Metabolic Effect of Dietary Taurine Supplementation on Nile Tilapia

Dec 7, 2017 - Fisheries College, Xiamen Key Laboratory for Feed Quality Testing and Safety Evaluation, Jimei University, Xiamen 361021, China. § Depa...
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Metabolic effect of dietary taurine supplementation on Nile tilapia (Oreochromis nilotictus) evaluated by NMR-based Metabolomics Guiping Shen, Ying Huang, Jiyang Dong, Xuexi Wang, KianKai Cheng, Jianghua Feng, Jingjing Xu, and Jidan Ye J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03182 • Publication Date (Web): 07 Dec 2017 Downloaded from http://pubs.acs.org on December 9, 2017

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REVISED MANUSCRIPT OF JF-2017-031823.R1

Metabolic effect of dietary taurine supplementation on Nile tilapia (Oreochromis nilotictus) evaluated by NMR-based Metabolomics

Guiping Shen,*, † Ying Huang,† Jiyang Dong,† Xuexi Wang,‡ Kian-Kai Cheng,§ Jianghua Feng,† Jingjing Xu,† and Jidan Ye*, ‡



Department of Electronic Science, Fujian Provincial Key Laboratory of Plasma and Magnetic

Resonance, Xiamen University, Xiamen 361005, China; ‡

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

Jimei University, Xiamen 361021, China; §

Department of Bioprocess & Polymer Engineering and Innovation Centre in Agritechnology,

University Teknologi Malaysia, Johor 81310, Malaysia;

* Corresponding authors at: †

422 Siming South Road, Xiamen University, Xiamen, Fujian Province 361005, China. Tel.: +86 592 2184026, Fax: +86 2189426. Email address: [email protected] (G. Shen)



43 Yindou Road, Jimei Univesity, Xiamen, Fujian Province 361021, China.

Tel./fax: +86 592 6181054. Email address: [email protected] (J. Ye)

Submitted to: Journal of Agricultural and Food Chemistry

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Abstract

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Taurine is indispensable in aquatic diets that are based solely on plant protein,

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and it promotes growth of many fish species. However, the physiological and

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metabolome effects of taurine on fish have not been well described. In this study, 1H

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NMR-based metabolomics approaches were applied to investigate the metabolite

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variations in Nile tilapia (Oreochromis nilotictus) muscle in order to visualize the

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metabolic trajectory and reveal the possible mechanisms of metabolic effects of

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dietary taurine supplementation on tilapia growth. After extraction using aqueous and

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organic solvents, nineteen taurine-induced metabolic changes were evaluated in our

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study. The metabolic changes were characterized by differences in carbohydrate,

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amino acid, lipid and nucleotide contents. The results indicate that taurine

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supplementation could significantly regulate the physiological state of fish and

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promote growth and development. These results provide a basis for understanding the

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mechanism of dietary taurine supplementation in fish feeding. 1H NMR spectroscopy,

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coupled with multivariate pattern recognition technologies, is an efficient and useful

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tool to map the fish metabolome and identify metabolic responses to different dietary

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nutrients in aquaculture.

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Keywords: NMR, Metabolomics, Taurine, Nile tilapia, Multivariate statistics

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Introduction

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Fish meal (FM) is the predominant protein source in aquafeed, due to its high

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protein quality and well-balanced amino acid profile.1-4 The demand for FM is high

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worldwide, and thus a partial or total replacement of FM with terrestrial plant

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ingredients in aquafeeds has been recognized as a potential solution to enhance

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aquaculture sustainability.2,

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variety of alternative plant protein (PP) sources,3-8 but the excessive replacement of

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FM resulted in inferior fish growth and feed utilization as well as physiological

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abnormalities, such as hemolytic anemia and green liver syndrome.9 This may be due

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to the endogenous antinutritional factors, such as protease inhibitors, glucosinolates

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and antivitamins in the alternative PP sources.8, 9 Moreover, PP sources have lower

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crude protein levels and are deficient in certain amino acids, such as

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sulphur-containing amino acids (e.g. taurine, methionine) and lysine.6

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Many studies have attempted to replace FM with a

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Taurine is rich in FM, and it plays an important physiological role in mammals

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and other animals, including fish.9, 10 Although taurine can be synthesized in fish from

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methionine and cysteine, the ability of taurine biosynthesis in different fish species

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varies greatly.11,

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physiological abnormalities and inferior performances.11 Recent studies have proven

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that taurine is necessary in aquatic diets that are strictly based on plant protein and

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promotes the growth of many freshwater and marine fishes, such as yellow catfish

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florida (Pelteobagrus fulvidraco),3 Nile tilapia (Oreochromis nilotictus),7 and white

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grouper (Epinephelus aeneus).11 However, taurine is considered nonessential for some

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It has been shown that taurine-deficiency will result in

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freshwater fishes, like grass carp (Ctenopharyngodon idellus).13 Thus, the essentiality

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of taurine for fish may depends on fish species and other factor, such as natural

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feeding habits and previous feeding history of the fish.3, 12 In addition, Salze and

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Davis estimated that the optimal requirements of dietary taurine for some cultured fish

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ranges from 0.20% to 1.66% of the diet.10 However, these studies mainly focused on

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the effect of dietary taurine on growth performance in fish, and the underlying

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physiological roles of taurine supplementation have yet to be established.

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Nile tilapia, a species of freshwater fish originally from Africa, has become the

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second largest farmed fish worldwide after carp.14,

15

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develop a nutritionally balanced and cost-effective diet to achieve a desired level of

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growth for this fish species.8, 16 Although the effects of dietary taurine on growth, feed

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utilization and reproductive performance of tilapia have been studied extensively in

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recent years,7, 16 limited information is available to understand the physiological or

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metabolic effects of taurine on tilapia.15 In addition, taurine is the most abundant free

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amino acid in animal tissues, accounting for 53% of the free amino acid pool in

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muscle.10 Muscle tissue is the main edible portion of fish and responsible for their

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nutritional value.17 Therefore, we focused on tilapia muscle in order to explore the

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metabolic effects of taurine supplementation.

There is an urgent need to

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Metabolomics is a systems biology approach to study complex biochemical

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processes of organisms in response to various biological factors at the metabolic

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scale.18 It allows investigation of global metabolic changes and disturbances in

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biochemical pathways due to dietary differences. To our knowledge, few studies have 4

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examined muscle metabolic variations so as to evaluate the effects of dietary taurine

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supplementation on fish growth performance and muscle quality.19-23 In the present

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study, global metabolic profiles of tilapia fed with four different taurine

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supplementations were studied, and the profiles were compared via an untargeted 1H

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NMR-based metabolomics technique. The aim of this work is to gain more insight

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into taurine-induced metabolic variations in tilapia, in terms of fish growth and

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nutrient utilization.

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Material and methods

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Experimental diets

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For aquafeed, casein and gelatin were used as a protein source (taurine free). Fish

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oil, soybean oil and soy lecithin were used as lipid sources, and corn starch (raw

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starch) was used as the carbohydrate source. Based on the proximate composition of

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the above ingredients and other feed ingredients (i.e. vitamin mix, mineral mix,

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vitamin

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microcrystalline cellulose), a basal diet (taurine free) was formulated to contain 37%

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crude protein and 6% crude lipid. Taurine (food-grade, Beijing Hui Kang Yuan

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Biological Technology Co., LTD) was incrementally added to the basal diet in ratios

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of 0.0%, 0.4%, 0.8% and 1.2%. Based on these feed ingredients, four experimental

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diets, which contained different taurine ratios, were prepared according to methods

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mentioned in our earlier study and tapped as CTRL, D1, D2 and D3, respectively.8

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The ingredients and proximate composition of the experimental diets are shown in

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Table S1 in the supporting information.

C,

monocalcium

phosphate,

choline

chloride,

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shrimp meal,

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Fish and experimental conditions

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All animal experiments were approved by the local animal ethics committee at

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Jimei University. Juvenile tilapias were obtained from a local commercial farm in

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Xiamen, China. The fish were transported to the aquaculture laboratory at Jimei

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University. Before the feeding trial began, fish were initially fed with a commercial

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tilapia feed and were acclimatized to the rearing conditions in a closed recirculating

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system consisting of two circular fiber glass tanks (0.85 m height × 1.22 m upper

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diameter, 1.04 m lower diameter) with a Polygeyser bead filter (Aquaculture Systems

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Technologies, LLC., USA) for two weeks.

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A total of 400 fish, initially weighing an average of 4.25 ± 0.12g (means ±

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SD), were randomly divided into four experimental groups and fed with the four

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different experimental diets, CTRL, D1, D2 and D3. Each group was divided into four

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identical 150-L cylindrical tanks (0.6 m in diameter and 0.75 m in height) at a density

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of 25 fish/tank (4 tanks/diet). During a feeding period of 84 d, the fish in each group

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were hand-fed one of the diets to satiation at 08:30 and 18:00 h per day under a

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natural photoperiod. Half an hour after each feeding, excess feed was collected by

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siphoning. The excess feed was dried at 70°C and weighed in order to calculate the

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feed intake. Dissolved oxygen and water temperature were measured daily at 12:00 h

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and nitrite-N was monitored twice weekly with a multi-parameter photome (HI83200;

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Hanna Instruments, Woonsocket, RI, USA). During the feeding trial, the water

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temperature, dissolved oxygen and nitrite-N ranged were 27.0 ± 2.0°C, 6.15 mg/L ±

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0.45 mg/L and 0.248 ± 0.15 mg/L, respectively. 6

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Sampling procedure and extraction

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On feed days (FD) 28, 56 and 84, three fish were randomly sampled from each

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tank (12 fish/diet, 48 fish per sampling event) after 12 h of fasting. All the sampled

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fish were anesthetized using MS 222 (tricaine methanesulfonate, Sigma-Aldrich

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Shanghai Trading Co. Ltd., Shanghai) solution. The fish were weighed individually,

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and their anterior spinal cord were severed. Then, the fish were dissected using sterile

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filleting tools under aseptic conditions. A 2-cm wide skinless muscle sample was

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taken above the dorsal line from each fish, using the insertion of the dorsal fin as the

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center line. The muscle samples were carefully wrapped in aluminum foil, flash

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frozen in liquid nitrogen, packed into a zip lock plastic bag with an identification card,

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and kept at -80°C.

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The fish carcasses were pooled, weighed, minced and then dried at 70°C for

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whole-body compositional analysis. The fish samples were ground into fine powder

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using a laboratory grinder. Moisture, crude protein, crude lipid, and ash in these

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samples were determined according to the methods of the Association of Official

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Chemists (AOAC).8 The fish weight gain rate (WGR, %) was calculated as follows:

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WGR (%) = 100×(Wf -Wi)/Wi

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where Wf (g) and Wi (g) are the final and initial body weight, respectively. Significant

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differences between taurine treatments were calculated using one-way ANOVA

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Tukey’s multiple-range test by SPSS Statistics for Windows (Version 17.0, SPSS Inc.,

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Chicago, IL, USA).

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Before the NMR analysis, the muscle samples were extracted using a modified 7

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Bligh-Dyer method.24, 25 In brief, fish muscle samples (100 mg) were homogenized

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for 30 s in 400 µL of methanol and 125 µL of deionized water at 4oC. The

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homogenates were transferred to a 2.5 mL tube. To each tube, 400 µL of chloroform

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and 400 µL of deionized water were added, and the mixture was vortexed for 60 s.

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(The use and disposal of chloroform and chloroform contaminated materials were in

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compliance with Xiamen University laboratory safety management regulations). After

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10 min partitioning on ice, the samples were centrifuged for 5 min (10000 × g, 4oC).

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The upper supernatants were transferred to 1.5 mL tubes, and lyophilized for 24 h to

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remove methanol, chloroform and water. Then, the dried extracts were stored at

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-80 °C for NMR experiments.

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Sample preparation and 1H NMR spectroscopy

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Aqueous freeze-dried powder of the fish muscle was mixed with 450 µL 99.9%

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D2O and 150 µL of 90 mM sodium phosphate buffer (pH 7.4) containing 0.02%

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sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4 (TSP), an internal chemical shift

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standard. The extracted muscle buffer mixture was kept at room temperature for 5 min,

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and then centrifuged for 10 min (6000 × g and 4°C) to remove suspended debris. Then,

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550 µL of the supernatant was transferred to a 5-mm NMR tube and stored at 4°C

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until the 1H NMR data acquisition.

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All 1H NMR spectra of the muscle samples were acquired at 298 K using a

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Varian NMR System 500 MHz spectrometer (Agilent, Santa Clara, CA) equipped

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with a 5 mm actively shielded x, y, z axis gradient indirect detection probe. Spectra

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were obtained with a one-dimensional pulse sequence based on a NOESY (nuclear 8

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Overhauser effect spectroscopy) pulse sequence (RD-90o-t1-90o-tm-90o-Acq) with

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water suppression (NOESYPR1D). The 90° pulse length was adjusted to

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approximately 10 µs, and 64 transients were collected, yielding 32 K data points for

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each spectrum with a spectral width of 10 K. Acquisition time was 1.8 s, and the

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relaxation delay was 4.0 s, with a fixed interval t1 of 4 µs. The water resonance was

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irradiated during relaxation delay, and the mixing time tm was 120 ms.

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Pre-processing of 1H NMR spectra and multivariate analysis

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All spectra were pre-processed with the software MestReNova (V7.1.0-9185,

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Mestrelab Research S.L.). Prior to Fourier transformation, the free induction decays

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(FIDs) were zero-filled to 64 K data points and multiplied by an exponential function

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of 0.3 Hz line-broadening factor. Afterwards, all spectra were phase- and

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baseline-corrected. Finally, the chemical shifts were referenced to the TSP signal at

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0.0 ppm. The spectral regions of the residual water resonance (5.0 ~ 4.7 ppm), TSP

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signal (0.0 ppm), residual methanol resonance (3.37 ~ 3.34 ppm), and peak-free

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baseline were removed from the spectra. Then, spectra over the range of 9.0 ~ 0.5

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ppm were binned into 226 buckets using the adaptive binning method.26 Using this

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method, each peak is binned as one bucket. Each spectrum was normalized using

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probabilistic quotient normalization (PQN) to account for sample dilution effects, thus,

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facilitating comparison analyses of samples.27 Interference factors, which were

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independent of the purpose of the study, were filtered out using analysis of

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variance-principal component analysis (ANOVA-PCA).28

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The NMR data were imported into SIMCA-P software (version14.0, Umetrics 9

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AB, Umeå, Sweden) for multivariate statistical analysis. The normalized bucket data

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were first subjected to principal component analysis (PCA) for the overview of the

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data distribution and potential outliers. Then, partial least squares discriminant

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analysis (PLS-DA) and orthogonal partial least squares discriminant analysis

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(OPLS-DA) were implemented on NMR data in order to identify specific

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metabolomic differences between the taurine-supplemented groups and the control

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group. All of the above analyses were carried out under a Pareto scaling pattern.

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Pairwise-comparisons were performed and validated with 7-fold cross validation and

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permutation test (permutation number n= 200) using PLS-DA and OPLS-DA methods.

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An additional validation method, CV-ANOVA was conducted to validate the models.

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In all instances, the level of statistical significance was p < 0.05.

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In addition, the relative concentrations of metabolites were compared using the

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fold-change and statistical analyzed p-value with Student’s t-test for better reliability

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of characteristic metabolites’ screening. In this study, we used a volcano plot to

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summarize both the t-test and fold-change criteria in a single plot. Typically, a scatter

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plot of -log10(p-value) against log2(fold-change) is used, which represent the y- and

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x-axes of the volcano plot, respectively. The metabolites that contributed to the

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metabolomic difference in pairwise-comparisons were marked with the circles size

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and color-coded based on the variable importance for the projection (VIP) and the

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corresponding absolute correlation coefficients (|r|) constructed from the OPLS-DA

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analysis. The larger circle size corresponds to a larger VIP value, and warm color

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corresponds to the significant difference between classes, while a cool color is 10

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opposite. In the volcano plots, the concentrations of metabolites located on the

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positive side of the horizontal axis are higher in the taurine-supplemented group than

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that in the control group. Thus, the four-dimensional volcano plots provide integrated

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information about metabolomic differences between the different treatment groups.

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Metabolites with significant changes were determined by combining restrictions of

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three dimensions: p < 0.05, |r| > 0.5 and VIP values above top 30%. Also, the

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metabolites with significant changes tended to be located at the upper zones of the

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plot, segmented by the horizontal threshold line p = 0.05, with larger circle sizes and

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warmer colors. The volcano plot was generated with MATLAB scripts (downloaded

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from http://www.mathworks.com) with some in-house modification.

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Metabolic correlation analysis and pathway analysis

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To determine the effect of exogenous taurine on metabolic correlations and

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pathways involved in the metabolomic difference between taurine treatment groups

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and the control group, a comprehensive metabolic correlation analysis and pathway

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analysis via KEGG and MetaboAnalyst online service (http://www.metaboanalyst.ca/)

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were conducted on the differential metabolites derived from different comparison

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models. Furthermore, inner relation plots were generated to explore the correlation

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between the concentrations of identified metabolites and the WGR value via PLS

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analysis with SIMCA-P software.

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Results

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Growth of tilapia under the effect of taurine supplementation

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The effects of dietary taurine on the whole-body composition of tilapia at day 84 11

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and the WGR at 28, 56 and 84 feed days are shown in Figures 1A and 1B, respectively.

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The data collected on day 84 shows that moisture content in muscle tissue decreased

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significantly in the taurine-supplemented groups (D1, D2 and D3 group), as compared

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to the control group. This was accompanied by significant increases in crude protein

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and crude lipid content. These results indicate that taurine supplementation probably

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influenced the moisture-holding capacity, the amino acid and protein metabolism and

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lipid metabolism of tilapia. Therefore, the growth of tilapia was also affected. In

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addition, WGR values were improved significantly in the taurine-supplemented

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groups for all data collection time points (FD28, 26 and 84) compared to the CTRL

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(see Figure 1B). The WGR was highest at FD28, and decreased with the increase of

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feed times. These results indicate that taurine supplementation indeed facilitated the

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growth of tilapia, and the growth of juvenile fish was improved most significantly at

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FD28. Figure 1

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Metabolic

profiles

of

muscle

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taurine-supplemented diets

from

tilapia

fed

with

four

different

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Typical 500 MHz 1H NMR spectra of tilapia muscle in all four groups (CTRL,

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D1, D2 and D3) at FD84 are shown in Figure 2. A total of 42 metabolites were

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assigned and labeled in NMR spectra with reference to published literature,21-23 public

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metabolite databases,29 and 2D NMR spectra (The 2D NMR spectra and metabolite

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assignment table were shown in Figure S1 and Table S2, respectively). Tilapia muscle

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from different groups shared similar spectral profiles. However, only a few 12

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differences can be observed by a visual comparison, including the high concentrations

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of taurine, adenosine monophosphate (AMP), and the low levels of alanime, glycine,

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and inosine in the taurine-supplemented groups when compared to the control group.

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Figure 2 Metabolic trajectory of tilapia muscle during the feed period

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First, PCA was performed to investigate the overall metabolic trajectory during

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feed periods and to identify possible outliers between the control group and the

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taurine-supplemented groups (Figure 3).

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Figure 3

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PCA scores plots (Figure 3) show an obvious separation between the control

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group (CTRL) and the taurine-supplemented groups (i.e. D1, D2 and D3) at FD28 and

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FD84, and a slight overlap between CTRL and D1 at FD56 was observed. In addition,

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the clusters from the taurine-supplemented groups D2 and D3 overlapped at these

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three feed periods, and they were clearly separated from D1 at FD28. This imply that

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the metabolomic profiles of D2 and D3 groups were partly similar, but they had

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different metabolic phenotypes from D1 at FD28 (Figure 3A). Further analysis found

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that the overlap among the three taurine-supplemented groups became more obvious

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with the increase in feed period. It should be note that two sample points appeared

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outside the T2 Hotelling Ellipses but not far from the 95% confidence interval.

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However, no significant anomalies occurred for these samples, when analyzed by the

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corresponding original NMR spectra. This might be due to the individual differences 13

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of fish, which are caused by the growth environment or other external stimuli.

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Therefore, the two sample points were not considered outliers and not removed from

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the model. PLS-DA highlight the distinctive inter-group separations (Figure S2).

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These results indicate that significant and nonuniform physiological changes in tilapia

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muscle were induced by the taurine supplementation, and the effect of exogenous

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taurine on tilapia metabolites was different at different feed periods, compared with

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the control group. Based on these results, the metabolic analyses were further

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evaluated via pairwise-comparisons between the taurine-supplemented and control

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groups.

270 271

Figure 4 Metabolic variations in response to different taurine content

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Pairwise-comparisons between the taurine-supplemented groups and the control

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group were carried out using PLS-DA and OPLS-DA analysis (Figures S3 and S4 in

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the supporting information). Then, volcano plots were used to identify the specific

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differential metabolites that contributed to the inter-group separation. In the volcano

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plots (right panel in Figure S4), taurine was found at a significantly higher than the

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other metabolites, which is a result of the exogenous taurine supplementation. In order

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to investigate the effect of exogenous taurine on other metabolites, a new OPLS-DA

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model was built by removing taurine from data (Figure S5). The differential

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metabolites were visually displayed in volcano plots (above the dash line in Figure 4)

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and summarized with the evaluating parameters of the models including R2X, R2Y

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and Q2 and p-value of the pairwise-comparisons groups in Table 1. 14

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Table 1

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The changes in metabolites in tilapia muscle can be summarized as follows: (1)

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The levels of proline, glycine and alanine decreased from D1, D2 to D3 at FD28. This

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trend was also presented in D2 and D3 at FD56. The changes were subtle in all

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taurine-supplemented groups at FD84, except for the decrease of glycine. Furthermore,

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only alanine decreased in D1 at FD56; (2) An irregular decline of methionine,

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histidine and lysine occurred in D3 at FD28, D1 at FD56 and D2 at FD28, and D1 at

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FD84, respectively. In addition, a slight decrease in leucine was found in both D1 and

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D3 groups at FD84; (3) During the 84 days feed period, the level of betaine decreased

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slightly in all taurine-supplemented groups when compared with the control, with the

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exception of D2 at FD28. Conversely, the level of carnosine decreased only in D2 at

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FD28. Moreover, glycerol increased in D1 at FD56 and FD84, and choline increased

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only in D2 and D3 groups at FD56 and FD84, respectively; (4) A relatively decrease

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in α- or β- glucose was observed in both D1 and D3 groups at FD84. The energy

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metabolites, such as AMP, increased in all the taurine-supplemented groups, with the

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exception of D1 at FD28. Lactate also increased in D1, D2 and D3 groups at FD28

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and FD84. A high level of creatine was detected in D2 at FD28 and D3 at FD28 and

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FD84. (5) Other metabolites, such as pantothenate and cholate, decreased slightly in

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D1 and D3 groups at FD84. Moreover, acetate slightly decreased in D1 at FD56 and

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D2 at FD28 but increased in D3 at FD56. These results imply that taurine

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supplementation leads to significant metabolic changes at the early stage of fish

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growth, and this effect weaken as time goes on. 15

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To further understand the metabolic differences between taurine-supplemented

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groups and the control group, the differential metabolites data were further analyzed

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using correlation analysis and a clustering heatmap (Figure S6). According to the

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strong correlations (absolute correlation coefficients |r| ≥ 0.5) detected and the cluster

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analysis, these differential metabolites can be classified into eight categories. The

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metabolites with similar correlation coefficients appear adjacent in the map, so that

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the dendrograms can indicate the relationships among metabolites. The significant

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correlations might suggest that metabolites may have similar functions or that the

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contents of both metabolites were simultaneously affected by the exogenous taurine.

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Adjacent and positive correlations were observed between lactate, AMP and creatine,

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which are involved in energy metabolism, and this indicates that they were regulated

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simultaneously by taurine supplementation to meet energy demands. Lactate was

317

negatively correlated with α-glucose, β-glucose and alanine, which participate in

318

glycolysis. Additionally, glycerol and choline, which are associated with lipid

319

metabolism, were adjacent and positively correlated. In addition, for some specific

320

amino acid metabolisms, proline and glycine; acetate, carnosine and histidine; betaine

321

and alanine; α-glucose, methionine and lysine were adjacent and had strong positive

322

correlations. Creatine had strong negative correlations with betaine and glycine. A

323

number of discriminant metabolic pathways between the taurine-supplemented groups

324

and control group were identified (Figure 5), including glycine, serine and threonine

325

metabolism, purine metabolism, arginine and proline metabolism, tricarboxylic acid

326

(TCA) cycle, urea cycle, glycolysis/gluconeogenesis, and alanine metabolism and 16

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histidine metabolism. Figure 5

328

329

In addition, PLS correlation analysis show that these differential metabolites are

330

correlated with fish development and growth (the inner relation plots were shown in

331

Figures S7 and S8 in supporting information). These correlation results indicate that

332

these taurine-induced metabolic variations played a positive role in promoting fish

333

growth at each feed period, which was also in agreement with the results shown in

334

Figure 1.

335

Discussion

336

Perturbation in metabolism

337

The

experimental

results

demonstrate

that

taurine

caused

significant

338

dietary-dependent and time/development-dependent metabolomic changes in tilapia

339

muscle, particularly regarding the TCA cycle and glycolysis/gluconeogenesis, amino

340

acid metabolism, lipid metabolism, and nucleotide-related metabolism (Figure 5).

341

Amino acid metabolism

342

Taurine supplementation led to a lower concentration of a number of amino acids

343

in muscle, including methionine, histidine, proline, glycine, lysine, alanine and

344

leucine. The changes can be correlated with perturbations in metabolism, such as

345

ABC transporters, biosynthesis of amino acids, mineral absorption, and protein

346

digestion and absorption.30, 31 In our study, proline, glycine and alanine decreased with

347

increasing taurine supplementation levels, and this result was consistent with previous

348

studies on red sea bream,31 yellowtail32 and milkfish (Chanos chanos).33 However, the 17

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349

extent of these reductions was found to decline slightly with an increase in the number

350

of feed days (Table 1). This might indicate that taurine supplementation in daily diet

351

would slightly decrease the content of some free amino acids in tilapia muscle tissue.

352

Additionally, the synthesis of protein or fat would be induced with the participation of

353

these amino acids, which would further improve the growth of fish. These results are

354

consistent with the increase in the levels of crude protein and crude lipid content in

355

tilapia body, as shown in Figure 1A, as well as with the WGR values shown in Figure

356

1B.

357

According to a previous study, taurine supplementation could act synergistically

358

with insulin or insulin-like substances and further promote the utilization of amino

359

acid and glucose uptake in cells.34 Consequently, taurine supplementation would

360

decrease the levels of some amino acids and accelerate the glycolysis/gluconeogenesis,

361

promoting the fish body growth. This would explain the decline of some amino acid

362

concentrations (i.e. glycine, alanine, proline and histidine) at FD28 and FD56 as well

363

as the lower level of glucose in D1 at FD84. This indicates that taurine

364

supplementation would promote rapid growth of fish in the early and middle growth

365

stages. This is supported by the WGR values (Figure 1B), in which the highest WGR

366

values was observed during the feed period of 1-28 days, and then WGR values

367

declined with an increase in the number of the feed days.

368

Energy metabolism

369

As shown in Table 1, the metabolites related to energy metabolism changed

370

differently. An obvious increase in AMP (except for the D1 group at FD28) and lactate 18

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371

(except for the D2 group at FD28) were observed for the taurine-supplemented groups,

372

when compared with the control. Additionally, a decrease in α-glucose, β-glucose (D1

373

and D3 group at FD84), alanine and glycine (at FD28 and FD56) were found in the

374

taurine-supplemented groups, when compared with the control. In fact, AMP, lactate,

375

glycine and alanine are also involved in glycolysis and gluconeogenesis pathways

376

(Figure 6). Energy metabolism is closely linked to glucose and ATP levels.35 In the

377

present study, the increase of AMP could be due to the increased hydrolysis of ATP,

378

which occurred in order to meet the energy demand for fish growth.36 The decline of

379

glucose at FD84 implied an intensive oxygenolysis of glucose or conversion to amino

380

acids or other intermediates via pyruvate in tricarboxylic acid (TCA) cycle (Figure

381

6).29 The increase of lactate indicated the acceleration of anaerobic glycolysis in fish

382

muscle, which might be due to the rapid growth of fish in urgent need of large

383

amounts of energy consumption for rapid growth of fish at early and later growth

384

stages (i.e. FD28 and FD84). These changes were also verified by the high WGR

385

values of fish that were fed the taurine-supplemented daily diet, particularly at the

386

period of 1-28 day (see Figure 1B).

387

In addition, alanine is a major glucogenic precursor, an important energy

388

substrate for fish, and a preferred carrier of nitrogen for inter-organ amino acids

389

metabolism in fish.23 Moreover, carnosine has the potential to suppress many of the

390

biochemical changes (e.g., protein oxidation, glycation) as well as provide an

391

important buffer in skeletal muscle of aquatic animals, especially migratory pelagic

392

marine fishes.37 Reduced levels of alanine and carnosine in fish muscle may be in 19

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393

response to acceleration of glycolysis, protein digestion and absorption. Furthermore,

394

significantly high levels of creatine, which functions as a cell energy shuttle, were

395

presented in D2 and D3 groups at the FD28 and FD84.35 This was accompanied by a

396

decline in glycine, possibly because the fish body used glycine to synthesize more

397

creatine for energy demands in the early and later feed periods. The change in energy

398

metabolism might suggest that the taurine supplementation possibly increased the

399

energy consumption of muscle tissue, and this is most likely due to the enhanced

400

metabolism, such as protein syntheses and lipid metabolism. This observation is in

401

agreement with the previous literature.12, 30

402

Lipid metabolism

403

Regarding changes in lipid metabolism, betaine significantly decreased in all

404

taurine-supplemented groups, except for D2 at FD28. The decrease of betaine might

405

be due to the following reasons. First, betaine donates methyl groups for the synthesis

406

of methionine and other compounds that play a key role in protein, energy and lipid

407

metabolism.21, 38 Betaine deficiency is associated with lipid disorders. Second, taurine

408

could improve the activity of the rate-limiting enzyme, CYP7A1, promoting

409

cholesterol to change into bile acid and further promoting the synthesis of lipids.39 As

410

a result, there is increased demand for energy, acceleration of protein synthesis and

411

lipid accumulation during the feed period. Additionally, betaine decreased more

412

markedly in the taurine-supplemented groups, as compared with the controls.

413

It should be noted that glycerol showed a significant increase in the D1 group,

414

compared with the control group at the middle and late feeding periods (i.e. at FD56 20

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415

and FD84). Glycerol is an important component of triglycerides (i.e. fats and oils) and

416

of phospholipids, and it can be used as a substrate in gluconeogenesis in fish. The

417

increase in glycerol potentially suggests that taurine supplementation would accelerate

418

glycolysis and promote lipid synthesis in muscle at FD56 and FD84, and this

419

assumption is supported by the decrease in glucose at FD84 (see Table 1) and the

420

increase of crude lipid in muscle (see Figure 1A).

421

The metabolic changes related to lipid metabolism include an increase of choline

422

in the pairwise-comparison of D2-CTRL (FD56) and D3-CTRL (FD84), and a

423

decrease of cholate in the pairwise-compare of D1-CTRL and D3-CTRL at FD84. It

424

should

425

phosphocholine/glycerophosphocholine

426

glycerophosphocholines, which are present in high concentrations in lipoprotein

427

particles. The increase in choline, phosphorylcholine, and glycerolphosphocholine

428

suggest increased lipid metabolism. Juvenile tilapia normally undergo fat storage for

429

body growth and development from their non-gravid state and early feed period into

430

the middle and late feed periods.40 Furthermore, cholate usually conjugates with

431

glycine or taurine to facilitate fat absorption and cholesterol excretion. The decline of

432

cholate in D1 and D3 group at FD84 also indirectly explained the accumulation of

433

lipids in the fish muscle. These metabolic changes indirectly suggest that taurine

434

could promote lipid and protein synthesis in fish, and this is in agreement with the

435

increase of crude protein and crude lipid in fish body (Figure 1A).

436

Tilapia muscle quality changes with taurine supplementation

be

noted

that

the

choline in

metabolites,

Figure

21

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2,

were

indicated actually

as from

Journal of Agricultural and Food Chemistry

437

As previously stated, the supplementation of taurine significantly affected the

438

metabolism of fish muscle and indirectly affected the fish meat quality, such as the

439

taste, tenderness and degree of freshness.

440

Betaine and carnosine are widely regarded as antioxidants. Intracellular

441

accumulation of betaine permits water retention in cells, thus, protecting cells from

442

the effects of dehydration.41 The decline of betaine in the taurine-supplemented

443

groups suggests that the moisture content in tilapia muscle would slightly decrease

444

with the effect of taurine supplementation. Carnosine suppress the accumulation of

445

lipid oxidation products, like malondialdehyde (MDA).42 A previous study reported

446

that lipid oxidation influences meat quality and leads to bad flavor and poor

447

nutritional value.43 In this study, no significant changes of carnosine (except for D2 at

448

FD28) and MDA imply that lipid oxidation was maintained in an inactive state.

449

Consequently, the variations of betaine and carnosine might affect the antioxidant

450

capacity of fish and help to keep the freshness of fish muscle.

451

According to the literature, glycine and proline are involved in the production of

452

collagen,44 and the improvement of texture parameters (hardness, springiness and

453

chewiness) of large yellow croaker (Larimichthys crocea) were highly correlated with

454

total collagen content in muscle.45 In this study, the reductions of both glycine and

455

proline are probably due to higher consumption of the amino acids for collagen

456

synthesis. Therefore, the additive taurine possibly caused a reduction in the amino

457

acids and indirectly facilitated the collagen synthesis, thus, improving the muscle

458

composition and the meat quality. The flavor and taste of fish meat are partially 22

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459

depend on the contents of delicious amino acids, like glycine, alanine and proline, in

460

the muscle.46 In the present study, the contents of the above three amino acids

461

decreased with increasing taurine supplementation.

462

All in all, the metabolite changes in tilapia muscle reflect both the physiological

463

state and growth potential in fish, indicating that taurine supplementation would

464

significantly affect the tilapia metabolome, improve energy utilization and amino acid

465

uptake, promote protein and lipid synthesis, accelerate the production of collagen and

466

further improve the muscle quality. The benefits of taurine were dependent on

467

concentration and feed period duration. The optimum condition for each taurine

468

treatment group was as follows: 1.0% (D2, at FD28), 1.2% (D3, at FD56) and 0.4%

469

(D1, at FD84). Our study might provide important insight into fish feed nutrition and

470

fish meat quality monitoring during the fish feed period as well as offer guidance for

471

the implementation of fish culture on metabolism. Further research is needed to

472

investigate the best supplementation strategy to simultaneously improve tilapia

473

growth and ensure good quality meat.

474

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ASSOCIATED CONTENT Supporting Information Figure S1: 2D NMR J-res, TOCSY and HSQC spectra of tilapia muscle; Fiugre S2: PLS-DA scores plots with four different taurine contents; Figure S3-S5: PLS-DA and OPLS-DA

scores

plots

for

the

pairwise-comparisons

between

one

taurine-supplemented group and the control group. Figure S6: Correlation analysis heatmap of differential metabolites; Figure S7-S8: Inner relation plots between differential metabolite; Table S1: Ingredients and proximate composition of tilapia experimental diets; Table S2: Metabolites identified from NMR spectra of tilapia muscle and the corresponding assignments.

AUTHOR INFORMATION Corresponding Author †

422 Siming South Road, Xiamen University, Xiamen, Fujian Province 361005, China. Tel.: +86 592 2184026, Fax: +86 2189426. Email address: [email protected] (G. Shen)



43 Yindou Road, Jimei Univesity, Xiamen, Fujian Province 361021, China.

Tel./fax: +86 592 6181054. Email address: [email protected] (J. Ye)

ORCID Guiping Shen: 0000-0002-0779-1859

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Funding This research was supported with the funding from the National Natural Science Foundation of China (Grant No. 31372546 and 81371639), the Natural Science Foundation of Fujian Province of China (Grant No. 2015Y0032) and the Fundamental Research Funds for the Central Universities (Grant No. 20720150018 and 20720160125).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We gratefully acknowledge Liubin Feng for technical support. We thank Lingli Deng and Xiangnan Xu for helpful discussions on the multivariate data analysis.

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(32) Matsunari, H.; Takeuchi, T.; Murata, Y.; Takahashi, M.; Ishibashi, N.; Chuda, H.; Arakawa, T. Changes in the taurine content during the early growth stages of artificially produced yellowtail compared with wild fish. Nippon Suisan Gakkaishi 2003, 69, 757-762. (33) Shiau, C. Y.; Pong, Y. J.; Chiou, T. K.; Chai, T. J. Effect of growth on the levels of free histidine and amino acids in white muscle of milkfish (Chanos chanos). J. Agric. Food Chem. 1997, 45, 2103-2106. (34) Nakamura, K.; Morimoto, K.; Shima, K.; Yoshimura, Y.; Kazuki, Y.; Suzuki, O.; Matsuda, J.; Ohbayashi, T. The effect of supplementation of amino acids and taurine to modified KSOM culture medium on rat embryo development. Theriogenology 2016, 86, 2083-2090. (35) Owen, L.; Sunram-Lea, S. I. Metabolic agents that enhance ATP can improve cognitive functioning: A review of the evidence for glucose, oxygen, pyruvate, creatine, and L-carnitine. Nutrients 2011, 3, 735-755. (36) Shao, Y. N.; Li, C. H.; Chen, X. C.; Zhang, P. J.; Li, Y.; Li, T. W.; Jiang, J. B. Metabolomic responses of sea cucumber Apostichopus japonicus to thermal stresses. Aquaculture 2015, 435, 390-397. (37) Blancquaert, L.; Baba, S. P.; Kwiatkowski, S.; Stautemas, J.; Stegen, S.; Barbaresi, S.; Chung, W. L.; Boakye, A. A.; Hoetker, J. D.; Bhatnagar, A.; Delanghe, J.; Vanheel, B.; Veiga-da-Cunha, M.; Derave, W.; Everaert, I. Carnosine and anserine homeostasis in skeletal muscle and heart is controlled by beta-alanine transamination. J. Physiol.-London 2016, 594, 4849-4863. (38) Lever, M.; Slow, S. The clinical significance of betaine, an osmolyte with a key role in methyl group metabolism. Clin. Biochem. 2010, 43, 732-744. (39) Russell, D. W. The enzymes, regulation, and genetics of bile acid synthesis. Annu. Rev. Biochem. 2003, 72, 137-174. (40) Gaylord, T. G.; Teague, A. M.; Barrows, F. T. Taurine supplementation of all-plant protein diets for rainbow trout (Oncorhynchus mykiss). J. Agric. Food Chem. 2006, 37, 509-517. (41) Bingul, I.; Basaran-Kucukgergin, C.; Aydin, A. F.; Coban, J.; Dogan-Ekici, I.; Dogru-Abbasoglu, S.; Uysal, M. Betaine treatment decreased oxidative stress, inflammation, and stellate cell activation in rats with alcoholic liver fibrosis. Environ. Toxicol. Pharmacol. 2016, 45, 170-178. (42) Boldyrev, A. A. Problems and perspectives in studying the biological role of carnosine Introduction of the guest-editor. Biochem.-Moscow 2000, 65, 751-756. 29

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(43) Baron, C. P.; Andersen, H. J. Myoglobin-induced lipid oxidation. A review. J. Agric. Food Chem. 2002, 50, 3887-3897. (44) Ottani, V.; Martini, D.; Franchi, M.; Ruggeri, A.; Raspanti, M. Hierarchical structures in fibrillar collagens. Micron 2002, 33, 587-596. (45) Wei, Z. H.; Ma, J.; Pan, X. Y.; Mua, H.; Li, J.; Shentu, J.; Zhang, W. B.; Mai, K. S. Dietary hydroxyproline improves the growth and muscle quality of large yellow croaker Larimichthys crocea. Aquaculture 2016, 464, 497-504. (46) Ruiz-Capillas, C.; Moral, A. Free amino acids in muscle of Norway lobster (Nephrops novergicus (L.)) in controlled and modified atmospheres during chilled storage. Food Chem. 2004, 86, 85-91.

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Figure Captions Figure 1 (A) The tilapia whole-body composition at FD84 and (B) the weight gain rate (WGR) at FD28, FD56 and FD84 with diets containing different taurine contents (CTRL: 0.0%, D1: 0.4%, D2: 0.8%, D3: 1.2%). Asterisk (*) indicates significant differences between taurine-supplemented groups (D1, D2 and D3) and the control group (CTRL): p < 0.05.

Figure 2 Representative 500 MHz water-suppressed 1H NMR spectra (δ0.5-9.2) of tilapia muscle from fish fed diets containing different taurine contents (CTRL: 0.0%, D1: 0.4%, D2: 0.8%, D3: 1.2%) at FD84. The region of δ5.1-9.2 was vertically magnified 10 times, in comparison with the region δ0.5-5.1 for the purpose of clarity.

Keys for the assignments of peaks: 1. Cholate, 2. Pantothenate, 3. Isoleucine, 4. Leucine, 5. Valine, 6. 3-Hydroxybutyrate, 7. Lactate, 8. Alanine, 9. Lysine, 10. Acetate, 11. Proline, 12. Methionine, 13. Succinate, 14. Glutamine, 15. Aspartate, 16. DMA: Dimethylamine, 17. Sarcosine, 18. TMA: Trimethylamine, 19. DMG: N,N-Dimethylglycine, 20. Creatine, 21. Ethanolamine, 22. β-Alanine, 23. Choline, 24. PC: Phosphocholine, 25. GPC: Glycerolphosphocholine, 26. Taurine, 27. Betaine, 28. β-Glucose, 29. α-Glucose, 30. Glycine, 31. Glycerol, 32. Histidine, 33. AMP: Adenosine monophosphate, 34. Inosine, 35. Carnosine, 36. Trehalose, 37. Glycogen, 38. Adenosine, 39. Fumarate, 40. Tyrosine, 41. Phenylalanine, 42. Nicotinate, 43, 44, 45. Unknown.

Figure 3 PCA scores plots of tilapia muscle from fish fed diets with four different taurine contents (CTRL, D1, D2 and D3) at different feeding days (FD). FD28: (A); FD56: (B) and FD84: (C).

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Figure 4 Volcano plots of multiple matrices for screening differential metabolites between taurine-supplemented groups and the control group after taurine was removed from NMR data. (A1, A2, A3), (B1, B2, B3) and (C1, C2, C3) correspond to FD28, FD56 and FD84, respectively. Each circle represents one metabolite. The point size of big and small indicate the VIP values of the top 30% and the remaining 70%. Differential metabolites marked in the plot were determined by combining restrictions of three dimensions: p < 0.05, |r|> 0.5 and the VIP values above the top 30% which are listed in Table 1.

Figure 5 Metabolic pathways affected by dietary taurine in tilapia muscle extracts. Metabolites in red and blue represent higher or lower levels in tilapia muscle extracts of the taurine-supplemented groups, when compared with the control group. Metabolites in green frames represent non-significant change and were detected by 1H NMR, and metabolites with no color marking were not detected.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Table 1 OPLS-DA coefficients derived from NMR data of tilapia fed diets containing different taurine contents during 84 feed days D1-CTRL FD28

D3-CTRL

FD56

FD84

FD28

FD56

FD84

FD28

FD56

FD84

a

0.585

0.498

0.624

0.278

0.539

0.669

0.617

0.578

R2Y = 0.746

0.589

0.81

0.862

0.689

0.667

0.862

0.755

0.734

0.248

0.579

0.757

0.4

0.223

0.748

0.665

0.415

2

R X = 0.583

Metabolites

D2-CTRL

2

Q = 0.547 p = 0.052 Foldb

rc

0.538 Fold

0.036 r

Fold

0.008 r

Fold

0.233 r

Fold

0.556 r

Fold

0.009 r

0.018

Fold

r

0.604

-0.556

0.172

Fold

r

Fold

r

0.552

-0.570

0.484

-0.641

0.487

-0.712

1.264

0.537

1.553

0.797

1.292

0.718

0.510

-0.651

Amino acid metabolism Methionine Histidine

0.756

-0.571

Proline

0.795

-0.559

0.725

-0.517

0.455

-0.861

0.720

-0.537

0.516

-0.856

0.652

-0.592

Glycine

0.642

-0.631

0.577

-0.703

0.478

-0.673

0.468

-0.833

0.530

-0.609

0.778

-0.651

0.728

-0.698

0.751

-0.560

0.789

-0.536

0.771

-0.541

Lysine Alanine

0.578 0.685

-0.508

-0.651

Leucine

0.557

-0.627

0.614

-0.601

Lipid metabolism Betaine

0.806

-0.627

0.675

-0.597

Carnosine

0.707

Glycerol

1.965

0.529

2.171

0.546

-0.768

1.331

0.548

1.496

0.609

0.561

-0.578

0.741

-0.755

0.354

-0.832

-0.644

0.691

Choline Energy metabolism AMP Lactate α-Glucose

1.327 1.135

0.585

0.634

1.288

0.618

1.560

0.904

1.235

0.613

1.536

0.584

0.418

-0.714

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1.353

0.631

1.339

0.819

1.259

0.642

1.172

0.665

1.859

0.816

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β-Glucose

0.633

-0.680

Creatine

1.120

0.597

0.733

-0.640

1.155

0.766

0.692

-0.621

1.204

0.638

Other Acetate

a

0.757

-0.551

1.245

0.563

Pantothenate

0.583

-0.548

0.429

-0.677

Cholate

0.247

-0.597

0.371

-0.502

The evaluating parameters of the models including R2X, R2Y, Q2 and p-value. b Fold-change values, numbers greater and less than 1 indicate that the metabolites are

more abundant or few in the taurine treatment group when compared with the control group. c Correlation coefficients, positive and negative signs indicate positive and negative correlation in the concentrations, respectively.

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TABLE OF CONTENTS GRAPHICS

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Figure 1 (A) The tilapia whole-body composition at FD84 and (B) the weight gain rate (WGR) at FD28, FD56 and FD84 with diets containing different taurine contents (CTRL: 0.0%, D1: 0.4%, D2: 0.8%, D3: 1.2%). Asterisk (*) indicates significant differences between taurine-supplemented groups (D1, D2 and D3) and the control group (CTRL): p < 0.05. 338x133mm (300 x 300 DPI)

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Figure 2 Representative 500 MHz water-suppressed 1H NMR spectra (δ0.5-9.2) of tilapia muscle from fish fed diets containing different taurine contents (CTRL: 0.0%, D1: 0.4%, D2: 0.8%, D3: 1.2%) at FD84. The region of δ5.1-9.2 was vertically magnified 10 times, in comparison with the region δ0.5-5.1 for the purpose of clarity. Keys for the assignments of peaks: 1. Cholate, 2. Pantothenate, 3. Isoleucine, 4. Leucine, 5. Valine, 6. 3Hydroxybutyrate, 7. Lactate, 8. Alanine, 9. Lysine, 10. Acetate, 11. Proline, 12. Methionine, 13. Succinate, 14. Glutamine, 15. Aspartate, 16. DMA: Dimethylamine, 17. Sarcosine, 18. TMA: Trimethylamine, 19. DMG: N,N-Dimethylglycine, 20. Creatine, 21. Ethanolamine, 22. β-Alanine, 23. Choline, 24. PC: Phosphocholine, 25. GPC: Glycerolphosphocholine, 26. Taurine, 27. Betaine, 28. β-Glucose, 29. α-Glucose, 30. Glycine, 31. Glycerol, 32. Histidine, 33. AMP: Adenosine monophosphate, 34. Inosine, 35. Carnosine, 36. Trehalose, 37. Glycogen, 38. Adenosine, 39. Fumarate, 40. Tyrosine, 41. Phenylalanine, 42. Nicotinate, 43, 44, 45. Unknown. 584x383mm (300 x 300 DPI)

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Figure 3 PCA scores plots of tilapia muscle from fish fed diets with four different taurine contents (CTRL, D1, D2 and D3) at different feeding days (FD). FD28: (A); FD56: (B) and FD84: (C). 123x262mm (300 x 300 DPI)

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Figure 4 Volcano plots of multiple matrices for screening differential metabolites between taurinesupplemented groups and the control group after taurine was removed from NMR data. (A1, A2, A3), (B1, B2, B3) and (C1, C2, C3) correspond to FD28, FD56 and FD84, respectively. Each circle represents one metabolite. The point size of big and small indicate the VIP values of the top 30% and the remaining 70%. Differential metabolites marked in the plot were determined by combining restrictions of three dimensions: p < 0.05, |r|> 0.5 and the VIP values above the top 30% which are listed in Table 1. 285x232mm (300 x 300 DPI)

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Figure 5 Metabolic pathways affected by dietary taurine in tilapia muscle extracts. Metabolites in red and blue represent higher or lower levels in tilapia muscle extracts of the taurine-supplemented groups, when compared with the control group. Metabolites in green frames represent non-significant change and were detected by 1H NMR, and metabolites with no color marking were not detected. 210x133mm (300 x 300 DPI)

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TABLE OF CONTENTS GRAPHICS 335x175mm (300 x 300 DPI)

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