 = 2). The calculation was

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then conducted every 5 minutes after the first exposure. The values of log survival rate were

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then plotted against every minute to determine the time needed for 50% mortality of the

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juveniles in each treatment. The equation is as follows:

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Y= aX+ b where Y = log10 (survival), X = time for individual juvenile death (min).

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LT50 (X) was obtained when Y = 1.7 since log10 (50) = 1.7. Each value obtained from the

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equation above was compared statistically.

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2.10. Statistical analysis

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All data were subjected to statistical verification using one-way ANOVA (Package

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Super ANOVA 1.11, Abacus Concepts, Berkeley, California, USA). Probabilities of P<0.05

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were considered significant. Significance differences between means were evaluated using

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the Turkey Kramer test. Weight gain (%) and lysozyme activity were subjected to broken line

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regression analysis [29, 30] with dietary AMP supplementation level.

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3. Results

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3.1 Growth performance and nutrient utilization

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Growth performance, nutrient utilization and survival of red sea bream fed

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experimental diets are presented in Table 3. In general dietary AMP supplementations tended

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to improve growth performances (final body weight, % weight gain (WG %) and specific

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growth rate (SGR)). One of the best ones was found in diet group AMP-0.2, followed by diet

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groups AMP-0.1, AMP-0.4 and AMP-0.8, respectively. Feed utilization parameters of fish

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also improved when fed the diets containing AMP although the values were not always

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statistically significant. Significantly higher feed intake observed in fish fed diet group AMP-

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0.1 but was not significantly different with other AMP supplemented groups. FCE and PER

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also improved in diet groups AMP-0.1 and AMP-0.2 and then decreased the performance.

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The poorest growth performance and feed utilization were obtained in an AMP

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supplementation-free control diet group. All treatments showed relatively high survival rates,

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between 91.6 % and 93.3%, and there were no significant differences among all diet groups

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(P>0.05).

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3.2. Hematological parameters

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Table 4 represents the blood parameters of juvenile red sea bream after 56 days of the

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feeding trial. Overall, dietary treatments had no significant effect on blood chemical

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parameters of fish among different treatments except in GOT. Fish fed AMP supplemented

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diets showed significantly reduced plasma GOT level compare to the control group.

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Meanwhile, plasma glucose, cholesterol, total bilirubin and GPT levels also numerically

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reduced with fish fed AMP supplemented diets compared to control. Hematocrit content

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increased with fish fed AMP supplemented diets and it was significantly higher (P<0.05) in

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fish fed diet group AMP-0.4.

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3.3. Oxidative stress parameters

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Fig. 1 indicates the status of oxidative stress conditions, in which the conditions of both

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stress intensity and tolerance ability were combined, of fish fed with test diets for 56 days.

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Dietary AMP supplementation significantly reduce d-ROMs values (P<0.05) and it was

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lower in diet groups AMP-0.2 and AMP-0.4. In contrast, BAP values were not significantly

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influenced by dietary supplementation (no data illustrated here). Combined effects of d-

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ROM and BAP showed that fish fed diet groups AMP-0.2 and AMP-0.4 were located in

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zone- A which was categorized as having a lower intensity of oxidative stress and higher

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tolerance ability against oxidative stress. Supplementation free-control group, lowest and

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highest AMP supplemented diet groups (AMP-0.1 and AMP-0.8, respectively) were located

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in zone D (Fig.1), indicates high oxidative stress and low antioxidant levels. Numerically

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lower values (P>0.05) for plasma cortisol levels (%) (Table 4) were obtained in the AMP

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supplemented groups compared to the control group.

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3.4. Low salinity stress test The results of a lethal stress test against fresh water shock on LT50 obtained by

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regression analysis are shown in Fig. 2. Supplementation increased the LT50 values of red

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sea bream compare to control and it was numerically higher in fish feed diet group AMP-0.2.

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However, LT50 was not significantly influenced (P>0.05) by dietary treatments.

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3.5. Innate immune responses

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ACCEPTED MANUSCRIPT Non-specific innate immune responses were positively affected by dietary AMP

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supplementations (Table 5). Fish fed diet groups AMP-0.2 or AMP-0.4 had significantly

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higher PA and BA than fish fed the control diet. NBT activity was found to be significantly

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(P < 0.05) greater in fish fed diet group AMP-0.4 and AMP-0.8. Although numerically higher

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TSP, LA and bacterial agglutination antibody titer were detected in AMP supplemented diet

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groups compared to the control but there were no significant differences among treatments.

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In contrast, CAT activity decreased (P>0.05) with AMP supplementations.

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3.6. Digestibility coefficients

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The apparent digestibility coefficients (ADCs) of dry matter (ADCDM), protein (ADC

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and lipid (ADC

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AMP supplementations and was highest (P<0.05) in fish fed diet group AMP-0.4.

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Supplementation also increased ADC

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However, the AMP supplementation free control group showed lowest values of ADC

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ADC Protein and ADC Lipid (Table 6).

are presented in Table 6. ADCDM significantly increased by the dietary

Protein

and ADC

lipid

but not to a significant level. DM,

TE D

340

Protein)

3.7. Whole body proximate analysis and biometric indices

EP

339

Lipid)

M AN U

332

The initial and final whole body proximate compositions of juvenile red sea bream are

342

shown in Table 7. All the fish showed a change in the analyzed parameters compared to those

343

of the initial values. In comparison with the control group, dietary treatments had no

344

significant influences on the whole body proximate composition and biometric indices except

345

those of whole body crude lipid and condition factor (CF). Fish fed AMP supplemented diet

346

showed a significant increase of whole body lipid content and it was highest in diet group

347

AMP-0.1. On the other hand, the supplementation-free control group showed significantly

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14

ACCEPTED MANUSCRIPT 348

lowest value. CF also followed the similar trend of whole body lipid content of the present

349

study.

350 351

3.8. Quantification of optimum supplementation of AMP nucleotide for P. major. The optimum supplementation levels of AMP nucleotide for % WG and LA were

353

estimated by broken line regression analysis (Fig. 3a, b). Based on % WG, the optimal

354

supplemental level of AMP was estimated to be 0.16 % in diet (y = 589.5x + 672.48; R² =

355

0.8267). On the other hand, the optimal supplemental level of AMP nucleotide based on LA

356

was estimated to be 0.41% in diet (y = 231.34x +87.64; R² = 0.9756).

SC

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352

M AN U

357

4. Discussion

359

In aquaculture, research to date on dietary nucleotides has mainly focused on mixtures of

360

nucleotides rather than individual nucleotides. In limited cases, inosine and IMP, uridine

361

monophosphate (UMP) and AMP have been used as feeding stimulants or feed enhancers [9,

362

2, 31]. Recently, Hossain et al. [3] also reported inosine and IMP as potential functional

363

nutrient materials in aquafeed. However, to the best of our knowledge, there is very limited

364

information available on dietary AMP supplementation with regard to potential growth and

365

health benefits for aquatic species. Under these circumstances, it is important to investigate

366

the supplementation effects of AMP as functional materials in aquafeed.

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358

The results obtained clearly demonstrated that feeding with AMP supplemented diets

368

significantly improves the growth performances of red sea bream compared to a

369

supplementation-free control group after 56 days feeding trial. This observation is in

370

agreement with the previous findings of Lin et al. [17] who reported that nucleotide AMP

371

significantly increased the growth performance of grouper (E. malabaricus). Similarly, the

372

growth enhancing effects of some other individual and mixed nucleotides have also been

15

ACCEPTED MANUSCRIPT reported in some previous studies [2, 3, 32, 33]. So far, there is no exact explanation of how

374

dietary nucleotide works to enhance growth. However, it is assumed that the growth-

375

enhancing effect of AMP in the present study resulted from improved feed intake (Table 4),

376

promoting more rapid feed intake that reduced nutrient leaching into the water or possibly

377

playing roles in metabolism [3, 34]. In the present study, comparison of significantly

378

improved feed intake in AMP supplemented diet groups and a supplementation free control

379

diet group, strongly substantiates the previous hypothesis. In contrast, supplementation of

380

AMP after a certain concentration in the present study showed reduced growth performance

381

and feed utilization. Reduced growth performance and feed utilization by high mixed or

382

individual nucleotide levels was also reported in some previous studies [2, 3, 35-37]. In case

383

of the Japanese flounder, Song et al. [32] reported that a high dietary concentration of IMP

384

(1.0%) resulted in depressed growth performance compared to lower levels of dietary IMP

385

(0.1 to 0.2%). Adamek et al. [37] also reported that high dietary nucleotide concentration

386

(5%) caused growth depression in rainbow trout (Oncorhynchus mykiss) during a 37 day

387

feeding trial. In our previous studies it was also found that a high dietary concentration of the

388

mixed nucleotides (≤0.2%), IMP and inosine nucleoside (<0.4%) caused reduced growth and

389

feed utilization performances of red sea bream. Similarly, in the present study

390

supplementation of AMP at concentration in excess of 0.2% caused reduced growth and feed

391

utilization performance of juvenile red sea bream.

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392

The enhanced growth performance and feed utilization of fish fed dietary AMP might be

393

also attributed to the enhanced digestibility coefficients. In the present study, the highest

394

growth performances in AMP-0.2 diet group might be partly due to the improved ADC protein,

395

ADC

396

reported by Hossain et al. [38] for juvenile amberjack fed diet supplemented with dietary

397

nucleoside, inosine. Increased digestibility in AMP supplemented diet groups in the present

lipid

and ADCDM digestibility in this group. Similar enhanced digestibility was also

16

ACCEPTED MANUSCRIPT study might be due to feeding stimulatory properties of AMP. It is well documented that

399

feeding stimulant increase the secretion of a different digestive enzyme [39-42]. These

400

increased secretions of different digestive enzyme due to AMP supplementation might help

401

to improve this digestibility. However, in the present study the secretion of different digestive

402

enzyme are not studied. So, further study on secretions of different digestive enzymes due to

403

nucleotide supplementation in fish is warranted to prove this hypothesis.

RI PT

398

Hematological parameters are being used as an effective and sensitive index for the

405

physiological and pathological condition as well as welfare of fish [2, 3]. Blood parameters

406

obtained in the present experiment are considered to be within the normal range for juvenile

407

red sea beam, compared to those of the previous findings [2, 3, 43]. Dietary AMP

408

supplementation significantly enhanced hematocrit of juvenile red sea bream as a general

409

health response towards nutritional strategies. In the present experiment, hematocrit content

410

was significantly highest in fish fed AMP-0.4 diet group. Other AMP supplemented groups

411

showed intermediate values whereas control group showed significantly lowest value. This

412

indicated that dietary AMP elevated the health status of fish. Similarly, Song et al. [32] and

413

Hossain et al. [2, 3] reported the enhanced hematocrit level by the supplementation of

414

nucleotides in Japanese olive flounder and red sea bream diets, respectively. Plasma glucose

415

is commonly considered to be one stress indicator in fish, high glucose levels often indicate

416

the higher stress status of fish [44] because the rise of cortisol level would cause dissimilation

417

of liver sugar and strengthen dissolution of liver glycogen in order to provide energy during

418

stress process [45]. The lower blood glucose content in fish fed AMP supplemented diet

419

groups indicated the possible efficacy of dietary nucleotides in stress reduction as well as

420

inducing an optimal physiological condition of the fish. Result of the present study also

421

revealed that plasma bilirubin, GOT and GPT levels showed lower values (P>0.05) in AMP

422

supplemented diet groups compared to supplementation-free control group. Plasma bilirubin,

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404

17

ACCEPTED MANUSCRIPT GOT (or aspartate aminotransferase, AST) and GPT (or alanine aminotransferase, ALT) are

424

often used for the evaluation of liver function as they are released into the blood during injury

425

or damage to the liver cells [46]. Lower values of these parameters in AMP supplemented

426

diet groups indicated that AMP induced an optimal physiological condition as well as better

427

liver health conditions when compared with the supplementation-free control group.

RI PT

423

Stress is one of the emerging factors in aquaculture activities that may affect hormonal

429

secretion rates, intermediary metabolism, immunity and nutrient utilization [2, 3]. Dietary

430

nucleotide increases the resistance of fish to a variety of stressors as is well documented [2, 3,

431

47-49]. In our study, red sea bream fed AMP supplemented diets showed an increase in stress

432

resistance of red sea bream after a low salinity stress test. Increased (P>0.05) LT50 in red sea

433

bream fed diet group AMP-0.2 indicates the healthy status of red sea bream [3, 50].

434

Oxidative stress is considered to be involved in many diseases and pathological states in fish

435

[51], and is the consequence of an imbalance between oxidants and antioxidants in which

436

oxidant activity exceeds the neutralizing capacity of antioxidants [52]. Simultaneous

437

measurements of d-ROMs with BAP can provide a suitable tool for measuring the oxidative

438

stress in humans, pigs, rabbits and dogs [53-55]. Recently, d-ROMs and BAP were reported

439

to be reliable parameters for determining the oxidative stress condition of fish [2, 3]. Fish

440

with higher d-ROM values indicate that they are under more oxidative stress conditions. On

441

the other hand, fish with higher BAP values indicate stronger tolerance against oxidation. As

442

shown in Fig. 1, it is interesting to note that diet groups AMP-0.2 and AMP-0.4 were located

443

in zone A, which represents the best condition with low oxidative stress and high antioxidant

444

levels, while the other diet groups located in zone D were considered to show the stressed

445

condition of fish. Similarly, Hossain et al. [2, 3] also illustrated that dietary supplementations

446

of inosine and nucleotide mixtures stimulated the oxidative status of red sea bream. In

447

humans positive effects of increased oxidative stress resistance due to nucleotide

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428

18

ACCEPTED MANUSCRIPT 448

supplementation has been reviewed by Hess and Norman [56]. However, to date, there

449

remains a lack of explanation about how these supplements work to affect these parameters,

450

so further study is needed. In the present study, among whole-body proximate composition and biometric indices,

452

the whole body lipid and condition factor (CF) were significantly influenced by dietary AMP

453

supplementation. Similarly, Hossain et al. [3] and Li et al. [57] observed the significantly

454

increased whole body lipid content of red sea bream and red drum, respectively, with dietary

455

nucleotide supplementations. The positive effect of nucleotide on lipid metabolism was

456

reported by Cosgrove [58]. This mechanism acts via enzymes or coenzymes involved in

457

desaturation and elongation of fatty acids by intestine bacteria, red blood cell and liver.

458

Similar positive effects of dietary nucleotide supplementation were also reported by Gil, et al.

459

[59], Fontana et al. [60] and Salimi Khorshidi et al. [61] regarding an increase in the levels of

460

docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human or rat plasma or

461

unsaturated fatty acids in the carcasses of rainbow trout. However, research on the effect of

462

exogenous nucleotides and nucleosides on the lipid metabolism of fishes is very limited,

463

although it is known that dietary nucleotides can influence levels of various lipids and/or

464

fatty acids in certain tissues, such as erythrocytes, plasma, liver or the brain [62, 63]. Our

465

observation on whole-body lipid of red sea bream is likely the first report on the response of

466

fish supplemented with dietary AMP. Information on changes in various lipids in tissues as

467

well as HSI data in response to dietary nucleotides and nucleoside associated with

468

physiological consequences may be important; therefore, further investigation is warranted.

469

Significant alternation of CF in the present study also reflects the improvement in body

470

weight together with body length of fish fed AMP supplemented diets when compared with

471

supplementation-free control diet.

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19

ACCEPTED MANUSCRIPT The innate immune system in fishes plays a key role in preservation of fish against infectious

473

diseases [64]. Lysozyme, being an enzyme with antimicrobial activity, can split

474

peptidoglycan in bacterial cell walls, especially in Gram positive species, and can cause lysis

475

of the cells [65]. In the present study, red sea bream fed diets supplemented with AMP

476

showed increased LA. Similar increases in LA with fish fed nucleoside and nucleotide

477

supplemented diet were also reported previously in common carp (Cyprinus carpio) [66],

478

rainbow trout [67], Japanese flounder [32] and red sea bream [2, 3]. BA is one of the most

479

important factors in host resistance against pathogenic bacteria. Our results revealed that

480

supplementations of AMP increased the BA and it was significantly higher in diet group

481

AMP-0.2 which did not differ significantly with diet group AMP-0.4. Similarly, Hossain et al.

482

[2, 3] reported that dietary nucleotide, nucleoside supplementations significantly increased

483

the BA of red sea bream. Agglutination antibody titer against different bacteria is another

484

mechanism of innate immune response which has high activity in fish Oriol Sunyer and Tort

485

[68]. In the present study, agglutination antibody titer, PA and NBT activity increased by

486

dietary AMP supplementations. Similarly increased PA and NBT was also observed by Song

487

et al. [32] in Japanese olive flounder fed diet supplemented with IMP. Our previous studies

488

on red sea bream fed diets with supplemented nucleotides (inosine, IMP, mixed nucleotides)

489

also showed increased PA which is in agreement with the present study. Proteins are the most

490

important compounds in the serum and are essential for sustaining a healthy immune system

491

[69]. In the present study supplementation of AMP significantly increased TSP compared to

492

the control. Increased TSP due to nucleotide supplementation were also reported in Catla

493

catla [70], red sea bream (P. major) [2, 3] and rainbow trout [49]. These findings are in

494

agreement with the present study. Overall, all the non specific immune response parameters

495

measured in the present study showed improved value in AMP supplemented diet groups

496

compared to non supplemented control group confirming the benefits of AMP nucleotide for

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20

ACCEPTED MANUSCRIPT the non-specific innate immunity of red sea bream. Similarly to fish, enhanced immune

498

responses due to nucleotide supplementation have been studied in other animals such as

499

humans, mice, swine and poultry [71-77]. Human infants fed milk formula fortified with

500

nucleotides had better responses to immunization as evidenced by an increase in humoral

501

antibody response [71, 72] and increased cytokine production [73]. Similar responses to

502

nucleotide supplementation were reported in vivo experiments with mice [74, 75]. The

503

dietary supplementation of purified nucleotides to milk replacers of newborn bull calves

504

challenged with lipopolysaccharide, resulted in calves that tended to have higher mean IgG

505

levels compared to the unsupplemented control calves [76].

506

humoral and cellular immune stimulation by dietary nucleotides supplementation has not

507

been elucidated. Dietary nucleotides contribute to the circulating pool of nucleosides

508

available to stimulate leukocyte production [77, 62] which are the important cells of the

509

immune system that protect the body against infectious disease and foreign invaders.

510

Moreover, it is also thought that, dietary nucleotides may be of importance for optimal

511

function of the cellular immune response such as increased interferon-γ (INF-γ), decreased

512

production of Interleukin-2 (IL-2) and natural killer cells, and increased resistance to bacterial

513

or fungal infection.

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497

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The exact mechanism of

Increased CAT activity rates were attributed to elevated levels of exogenous hydrogen

515

peroxide (H2O2 ) which is the main cellular precursor of the hydroxyl radical (HO-), a highly

516

reactive and toxic form of ROS [2, 3]. In humans increased serum CAT activity is used as a

517

diagnostic tool in acute pancreatitis [78], hemolytic disease [79] and some liver diseases [80].

518

In the present study fish fed AMP supplemented diet groups showed reduced mean CAT

519

activity in comparison to the control indicated that AMP supplementation helps to maintain

520

lower level of exogenous H2O2 production and simultaneously increase the resistance against

521

oxidative stress. Similar observations were also reported for red sea bream [2, 3] where

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ACCEPTED MANUSCRIPT 522

nucleotide supplementation reduces CAT activity. Results of the CAT activity as well as the

523

BAP and d-ROMs measured in the present study reconfirmed the positive supplemental

524

effects of AMP associated with the conditions on oxidative stress and immunity. WG and LA activity are often used to estimate the optimum supplementation level of

526

nutrient components in fish [29, 30]. Based on the broken line regression analysis of % WG

527

and LA, the present study illustrated that the optimal levels of dietary AMP for juvenile red

528

sea bream might be 0.16% and 0.41 % respectively. Similarly, Hossain et al. [3] reported that

529

optimum inosine and IMP supplementation level based on growth performances and

530

immunity and intestinal health 0.4% for red sea bream; while Song et al. [32] suggested a

531

supplementation of 0.1–0.4% IMP could enhance innate immunity and disease resistance of

532

olive flounder. Results of the present study indicated for the first time that optimum AMP

533

supplementation level in red sea bream diets for enhanced immunity was higher than that for

534

growth. Similar results for other micronutrients like myoinositol and pantothenic acids have

535

been reported in Jian carp [81, 82] and riboflavin for grass carp [83].

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525

Therefore, the present study demonstrated that the dietary supplementation of AMP

537

positively influences the growth performances, feed utilization, innate immune responses,

538

hematological parameters and oxidative stress responses of fish. Based on the present

539

experimental condition, it can be concluded that the optimal levels of dietary AMP

540

supplementation appear to be between 0.2 to 0.4% for juvenile red sea bream, which is also

541

in line with the most of the growth performance and health parameters of the fish.

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543

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544

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ACCEPTED MANUSCRIPT 546

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ACCEPTED MANUSCRIPT [38] Hossain MS, Koshio S, Ishikawa M, Yokoyama S, Sony NM, Mayumi M, Fujieda T.

646

Inosine supplementation in low fishmeal based diets for juvenile amberjack seriola dumerili:

647

effects on growth, digestibility, immune response, stress resistance and gut morphology.

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Book of abstract Aquaculture America 2015, page 55, February 19-22, New Orleans, USA.

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protein digestion at low water temperatures. Aquac Res 2006;37: 366-73.

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[42] Satoh K. Effects of supplement with krill extract and krill meal to diet on the growth

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performance and protein digestibility of yellowtail during low water temperature.

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Suisanzoshoku 2003; 51: 93-9.

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[43] Aoki H, Furuichi M, Viyakarn V, Yamagata Y, Watanabe T. Feed protein ingredients

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for red sea bream. Suisanzoshoku 1998;46: 121–7.

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[44] Eslamloo K, Falahatkar B, Yokoyama S. Effects of dietary bovine lactoferrin on growth,

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physiological performance, iron metabolism and non-specific immune responses of Siberian

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31

ACCEPTED MANUSCRIPT

1 2

Table 1

3

Basal diet formulation of juvenile red sea bream. Ingredients

Composition

Fishmeala

20

Caseinb

32

Pollack liver oilc

3

Mineral mixturef

3

a

L-arginineg

1.5

Stay-Ch

0.08

Dextrin

7

α-starch

6

Activated gluteni

5

α- cellulose

12.42

AMPj

0-0.8

SC

Vitamin mixturee

M AN U

3

EP

TE D

Nippon Suisan Co. Ltd., Tokyo, Japan Waco Pure Chemical Industries Inc. (Osaka, Japan) c Riken Vitamin, Tokyo, Japan. d Kanto Chemical Co., Inc. (Tokyo, Japan) e Vitamin mixture (g kg−1 diet): β-carotene, 0.10; Vitamin D3, 0.01;Menadione NaHSO3.3H2O (K3), 0.05; DL-α-Tochopherol Acetate (E), 0.38; Thiamine-Nitrate (B1), 0.06; Riboflavin (B2), 0.19; Pyridoxine-HCl (B6), 0.05; Cyanocobalamin (B12), 0.0001; Biotin, 0.01; Inositol, 3.85; Niacine (Nicotic acid), 0.77; Ca Panthothenate, 0.27; Folic acid, 0.01; Choline choloride, 7.87; ρAminobenzoic acid, 0.38; cellulose, 1.92. f Mineral mixture (g kg−1 diet): MgSO4, 5.07; Na2HPO4, 3.23; K2HPO4, 8.87; Fe citrate, 1.10; Ca lactate,12.09; Al (OH)3, 0.01; ZnSO4, 0.13; CuSO4, 0.004; MnSO4, 0.03; Ca (IO3)2, 0.01; CoSO4, 0.04. g Nacalai Tesque, Kyoto, Japan. h Stay-C 35. i Glico Nutrition Company Ltd. Osaka, Japan. Commercial name “A-glu SS”. j Sigma Aldrich Co., St. Louis, MO, USA. (AMP added to diets at the expense of α- cellulose). b

AC C

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

7 d

Soybean lecithin

RI PT

(%)

22 23 1

ACCEPTED MANUSCRIPT

24 25 26

Table 2

27

Proximate composition of the experimental diets (% dry matter basis). Diet groups

Parameters

AMP-0.1

AMP-0.2

AMP-0.4

AMP-0.8

7.9

7.5

8.2

7.5

8.3

Crude protein

54.4

54.8

54.2

54.2

54.3

Crude lipid

11.4

11.2

11.2

11.2

11.8

Ash

6.53

6.53

6.45

6.60

6.50

Gross energy (KJg-1)*

22.1

22.1

22.1

22.0

22.2

28

SC

M AN U

Moisture

RI PT

Control

*Calculated using combustion values for protein, lipid and carbohydrate of 236, 395 and 172 KJ

30

kg-1, respectively.

31

Carbohydrate was calculated by the difference: 100-(protein + lipid + ash).

EP AC C

32

TE D

29

2

ACCEPTED MANUSCRIPT

33

Table 3

35

Growth performance and feed utilization parameters of red sea bream fed test diets for 56 days.

36

Values are means of triplicate groups ± S.E.M. Within a row, means with the same letters are not

37

significantly different (P>0.05). Parameters

Diet groups

RI PT

34

AMP-0.1

AMP-0.2

IBWa

3.38±0.01

3.36±0.01

3.34±0.01

FBWb

25.56±0.48a

28.94±1.02ab

29.19±0.41b

27.61±0.55ab

27.70±1.22ab

WG (%)c

656.9±15.2a

762.6±29.12ab

774.8±14.8b

725.2±19.9ab

719.6±34.6ab

SGRd

3.61±0.04a

3.85±0.06ab

3.87±0.03b

3.77±0.04ab

3.75±0.08ab

FIe

20.14±0.37a

23.18±0.88 b

22.85±0.25b

22.67±044b

22.90±0.54b

FCEf

1.10±0.04

1.13±0.00

1.13±0.03

1.07±0.01

1.06±0.03

PERg

1.99±0.06

2.02±0.00

2.09±0.06

1.94±0.01

1.96±0.05

Surh

93.33±1.67

92.50±2.50

91.67±1.67

93.33±1.67

93.33±1.67

M AN U

TE D

a

IBW: initial body weight (g).

39

b

FBW: final body weight (g).

40

c

WG: percent weight gain (%).

41

d

SGR: specific growth rate (% day−1).

42

e

FI: feed intake (g fish−1 50 days−1).

43

f

FCE: feed conversion efficiency.

44

g

PER: protein efficiency ratio.

45

h

Sur: survival (%).

AC C

EP

38

AMP-0.4

AMP-0.8

3.35±0.03

3.38±0.02

SC

Control

46 47 48 49 3

ACCEPTED MANUSCRIPT

50

Table 4

51

Blood parameters of juvenile red sea bream fed test diets for 56days. Values are means±SE of

52

triplicate groups. Within a row, means with the same letters are not significantly different (P>0.05). Parameters

Diet groups Control

AMP -0.4

37.0±3.6ab

39.0±1.7ab

Glucose (mg/dl)

73.5±9.5

51.0±4.0

56.0±11.0

T-Cho (mg/dl)a

149.3±5.9

146.3±9.6

141.5±10.5

BUN (mg/dl)b

5.33±0.3

6.0±0.6

6.67±0.9

T-Bill (mg/dl)c

0.23±0.03

<0.2

<0.2

GOT (IU/l)d

78.0±2.0b

30.5±6.5a

GPT(IU/L)e

78.3±7.0

TG (mg/dl)f

135.0±35.5

b

BUN: blood urea nitrogen.

55

c

56

d

57

e

58

f

T- Bill: Total bilirubin

37.67±1.3ab

57.3±7.2

50.7±12.2

132.7±12.4

142.0±10.2

7.5±2.5

6.33±0.9

<0.2

<0.2

43.0±10.0a

36.0±5.5a

46.0±1.0ab

36.5±16.5

44.3±3.2

45.7±18.9

28.7±3.7

180.7±21.1

200.0±15.0

152.67±31.7

156.0±26.5

EP

GOT: glutamyl oxaloacetic transaminase.

GPT: glutamic pyruvate transaminase.

AC C

TG: triglyceride.

SC

M AN U

54

41.0±1.5b

TE D

T-Cho: total cholesterol.

AMP -0.8

RI PT

30.3±1.7a

a

60

AMP-0.2

Hematocrit (%)

53

59

AMP-0.1

4

ACCEPTED MANUSCRIPT 61

Table 5

63

Non-specific immune response of juvenile red sea bream fed test diet for 50 days. Values are

64

means±SE of triplicate groups. Within a row, means with the same letters are not

65

significantly different (P>0.05). Parameters

Diet groups Control

AMP-0.1

AMP-0.2

RI PT

62

AMP-0.4

AMP-0.8

3.70±0.25

3.50±0.10

2.90±0.50

3.53±0.32

3.20±0.40

LA (unit/mL)b

87.5±37.5

116.7±36.3

125.0±50

183.3±16.7

141.7± 46.4

PAc

1.61±0.05a

1.65±0.05a

1.97±0.02b

1.78±0.1ab

1.89± 0.03ab

BA (%)d

56.6±1.8a

67.7±3.5ab

71.7±2.6b

75.3±2.6b

68.3±2.3ab

NBTe

0.35±0.02a

0.36±0.01a

0.41±0.02ab

0.43±0.01b

0.45± 0.01b

CATf

87.3±10.4

72.3±10.5

55.8±16.7

43.68±20.2

79.4± 6.3

B-Agglug

0.70±0.1

0.90±0.2

1.00±0.1

1.00±0.1

0.80± 0.1

a

67

b

68

c

69

d

70

group.

71

e

72

f

73

g

TSP: Total serum protein LA: lysozyme activity

M AN U

TE D

66

SC

TSP (g/dl)a

EP

PA: Peroxidase activity (measured at OD of 450nm).

AC C

BA: Bactericidal activity=100× (CFU of blank group-CFU of each group)/CFU of blank

NBT: Nitro-blue-tetrazolium activity

CAT: Catalase activity

B-Agglu: Bacterial agglutination antibody titer

74 75 76

5

ACCEPTED MANUSCRIPT 77

Table 6

78

Digestibility of juvenile red sea bream fed different experimental diets for 56 days.*Values

79

are means ± SE of triplicate groups. Within a row, means with the same letters are not

80

significantly different (P>0.05).

AMP-0.1

AMP-0.2

ADCDMa

76.5±0.6a

80.3±0.3b

82.2±0.6b

ADCprotein (%)b

91.8±0.3

93.0±0.01

93.0±0.1

ADClipid (%)c

92.0±0.6

93.1±0.3

94.0±0.1

(

85

c

86

(

89

ADCProtein (Apparent protein digestibility, %) = 100- 100

% marker in diet % protein in faeces × ) % marker in faeces % protein in diet

TE D

84

% marker in diet ) % marker in faeces

M AN U

( b

88

AMP-0.8

81.0±0.7b

81.5±0.4b

93.1±0.01

92.6±0.6

93.7±0.6

93.8±0.2

ADCDM (Apparent digestibility coefficient of dry matter) = 100 – 100

83

87

AMP-0.4

SC

a

ADCLipid (Apparent lipid digestibility, %) = 100- 100

% marker in diet % lipid in faeces × ) % marker in faeces % lipid in diet

EP

82

Control

AC C

81

Diet groups

RI PT

Parameters

90 91 92 93 94 6

ACCEPTED MANUSCRIPT Table 7

96

Whole body proximate analysis (% wet basis) and biometric indices in juvenile red sea bream

97

fed test diets for 56 days. Values are means of triplicate groups ± S.E.M. Within a row, means

98

with the same letters are not significantly different (P>0.05). Crude protein, crude lipid and ash

99

are expressed on a wet weight basis. Initial1

Diet group Control

AMP-0.1

AMP-0.2

AMP -0.4

AMP -0.8

75.7±0.5

74.4±0.4

76.4±0.4

13.5±0.2

13.5±1.3

13.5±0.2

6.2±0.2ab

6.2±0.1ab

5.6±0.3ab

3.5±0.3

3.4±0.1

3.6±0.1

3.6±0.1

2.21±0.03b

2.13±0.02a

2.11±0.06a

2.09±0.05a

b

b

b

1.61±0.09

1.49±0.10

1.49±0.18

7.33±0.16

7.36±0.07

6.64±0.33

83.6

76.2±0.4

74.5±1.6

Crude protein

12.1

13.4±0.1

13.6±1.1

Crude lipid

0.7

5.4±0.2a

6.6±0.2b

Crude ash

3.4

3.5±0.04

CF2

-

1.97±0.05a

-

VSI4

-

1.10±0.14

1.74±0.18

TE D

HSI3

M AN U

Moisture

SC

Parameters

RI PT

95

7.14±0.35

7.23±0.09

1

Initial values are not included in the statistical analysis.

101

2

CF (condition factor)=100 × fish weight/(fish length)3

102

3

HSI (hepatosomatic index)=100 × liver weight/fish weight

103

4

VSI (viscerasomatic index)=100 × viscera weight/fish weight

105

AC C

104

EP

100

7

ACCEPTED MANUSCRIPT 1

3550 3500 3450 3400 3350 3300 3250 3200 3150 3100 3050 3000

AMP-0.4

A

B

(114.5, 3486.5)

AMP-0.8 (172, 3255.5)

Control

(191.3, 3149.7)

AMP-0.1

C 50

D

(164, 3117.5)

100

150

200

M AN U

d-ROMs (U. Carr.) 3 4

250

EP

TE D

Fig. 1. Oxidative stress parameters in red sea bream (P. major) fed test diets for 56 days. Values (d-ROMs, BAP) are means of triplicate groups. (The abbreviations of experimental treatments are illustrated in the text. Central axis based on mean values of d-ROM and BAP from each treatment.) Zone A: High antioxidant potential and low reactive oxygen metabolites (good condition). Zone B: High antioxidant potential and high reactive oxygen metabolites (acceptable condition). Zone C: Low antioxidant potential and low reactive oxygen metabolites (acceptable condition). Zone D: Low antioxidant potential and high reactive oxygen metabolites (stressed condition).

AC C

5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

RI PT

AMP-0.2 (113, 3426.5)

SC

BAP (μ Mol L-1)

2

ACCEPTED MANUSCRIPT

35.32

Control

AMP-0.1

32.49 32.48

RI PT

31.72

32.43

SC

45 40 35 30 25 20 15 10 5 0

AMP-0.2 AMP-0.4 Diet groups

AMP-0.8

M AN U

LT50 (Min.)

33 34 35 36

Fig. 2. LT50 (min) calculated from the lethal time of red sea bream exposed to fresh water

41

(see the text). Data were expressed as mean ± S.E.M. from triplicate groups. The

42

abbreviations of experimental diets are illustrated in the text.

46 47 48 49 50 51 52 53 54 55 56

EP

45

AC C

43 44

TE D

37 38 39 40

1000 900 800 700 600 500 400 300 200 100 0

Ymax = 769.25

X=0.16

0

0.2 0.4 0.6 0.8 Dietary AMP supplementation level (%)

57

Fig. 3a.

59 60

300

y = 231.34x + 87.64 R² = 0.9756

200

TE D

150

Y max= 182.49

100

X=0.41 0.41

50 0

62 63 64 65 66 67 68

0.2 0.4 0.6 0.8 Dietary AMP supplementation level (%)

AC C

0

EP

LA (unit ml-1)

250

61

1

M AN U

58

RI PT

y = 589.5x + 672.48 R² = 0.8267

SC

(% WG)

ACCEPTED MANUSCRIPT

1

Fig. 3b

Fig. 3a,b. Broken line regression analysis of percent weight gain (%WG), Fig. 3a and lysozyme activity (LA), Fig. 3b for juvenile red sea bream (P. major) fed diets supplemented with graded levels of AMP for 56 days.

ACCEPTED MANUSCRIPT

1

Highlights (for review)

3

1. No previous work has addressed the effects of adenosine monophosphate (AMP) as

4

functional nutrient for Pagrus major.

5

2. Fish growth performances were significantly improved by dietary AMP administration.

6

3. Enhancement of P. major innate immune responses by AMP supplementation.

7

4. AMP supplementations enhanced digestibility and antioxidant status of P. major.

8

5. The optimal levels of dietary AMP for P. major ranged between 0.16 and 0.41 % in the diet.

SC

RI PT

2

AC C

EP

TE D

M AN U

9