Metabolic Effect of Dietary Taurine Supplementation on Nile Tilapia

Subscriber access provided by READING UNIV

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 46

Journal of Agricultural and Food Chemistry

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

1

ACS Paragon Plus Environment


1

Abstract

2

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

3

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

4

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

5

NMR-based metabolomics approaches were applied to investigate the metabolite

6

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

7

metabolic trajectory and reveal the possible mechanisms of metabolic effects of

8

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

9

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

10

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

11

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

12

supplementation could significantly regulate the physiological state of fish and

13

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

14

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

15

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

16

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

17

nutrients in aquaculture.

18 19

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

2


Page 2 of 46

Page 3 of 46


20

Introduction

21

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

22

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

23

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

24

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

25

aquaculture sustainability.2,

26

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

27

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

28

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

29

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

30

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

31

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

32

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

4

Many studies have attempted to replace FM with a

33

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

34

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

35

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

36

varies greatly.11,

37

physiological abnormalities and inferior performances.11 Recent studies have proven

38

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

39

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

40

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

41

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

12

It has been shown that taurine-deficiency will result in

3



42

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

43

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

44

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

45

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

46

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

47

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

48

physiological roles of taurine supplementation have yet to be established.

49

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

50

second largest farmed fish worldwide after carp.14,

15

51

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

52

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

53

utilization and reproductive performance of tilapia have been studied extensively in

54

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

55

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

56

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

57

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

58

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

59

metabolic effects of taurine supplementation.

There is an urgent need to

60

Metabolomics is a systems biology approach to study complex biochemical

61

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

62

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

63

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


Page 4 of 46

Page 5 of 46


64

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

65

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

66

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

67

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

68

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

69

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

70

nutrient utilization.

71

Material and methods

72

Experimental diets

73

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

74

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

75

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

76

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

77

vitamin

78

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

79

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

80

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

81

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

82

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

83

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

84

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

85

Table S1 in the supporting information.

C,

monocalcium

phosphate,

choline

chloride,

5


shrimp meal,

and


86

Fish and experimental conditions

87

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

88

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

89

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

90

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

91

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

92

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

93

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

94

Technologies, LLC., USA) for two weeks.

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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


Page 6 of 46

Page 7 of 46


108

Sampling procedure and extraction

109

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

110

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

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

and kept at -80°C.

119

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

120

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

121

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

122

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

123

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

124

WGR (%) = 100×(Wf -Wi)/Wi

125

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

126

differences between taurine treatments were calculated using one-way ANOVA

127

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

128

Chicago, IL, USA).

129

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



130

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

131

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

132

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

133

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

134

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

135

compliance with Xiamen University laboratory safety management regulations). After

136

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

137

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

138

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

139

-80 °C for NMR experiments.

140

Sample preparation and 1H NMR spectroscopy

141

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

142

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

143

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

144

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

145

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

146

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

147

until the 1H NMR data acquisition.

148

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

149

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

150

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

151

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


Page 8 of 46

Page 9 of 46


152

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

153

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

154

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

155

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

156

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

157

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

158

Pre-processing of 1H NMR spectra and multivariate analysis

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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



174

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

175

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

176

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

177

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

178

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

179

metabolomic differences between the taurine-supplemented groups and the control

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

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

195

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


Page 10 of 46

Page 11 of 46


196

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

197

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

198

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

199

information about metabolomic differences between the different treatment groups.

200

Metabolites with significant changes were determined by combining restrictions of

201

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

202

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

203

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

204

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

205

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

206

Metabolic correlation analysis and pathway analysis

207

To determine the effect of exogenous taurine on metabolic correlations and

208

pathways involved in the metabolomic difference between taurine treatment groups

209

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

210

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

211

were conducted on the differential metabolites derived from different comparison

212

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

213

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

214

analysis with SIMCA-P software.

215

Results

216

Growth of tilapia under the effect of taurine supplementation

217

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



Page 12 of 46

218

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

219

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

220

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

221

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

222

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

223

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

224

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

225

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

226

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

227

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

228

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

229

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

230

FD28. Figure 1

231

232

Metabolic

profiles

of

muscle

233

taurine-supplemented diets

from

tilapia

fed

with

four

different

234

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

235

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

236

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

237

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

238

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

239

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


Page 13 of 46


240

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

241

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

242

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

243

244

Figure 2 Metabolic trajectory of tilapia muscle during the feed period

245

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

246

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

247

taurine-supplemented groups (Figure 3).

248

Figure 3

249

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

250

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

251

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

252

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

253

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

254

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

255

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

256

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

257

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

258

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

259

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

260

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



261

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

262

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

263

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

264

These results indicate that significant and nonuniform physiological changes in tilapia

265

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

266

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

267

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

268

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

269

groups.

270 271

Figure 4 Metabolic variations in response to different taurine content

272

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

273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

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

282

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


Page 14 of 46

Page 15 of 46


283

Table 1

284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

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

299

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

300

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

301

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

302

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

303

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

304

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



305

To further understand the metabolic differences between taurine-supplemented

306

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

307

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

308

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

309

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

310

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

311

the dendrograms can indicate the relationships among metabolites. The significant

312

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

313

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

314

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

315

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

316

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


Page 16 of 46

Page 17 of 46


327

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



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


Page 18 of 46

Page 19 of 46


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



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


Page 20 of 46

Page 21 of 46


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


2,

were

indicated actually

as from


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


Page 22 of 46

Page 23 of 46


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

23



Page 24 of 46

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

24


Page 25 of 46


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.

25



REFERENCES (1) Taher, S.; Romano, N.; Arshad, A.; Ebrahimi, M.; Teh, J. C.; Ng, W. K.; Kumar, V. Assessing the feasibility of dietary soybean meal replacement for fishmeal to the swimming crab, Portunus pelagicus, juveniles. Aquaculture 2017, 469, 88-94. (2) Lin, Y. H.; Mui, J. J. Comparison of dietary inclusion of commercial and fermented soybean meal on oxidative status and non-specific immune responses in white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol. 2017, 63, 208-212. (3) Li, M.; Lai, H.; Li, Q.; Gong, S. Y.; Wang, R. X. Effects of dietary taurine on growth, immunity and hyperammonemia in juvenile yellow catfish Pelteobagrus fulvidraco fed all-plant protein diets. Aquaculture 2016, 450, 349-355. (4) Kissinger, K. R.; Garcia-Ortega, A.; Trushenski, J. T. Partial fish meal replacement by soy protein concentrate, squid and algal meals in low fish-oil diets containing Schizochytrium limacinum for longfin yellowtail Seriola rivoliana. Aquaculture 2016, 452, 37-44. (5) Gajardo, K.; Jaramillo-Torres, A.; Kortner, T. M.; Merrifield, D. L.; Tinsley, J.; Bakke, A. M.; Krogdahl, A. Alternative protein sources in the diet modulate microbiota and functionality in the distal intestine of atlantic salmon (Salmo salar). Appl. Environ. Microbiol. 2017, 85. (6) Rhodes, L. D.; Johnson, R. B.; Myers, M. S. Effects of alternative plant-based feeds on hepatic and gastrointestinal histology and the gastrointestinal microbiome of sablefish (Anoplopoma fimbria). Aquaculture 2016, 464, 683-691. (7) Al-Feky, S. S. A.; El-Sayed, A. F. M.; Ezzat, A. A. Dietary taurine enhances growth and feed utilization in larval Nile tilapia (Oreochromis niloticus) fed soybean meal-based diets. Aquac. Nutr. 2016, 22, 457-464. (8) Ye, J. D.; Chen, J. C.; Wang, K. Growth performance and body composition in response to dietary protein and lipid levels in Nile tilapia (Oreochromis niloticus Linnaeus, 1758) subjected to normal and temporally restricted feeding regimes. J. Appl. Ichthyol. 2016, 32, 332-338. (9) Goto, T.; Takagi, S.; Ichiki, T.; Sakai, T.; Endo, M.; Yoshida, T.; Ukawa, M.; Murata, H. Studies on the green liver in cultured red sea bream fed low level and non-fish meal diets: Relationship between hepatic taurine and biliverdin levels. Fish. Sci. 2001, 67, 58-63.

26


Page 26 of 46

Page 27 of 46


(10) Salze, G. P.; Davis, D. A. Taurine: a critical nutrient for future fish feeds. Aquaculture 2015, 437, 215-229. (11) Koven, W.; Peduel, A.; Gada, M.; Nixon, O.; Ucko, M. Taurine improves the performance of white grouper juveniles (Epinephelus Aeneus) fed a reduced fish meal diet. Aquaculture 2016, 460, 8-14. (12) Wang, X.; He, G.; Mai, K. S.; Xu, W.; Zhou, H. H. Differential regulation of taurine biosynthesis in rainbow trout and Japanese flounder. Sci Rep 2016, 6. (13) Yang, H. J.; Tian, L. X.; Huang, J. W.; Liang, G. Y.; Liu, Y. J. Dietary taurine can improve the hypoxia-tolerance but not the growth performance in juvenile grass carp Ctenopharyngodon idellus. Fish Physiol. Biochem. 2013, 39, 1071-1078. (14) Teoh, C. Y.; Ng, W. K. Evaluation of the impact of dietary petroselinic acid on the growth performance, fatty acid composition, and efficacy of long chain-polyunsaturated fatty acid biosynthesis of farmed Nile tilapia. J. Agric. Food Chem. 2013, 61, 6056-6068. (15) Wang, M.; Lu, M. X. Tilapia polyculture: a global review. Aquac. Res. 2016, 47, 2363-2374. (16) Koch, J. F.; Rawles, S. D.; Webster, C. D.; Cummins, V.; Kobayashi, Y.; Thompson, K. R.; Gannam, A. L.; Twibell, R. G.; Hyde, N. M. Optimizing fish meal-free commercial diets for Nile tilapia, Oreochromis niloticus. Aquaculture 2016, 452, 357-366. (17) Watson, A. M.; Barrows, F. T.; Place, A. R. Effects of graded taurine levels on juvenile cobia. N. Am. J. Aqualcult. 2014, 76, 190-200. (18) Nicholson, J. K.; Lindon, J. C. Systems biology - metabonomics. Nature 2008, 455, 1054-1056. (19) Wei, Y. L.; Liang, M. Q.; Mai, K. S.; Zheng, K. K.; Xu, H. G. The effect of ultrafiltered fish protein hydrolysate levels on the liver and muscle metabolic profile of juvenile turbot (Scophthalmus maximus L.) by 1H NMR-based metabolomics studies. Aquac. Res. 2017, 48, 3515-3527. (20) Bankefors, J.; Kaszowska, M.; Schlechtriem, C.; Pickova, J.; Brannas, E.; Edebo, L.; Kiessling, A.; Sandstrom, C. A comparison of the metabolic profile on intact tissue and extracts of muscle and liver of juvenile Atlantic salmon (Salmo salar L.) - Application to a short feeding study. Food Chem. 2011, 129, 1397-1405. (21) Schock, T. B.; Newton, S.; Brenkert, K.; Leffler, J.; Bearden, D. W. An NMR-based metabolomic assessment of cultured cobia health in response to dietary manipulation. Food Chem. 2012, 133, 90-101. 27



(22) Allen, P. J.; Wise, D.; Greenway, T.; Khoo, L.; Griffin, M. J.; Jablonsky, M. Using 1D 1H and 2D 1

H J-resolved NMR metabolomics to understand the effects of anemia in channel catfish (Ictalurus

punctatus). Metabolomics 2015, 11, 1131-1143. (23) Li, M. H.; Wang, J. S.; Lu, Z. G.; Wei, D. D.; Yang, M. H.; Kong, L. Y. NMR-based metabolomics approach to study the toxicity of lambda-cyhalothrin to goldfish (Carassius auratus). Aquat. Toxicol. 2014, 146, 82-92. (24) Manirakiza, P.; Covaci, A.; Schepens, P. Comparative study on total lipid determination using Soxhlet, Roese-Gottlieb, Bligh & Dyer, and modified Bligh & Dyer extraction methods. J. Food Compos. Anal. 2001, 14, 93-100. (25) Wu, H. F.; Southam, A. D.; Hines, A.; Viant, M. R. High-throughput tissue extraction protocol for NMR- and MS-based metabolomics. Anal. Biochem. 2008, 372, 204-212. (26) Davis, R. A.; Charlton, A. J.; Godward, J.; Jones, S. A.; Harrison, M.; Wilson, J. C. Adaptive binning: An improved binning method for metabolomics data using the undecimated wavelet transform. Chemometrics Intell. Lab. Syst. 2007, 85, 144-154. (27) Dieterle, F.; Ross, A.; Schlotterbeck, G.; Senn, H. Probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures. Application in 1H NMR metabonomics. Anal. Chem. 2006, 78, 4281-4290. (28) Climaco-Pinto, R.; Barros, A. S.; Locquet, N.; Schmidtke, L.; Rutledge, D. N. Improving the detection of significant factors using ANOVA-PCA by selective reduction of residual variability. Anal. Chim. Acta 2009, 653, 131-142. (29) Wishart, D. S.; Jewison, T.; Guo, A. C.; Wilson, M.; Knox, C.; Liu, Y. F.; Djoumbou, Y.; Mandal, R.; Aziat, F.; Dong, E.; Bouatra, S.; Sinelnikov, I.; Arndt, D.; Xia, J. G.; Liu, P.; Yallou, F.; Bjorndahl, T.; Perez-Pineiro, R.; Eisner, R.; Allen, F.; Neveu, V.; Greiner, R.; Scalbert, A. HMDB 3.0-the human metabolome database in 2013. Nucleic Acids Res. 2013, 41, D801-D807. (30) Kim, Y. S.; Sasaki, T.; Awa, M.; Inomata, M.; Honryo, T.; Agawa, Y.; Ando, M.; Sawada, Y. Effect of dietary taurine enhancement on growth and development in red sea bream Pagrus major larvae. Aquac. Res. 2016, 47, 1168-1179. (31) Matsunari, H.; Yamamoto, T.; Kim, S. K.; Goto, T.; Takeuchi, T. Optimum dietary taurine level in casein-based diet for juvenile red sea bream Pagrus major. Fish. Sci. 2008, 74, 347-353.

28


Page 28 of 46

Page 29 of 46


(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



(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.

30


Page 30 of 46

Page 31 of 46


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).

31



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.

32


Page 32 of 46

Page 33 of 46


Figure 1

33



Figure 2

34


Page 34 of 46

Page 35 of 46


Figure 3

35



Figure 4

36


Page 36 of 46

Page 37 of 46


Figure 5

37



Page 38 of 46

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

38


1.353

0.631

1.339

0.819

1.259

0.642

1.172

0.665

1.859

0.816

Page 39 of 46


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.

39



TABLE OF CONTENTS GRAPHICS

40


Page 40 of 46

Page 41 of 46


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)



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)


Page 42 of 46

Page 43 of 46


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)



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)


Page 44 of 46

Page 45 of 46


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)



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


Page 46 of 46

Metabolic Effect of Dietary Taurine Supplementation on Nile Tilapia

Recommend Documents