Effects of Dietary n-6:n-3 PUFA Ratios on Lipid Levels and Fatty Acid

Oct 30, 2017 - The objective of this study was to investigate the effects of dietary n-6:n-3 PUFA ratio on growth performance, serum and tissue lipid ...
0 downloads 8 Views 451KB Size
Subscriber access provided by READING UNIV

Article

Effects of dietary n-6:n-3 PUFA ratios on lipid levels and fatty acid profile of Cherry valley ducks at 15–42 days of age mengmeng li, shuangshuang zhai, qiang xie, lu tian, xiaocun li, jiaming zhang, hui ye, yongwen zhu, lin yang, and wence wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02918 • Publication Date (Web): 30 Oct 2017 Downloaded from http://pubs.acs.org on November 4, 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 32

Journal of Agricultural and Food Chemistry

1

Effects of dietary n-6:n-3 PUFA ratios on lipid levels and fatty acid

2

profile of Cherry valley ducks at 15–42 days of age

3

Mengmeng Li†, Shuangshuang Zhai†, Qiang Xie†, Lu Tian†, Xiaocun Li‡, Jiaming Zhang‡,

4

Hui Ye†, Yongwen Zhu†, Lin Yang†*, Wence Wang†*

5



6

University, Guangzhou 510642, China

7



8

*

9

Phone: +86 20 85285232. Fax: +86 20 85285232.

College of Animal Science & College of Marine Sciences, South China Agricultural

Henan Huaying Agriculture development Co., Ltd, Xinyang 464000, China Correspondence should be addressed to [email protected] and [email protected].

10

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

11

Abstract

12

The objective of this study was to investigate the effects of dietary n-6:n-3 PUFA

13

ratio on growth performance, serum and tissue lipid levels, fatty acid profile, and hepatic

14

expression of fatty acid synthesis genes in ducks. A total of 3,168 15-day old ducks were

15

fed different n-6:n-3 PUFA ratios: 13:1 (control), 10:1, 8:1, 6:1, 4:1, and 2:1. The feeding

16

trial lasted four weeks. Our results revealed that dietary n-6:n-3 PUFA ratios had no

17

effects on growth performance. The 2:1 group had the highest serum triglyceride levels.

18

Serum total cholesterol and HDL levels were higher in the 13:1 and 8:1 groups than in

19

the 6:1 and 2:1 groups. The concentration of C18:3n-3 in serum and tissues (liver and

20

muscle) increased with decreasing dietary n-6:n-3 PUFA ratios. The hepatic expression of

21

FADS2, ELOVL5, FADS1, and ELOVL2 increased on a quadratic function with

22

decreasing dietary n-6:n-3 PUFA ratios. These results demonstrate that lower dietary

23

n-6:n-3 PUFA ratios had strong effects on the fatty acid profile of edible parts and the

24

deposition of n-3 PUFAs in adipose tissue of ducks.

25

Keywords: n-6:n-3 PUFA ratio, serum lipid level, fatty acid profile, gene mRNA

26

expression, duck

27

ACS Paragon Plus Environment

Page 2 of 32

Page 3 of 32

28

Journal of Agricultural and Food Chemistry

Introduction

29

Based on their chemical structure, essential fatty acids are classified into n-3 and n-6

30

polyunsaturated fatty acids (PUFAs). The n-3 and n-6 PUFAs are not inter-convertible

31

and have opposite physiological functions1. Specifically, n-3 PUFAs decrease

32

inflammatory signaling and fatty acid synthesis and increase lipid oxidation2. Fatty acid

33

composition, especially the n-6:n-3 PUFA ratio of cell and organelle membranes, affect

34

membrane function and cellular processes such as cell death and survival3. Metabolism

35

and organ function are dependent on a balanced concentration of n-6 and n-3 PUFAs4, 5.

36

Studies have reported that adequate dietary n-6:n-3 PUFA ratios are 6:1 and 3:1 in geese6

37

and 2.5:1 and 5:1 in chickens7. High n-6 PUFA levels and imbalanced n-6:n-3 PUFA

38

ratios are associated with the development of fatty acid deposition, adverse sperm quality,

39

and disease8, 9.

40

Dietary n-6:n-3 PUFA ratios decreased with increasing n-3 PUFA levels in animal

41

tissues and products, including eggs, milk, and meat10. There is a large variation in

42

n-6:n-3 PUFA ratios among commercial fattening duck feeds11. Conventional diets

43

contain high n-6 PUFA levels and low n-3 PUFA levels12. Vegetable and seed oils with

44

high levels of alpha-linolenic acid (ALA; C18:3n-3) have been used in livestock feed13.

45

Linseed oil, an n-3 PUFA source, increases n-3 PUFA content and decreases n-6:n-3

46

PUFA ratios in diets14. Previous studies in poultry and monogastric animals revealed that,

47

compared with tallow- and rapeseed-supplemented diets, linseed oil-supplemented diets

48

increase n-3 PUFA levels and decrease n-6:n-3 PUFA ratios in muscle of broiler and in

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

49

longissimus dorsi of swine7,

15

50

contributing factors to cardiovascular diseases in humans16. In humans, the optimal

51

n-6:n-3 PUFA ratio is < 517, 18. Therefore, increasing public awareness of the health

52

benefits of low n-6:n-3 PUFA ratios has prompted researchers to improve the fatty acid

53

profile of commonly consumed animal products.

. High n-6:n-3 PUFA ratios in animal products are

54

Dietary n-6:n-3 PUFA ratios affect the regulation of genes involved in the

55

metabolism of n-3 and n-6 fatty acids19-21. The conversion of ALA into eicosapentaenoic

56

(EPA; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3) occurs through the sequential

57

actions of ∆-6 desaturase (encoded by FADS2), elongases (encoded by ELOVL5 and

58

ELOVL2), and ∆-5 desaturase (encoded by FADS1), which are susceptible to the

59

nutritional status of the organism22-24. Fatty acid composition analysis have revealed that

60

ALA-supplemented diets increased n-3 PUFA levels in broilers and pigs25, 26, but this

61

effect is weak in geese6, 27. Therefore, it is important to investigate the effect of dietary

62

n-6:n-3 PUFA ratios on gene expression and n-3 PUFA content in duck tissues. In this

63

study, we assessed the effect of n-6:n-3 PUFA ratios on the fatty acid composition of

64

duck meat and evaluated the feasibility in producing n-3 fatty-acid-enriched ducks.

65

ACS Paragon Plus Environment

Page 4 of 32

Page 5 of 32

Journal of Agricultural and Food Chemistry

66

Materials and Methods

67

Experimental design and diets

68

A total of 3,168 15-day old Cherry valley ducks of the commodity generation

69

(average weight: 728.56 ± 1.83 g) were randomly divided into six treatment groups. Each

70

treatment group had six replicates (males and females), each consisting of 88 ducks. This

71

ducks from one batch were fed the same dietary at 1-14 days of age. The six treatment

72

groups were fed diets supplemented with linseed oil and containing different n-6:n-3

73

PUFA ratios: 13:1 (control), 10:1, 8:1, 6:1, 4:1, and 2:1. The feeding trial lasted four

74

weeks. The isonitrogenous and isocaloric experimental diets (dietary ME and CP levels

75

were 12.50 MJ/kg and 17%, respectively) were formulated to meet or exceed the nutrient

76

requirements of ducks based on NRC (1994) recommendations (Table 1). The nutrient

77

levels and fatty acid composition of the experimental diets are presented in Table 1. All

78

ducks had ad libitum access to the diets and water.

79

Sample collection

80

Animal handling and sampling protocols were approved by the Animal Care and

81

Use Committee of South China Agricultural University; all efforts were made to

82

minimize the suffering of animals according to recommendations proposed by the

83

European Commission (1997). The study was performed according to the protocol

84

D201611160945519 and conducted in accordance with relevant guidelines. Following a

85

12-h fast, body weight (BW) and feed intake of the ducks (42 d of age) were recorded,

86

and average daily gain (ADG), average daily feed intake (ADFI), and average feed: gain

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

87

(F/G) were calculated. Six ducks from each replicate were selected and sacrificed at the

88

end of the experiment (three male and three female). Blood samples were collected via

89

jugular vein puncture, and serum was obtained after centrifugation of blood samples at

90

2,000 g for 15 min at 4°C and stored at –20°C. After bleeding, a small portion of the liver

91

was collected, immediately placed in liquid N2, and stored at −80°C for hepatic gene

92

expression analysis. A large portion of the liver, breast, and thigh muscle were collected

93

and stored at −30°C for fatty acid analysis.

94

Serum biochemical parameters

95

Serum total triglyceride (TG), total cholesterol (TC), low-density lipoprotein

96

cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) concentrations were

97

determined spectrophotometrically (Bayer Diagnostics Manufacturing Ltd., Dublin,

98

Ireland) using commercial kits (Biosino Biotechnology and Science Inc., Beijing, China;

99

catalog numbers 0180, 0221, 0215, and 0200 for the analysis of TG, TC, LDL, and HDL,

100

respectively). These indicators were determined by Guangzhou LabGene Biotech Co.,

101

Ltd. There were six replicates per sample.

102

Fatty acid composition

103

Fatty acid composition was determined as previously reported28. Briefly, 2 g of

104

sample was extracted with chloroform: methanol (2:1; v/v) according to the method by

105

Folch29. Total fat was converted into fatty acid methyl esters (FAMEs) using a mixture of

106

boron-trifluoride, hexane, and methanol (35:20:45 v/v)30. FAME profiles were

107

determined by gas chromatography (GC; Model 7890A, Agilent Technologies, Palo Alto,

ACS Paragon Plus Environment

Page 6 of 32

Page 7 of 32

Journal of Agricultural and Food Chemistry

108

CA) as reported by Sukhija and Palmquist31. The gas chromatograph was equipped with a

109

capillary column (60 m × 0.25 mm DB-23, 0.25 film thickness; Agilent Technologies,

110

Palo Alto, CA). Nitrogen (1.1 mL/min) was used as the carrier gas, and the split/splitless

111

injector was used at a split/splitless ratio of 30:1. Injector and detector temperatures were

112

250°C and 300°C, respectively. The column oven temperature was maintained at 140°C

113

for 5 min after sample injection and was programmed to increase from 140°C to 220°C at

114

5°C/min and kept at 220°C for 16 mins. FAME separation was recorded using the GC

115

Chem Station software (Agilent Technologies, Palo Alto, CA). By comparing the FAME

116

profile of the samples with those of FAME standards (Supelco, 37 Component FAME

117

mix C4-C24, Catalog No. 47885-U, Supelco, Bellefonte, PA), we identified the fatty acids

118

in serum and tissues. The results were recorded as percentage of the total fatty acids.

119

Real-time PCR

120

Total RNA was extracted from the frozen tissues using Trizol reagent (Invitrogen,

121

Carlsbad, CA). RNA was digested with DNase I (RNase-Free DNase set; Qiagen)

122

(Invitrogen, Carlsbad, CA, USA). First-strand cDNA was synthesized using an equal

123

amount of total RNA and High Capacity RNA-to-cDNA Kit (Applied Bio-systems,

124

Madrid, Spain). The sequence of primers used in real-time PCR assays are shown in

125

Table 2. Real-time PCR was performed in an ABI 7500 (Applied Bio-systems, Foster

126

City, CA) using SYBR Green Quantitative PCR kit (TaKaRa). The thermal cycling

127

conditions consisted of one cycle at 95°C for 30 s followed by 40 cycles at 95°C for 5 s

128

and 60°C for 34 s. The target genes and reference genes (GAPDH and β-actin) were used

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

129

to calculate PCR efficiency. The mRNA expression of each gene was calculated by the

130

2−∆∆Ct method32.

131

Statistical analysis

132

Data were analyzed by one-way ANOVA and Tukey test using SAS for Windows

133

version 9.2 (SAS Institute Inc., Cary, NC). Data were expressed as mean ± standard

134

deviation. Differences among the groups were considered significant at P < 0.05.

135

ACS Paragon Plus Environment

Page 8 of 32

Page 9 of 32

Journal of Agricultural and Food Chemistry

136

Results

137

Growth performance

138

Table 3 shows the growth performance results. Dietary n-6:n-3 PUFA ratios had no

139

effects on BW of ducks at 42 d of age. Additionally, dietary n-6:n-3 PUFA ratios had no

140

effects on ADG, ADFI, or F/G of ducks at 15–42 d of age (P > 0.05).

141

Serum lipid concentrations

142

Table 4 shows the serum TC, TG, HDL, and LDL concentrations of ducks at 15–42 d

143

of age. Dietary n-6:n-3 PUFA ratios had no effects on serum LDL concentrations (P >

144

0.05). The 2:1 treatment group had higher serum TG concentrations than ducks fed the

145

other diets (P < 0.05). Serum TC and HDL concentrations were higher in the 13:1 and 8:1

146

groups than in the 6:1 and 2:1 groups (P < 0.05). Serum lipid concentrations (TG, TC,

147

and HDL) were affected by dietary n-6:n-3 PUFA ratios.

148

Tissue fat content

149

Dietary n-6:n-3 PUFA ratios had an effect (P < 0.05) on liver, breast, and thigh

150

muscle fat of ducks from 15 d to 42 d of age (Table 5). Ducks fed the 6:1 treatment had

151

lower liver fat content than ducks fed the 4:1 treatment (P < 0.05). The lowest fat content

152

in breast muscle and thigh muscle was observed in the 4:1 (P < 0.05) and 6:1 (P < 0.05)

153

groups, respectively.

154

Fatty acid composition

155

Tables 6–9 show the fatty acid composition of serum, liver, breast, and thigh muscle.

156

Serum arachidonic acid (AA; C20:4 n-6) was significantly (P < 0.05) lower in the 4:1 and

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

157

2:1 treatment groups than in the control group (13:1; Table 6). Ducks fed the 13:1 and

158

10:1 treatments had higher AA concentrations of liver than birds fed the 6:1, 4:1, or 2:1

159

treatments (P < 0.01); the 8:1 group reached a plateau (Table 7). However, dietary

160

n-6:n-3 PUFA ratios had no effect on AA concentrations of breast and thigh muscle

161

(Tables 8 and 9, P > 0.05). The EPA concentrations in serum and liver of the 2:1

162

treatment group were higher (P < 0.01) than those in other groups (Tables 6 and 7).

163

Additionally, the 4:1 treatment group had higher (P < 0.05) serum EPA concentrations

164

than other groups (Table 6). The EPA concentrations in breast muscle were higher in the

165

8:1 and 2:1 groups than in the 10:1 group (Table 8; P < 0.05). However, there were no

166

significant differences in DHA concentrations among all groups (Tables 7 and 8; P >

167

0.05). EPA and DHA were not detected in thigh muscle (Table 9). The proportions of

168

C18:3n3 and n-3 PUFAs (C18:3n3, C20:5n3, and C22:6n3) in serum and tissues (liver,

169

breast, and thigh muscle) increased linearly (P < 0.01) with decreasing dietary n-6:n-3

170

PUFA ratios (Tables 6, 7, 8, and 9). In contrast, the proportions of n-6 PUFAs (C18:2,

171

C20:3n6, and C20:4n6) and the n-6:n-3 PUFA ratios in serum and tissues decreased

172

linearly (P < 0.01) with decreasing dietary n-6:n-3 PUFA ratios (Tables 6, 7, 8, and 9) .

173

Hepatic gene expression

174

Figure 1A–D shows the relative gene expression in the liver of ducks. Hepatic

175

FADS2 mRNA expression was higher in the 8:1 group than in the 13:1 and 2:1 groups

176

(Figure 1A; P < 0.05). ELOVL5 mRNA expression was higher in the 6:1 group than in

177

the 13:1, 10:1, and 2:1 groups (Figure 1B; P < 0.05). FADS1 mRNA expression

ACS Paragon Plus Environment

Page 10 of 32

Page 11 of 32

Journal of Agricultural and Food Chemistry

178

increased in the 8:1 and 4:1 groups (Figure 1C; P < 0.05), and ELOVL2 mRNA

179

expression was the highest in the 4:1 group (Figure 1D). The hepatic expression of

180

FADS2, ELOVL5, FADS1, and ELOVL2 increased on a quadratic function with

181

decreasing dietary n-6:n-3 PUFA ratios; however, the 13:1 group had lower mRNA

182

expression levels (P < 0.05) than the other groups.

183

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

184

Discussion

185

Previous studies have shown that n-6 and n-3 PUFA supplementation improves the

186

serum lipid concentrations and muscle fatty acid composition of pigs and chickens7, 32.

187

Our study investigated the effect of dietary n-6:n-3 PUFA ratios on growth performance,

188

lipid levels, and fatty acid profile of ducks at 15–42 days of age.

189

In this study, dietary n-6:n-3 PUFA ratios had no effects on growth performance of

190

ducks. Dietary LA:ALA ratios of 17:1, 8:1, 4:1, and 2:1 have no effects on the growth

191

performance of chicken over 16 weeks of age33. In poultry, linseed oil does not affect

192

feed conversion rate34, and the effects of dietary oil supplements on the growth

193

performance of broilers are inconclusive35-37. This result could be attributed to similar fat

194

content in the experimental diets or similar feed intake during the trial.

195

A recent study revealed that dietary ALA does not affect serum TG concentrations38.

196

Some studies have reported that long chain n-3 PUFAs supplementation would reduce

197

serum TG concentrations39 and ALA-rich vegetable oils lower serum TG levels in rats40.

198

However, our findings showed that ducks fed the 2:1 treatment had higher serum TG

199

concentrations than other groups. The results were not consistent with previous findings,

200

which suggested that diets with high enough levels C18:3n-3 may contribute to glycerol

201

esterification in the liver, thereby increasing serum TG levels. On the other hand, serum

202

TC and HDL concentrations were higher in the 13:1 and 8:1 groups than those of 6:1 and

203

2:1 groups, consistent with previous reports41, 42. Low n-6:n-3 PUFA ratios may reduce

204

TC synthesis by inhibiting HMG-CoA reductase activity and changing the fatty acid

ACS Paragon Plus Environment

Page 12 of 32

Page 13 of 32

Journal of Agricultural and Food Chemistry

205

composition of biofilms and lipoproteins to enhance the metabolic activity of lipoprotein,

206

thereby promoting the breakdown of lipoproteins. A certain n-6:n-3 PUFA ratio may

207

reduce blood lipids. The effect of n-6:n-3 PUFA ratios on animal lipid metabolism

208

requires further studies.

209

In our experiments, ducks fed the 6:1 treatment had lower hepatic fat content than

210

those fed the 4:1 treatment. The lowest fat contents in breast muscle and thigh muscle

211

were observed in the 4:1 and 6:1 groups, respectively. Studies have shown that low

212

proportions of fatty acids (6:1 and 3:1) contribute to reduced fat deposition in geese6. In a

213

recent study, dietary n-6:n-3 PUFA ratios of 5:1 facilitated the absorption and utilization

214

of fatty acids in pigs32. This difference may be due to the species and fat sources used.

215

Adipose fatty acid composition reflects the type of lipid and fatty acid fed to

216

animals43. It should be noted that the conversion of ALA into long-chain PUFAs could be

217

affected by the relative amounts of LA and ALA in diets44. Therefore, LA:ALA was

218

important in the conversion of ALA into long-chain PUFAs45. The proportions of

219

C18:3n3 significantly increased in the serum and muscle of ducks with decreasing dietary

220

n-6:n-3 PUFA ratios, consistent with previous reports46, 47. In this study, n-6 PUFAs and

221

n-6:n-3 PUFA ratios decreased and n-3 PUFA increased in duck serum and tissue with

222

decreasing dietary n-6:n-3 PUFA ratios. Reducing dietary n-6 and increasing dietary n-3

223

PUFA could increase C18:3n-3 and n-3 PUFA concentrations in livestock and poultry7, 48.

224

Plant oils rich in ALA have been used to enhance the n-3 PUFA incorporation into duck

225

meat. Therefore, dietary supplementation with different n-6:n-3 PUFA ratios may be an

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

226

effective method of changing the fatty acid profile of meat ducks. The n-6 PUFA and n-3

227

PUFA sources should be taken into account. Based on our findings, hepatic DHA was not

228

significantly different among treatment groups; however, the 2:1 treatment group had the

229

highest hepatic EPA levels. In Shaoxing ducks, sunflower oil-supplemented diets

230

significantly increased leg muscle EPA contents, but had no significant effects on DHA43,

231

consistent with our results. Linseed oil increased both EPA and docosapentaenoic acid in

232

the breast muscle of chickens7 and n-3 PUFAs (especially EPA and DHA) in muscle of

233

broilers49. The limited accumulation of EPA and DHA observed in ducks may be

234

explained by some factors. EPA and DHA contents and poultry diets had the result of the

235

relationship between the relevant content50. The longer-chain PUFAs could compete with,

236

or be displaced by, increased ALA in the phospholipid fraction, which could prevent

237

PUFAs from accumulating. There is little information available on the differences among

238

meat-producing ducks in desaturase and elongase activities and in the expression of

239

related genes responsible for desaturation and extension during the synthesis of PUFAs

240

(C20-C24) from dietary ALA and LA.

241

PUFAs represent the main dietary regulator of desaturase and elongase enzymes23, 51.

242

The ∆-5 and ∆-6 desaturases are the rate-limiting enzymes in the synthesis of long-chain

243

PUFAs52; Elongases enzymes are required for the synthesis of EPA and DHA from ALA.

244

The FADS cluster and the ELOVL family may play an important role in the activities of

245

desaturases and elongases53. FADS1 and FADS2 were strongly associated with LC-PUFAs

246

and desaturase activity54. Our findings showed that the hepatic expression of FADS2,

ACS Paragon Plus Environment

Page 14 of 32

Page 15 of 32

Journal of Agricultural and Food Chemistry

247

ELOVL5, FADS1, and ELOVL2 increased on a quadratic function with decreasing dietary

248

n-6:n-3 PUFA ratios. However, there were no significant differences in hepatic n-3 long

249

chain PUFAs (especially EPA and DHA) between the 8:1 and 6:1 treatment groups. In

250

these groups, the genes were highly expressed but the enzymatic activities were not

251

enhanced. Interestingly, the conversion of n-3 and n-6 fatty acids shared the same

252

enzymes series (desaturase and elongase). There was competition between the n-3 and

253

n-6 fatty acid families; an excess of one leads to a significant reduction in the conversion

254

of the other55, 56. High FADS and ELOVL expression levels provide a target for breeding

255

poultry that readily synthesizes EPA and DHA from ALA.

256

In conclusion, dietary n-6:n-3 PUFA ratios affect serum lipid concentrations, tissue

257

fat content, and fatty acid profile and regulate the expressions of FADS and ELOVL in

258

ducks of 15–42 days of age. Additionally, decreasing dietary n-6:n-3 PUFA ratios resulted

259

in a nutritionally-enriched meat with a higher content of beneficial n-3 PUFAs, especially

260

C18:3n-3, and a lower n-6:n-3 PUFA ratio. Further studies should explore the effect of

261

n-6:n-3 PUFA ratios on the relationship between the liver cells and lipid metabolism.

262

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

263

Funding

264

This work was supported by the Special Fund for Agro-scientific Research in the

265

Public Interest, China (201303143-07), the China Agriculture Research System

266

(CARS-43-14), the National Youth Fund Project of China (31501959), the National Key

267

Research Program (2016YFD0500509-07).

268

ACS Paragon Plus Environment

Page 16 of 32

Page 17 of 32

Journal of Agricultural and Food Chemistry

269

References

270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308

(1) Calder, P. C., n-3 Polyunsaturated fatty acids and inflammation: from molecular biology to the clinic. Lipids 2003, 38, 343-52. (2) Arita, M.; Bianchini, F.; Aliberti, J.; Sher, A.; Chiang, N.; Hong, S.; Yang, R.; Petasis, N. A.; Serhan, C. N., Stereochemical assignment, antiinflammatory properties, and receptor for the omega-3 lipid mediator resolvin E1. J Exp Med 2005, 201, 713-22. (3) Schmitz, G.; Ecker, J., The opposing effects of n-3 and n-6 fatty acids. Prog Lipid Res 2008, 47, 147-55. (4) DeMar, J. C.; Ma, K.; Bell, J. M.; Igarashi, M.; Greenstein, D.; Rapoport, S. I., One generation of n-3 polyunsaturated fatty acid deprivation increases depression and aggression test scores in rats. J Lipid Res 2006, 47, 172-80. (5) Igarashi, M.; Ma, K.; Chang, L.; Bell, J. M.; Rapoport, S. I., Rat heart cannot synthesize docosahexaenoic acid from circulating α-linolenic acid because it lacks elongase-2. J Lipid Res 2008, 49, 1735-45. (6) Lihuai, Y. The study of influence for dietary n-6/n-3 fatty acid regulation of fatty metabalism and it's molecular mechanism in goose. Yangzhou university, 2012. (7) Qi, K. K.; Chen, J. L.; Zhao, G. P.; Zheng, M. Q.; J., W., Effect of dietary ω6/ω3 on growth performance, carcass traits, meat quality and fatty acid profiles of Beijing-you chicken. J Anim Physiol An N 2010, 94, 474-85. (8) Williams, C. D.; Whitley, B. M.; Hoyo, C.; Grant, D. J.; Iraggi, J. D.; Newman, K. A.; Gerber, L.; Taylor, L. A.; McKeever, M. G.; Freedland, S. J., A high ratio of dietary n-6/n-3 polyunsaturated fatty acids is associated with increased risk of prostate cancer. Nutr Res 2011, 31, 1-08. (9) Dannenberger, D.; Nuernberg, G.; Renne, U.; Nuernberg, K.; Langhammer, M.; Huber, K.; Breier, B., High-fat diets rich in ω-3 or ω-6 polyunsaturated fatty acids have distinct effects on lipid profiles and lipid peroxidation in mice selected for either high body weight or leanness. Nutrition 2013, 29, 765-71. (10) Mourot, J.; Hermier, D., Lipids in monogastric animal meat. Reproduction Nutrition Development 2001, 41, 109-18. (11) Mengmeng L; Shuangshuang Z, Analysis on Fatty acid Composition of Commercial Feed in Fattening ducks. Chinese Journal of Animal Science 2017, 53, 88-92. (12) Simopoulos, A. P., Evolutionary aspects of diet, the omega-6/omega-3 ratio and genetic variation: nutritional implications for chronic diseases. Biomed Pharmacother 2006, 60, 502-07. (13) Rebolé, A.; Rodriguez, M. L.; Ortiz, L. T.; Alzueta, C.; Centeno, C.; Viveros, A.; Brenes, A.; Arija, I., Effect of dietary high-oleic acid sunflower seed, palm oil and vitamin E supplementation on broiler performance, fatty acid composition and oxidation susceptibility of meat. Brit Poultry Sci 2006, 47, 581-91. (14) Williams, C. M.; Burdge, G., Long-chain n-3 PUFA: plant v. marine sources. P Nutr Soc 2006, 65, 42-50.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349

(15) Raes, K.; Smet, S. D.; Demeyer, D., Effect of dietary fatty acids on incorporation of long chain polyunsaturated fatty acids and conjugated linoleic acid in lamb, beef and pork meat: a review. Animal Feed Science & Technology 2004, 113, 199-221. (16) Calder, P. C., n-3 fatty acids, inflammation and immunity: new mechanisms to explain old actions. P Nutr Soc 2013, 72, 326-36. (17) Kouba, M.; Enser, M.; Whittington, F. M.; Nute, G. R.; Wood, J. D., Effect of a high-linolenic acid diet on lipogenic enzyme activities, fatty acid composition, and meat quality in the growing pig. J Anim Sci 2003, 81, 1967-79. (18) Wood, J. D.; Richardson, R. I.; Nute, G. R.; Fisher, A. V.; Campo, M. M.; Kasapidou, E.; Sheard, P. R.; Enser, M., Effects of fatty acids on meat quality: a review. Meat Sci 2004, 66, 21-32. (19) Yao, W.; Li, J.; Jun Wang, J.; Zhou, W.; Wang, Q.; Zhu, R.; Wang, F.; Thacker, P., Effects of dietary ratio of n-6 to n-3 polyunsaturated fatty acids on immunoglobulins, cytokines, fatty acid composition, and performance of lactating sows and suckling piglets. Journal of animal science and biotechnology 2012, 3, 1. (20) Lin, Y.; Cheng, X.; Mao, J.; Wu, D.; Ren, B.; Xu, S.; Fang, Z.; Che, L.; Wu, C.; Li, J., Effects of different dietary n-6/n-3 polyunsaturated fatty acid ratios on boar reproduction. Lipids Health Dis 2016, 15, 1. (21) Schmitz, G.; Ecker, J., The opposing effects of n-3 and n-6 fatty acids. Prog Lipid Res 2008, 47, 147-55. (22) Cormier, H.; Rudkowska, I.; Lemieux, S.; Couture, P.; Julien, P.; Vohl, M. C., Effects of FADS and ELOVL polymorphisms on indexes of desaturase and elongase activities: results from a pre-post fish oil supplementation. Genes & Nutrition 2014, 9, 1-15. (23) Nakamura, M. T.; Nara, T. Y., Structure, function, and dietary regulation of ∆6, ∆5, and ∆9 desaturases. Annu Rev Nutr 2004, 24, 345-76. (24) Marquardt, A.; Stöhr, H.; White, K.; Weber, B. H., cDNA cloning, genomic structure, and chromosomal localization of three members of the human fatty acid desaturase family. Genomics 2000, 66, 175-83. (25) Gatrell, S. K.; Kim, J.; Derksen, T. J.; O Neil, E. V.; Lei, X. G., Creating ω-3 Fatty-Acid-Enriched Chicken Using Defatted Green Microalgal Biomass. J Agr Food Chem 2015, 63, 9315-22. (26) Lopez-Ferrer, S.; Baucells, M. D.; Barroeta, A. C.; Galobart, J.; Grashorn, M. A., n-3 enrichment of chicken meat. 2. Use of precursors of long-chain polyunsaturated fatty acids: Linseed oil. Poultry Sci 2001, 80, 753-61. (27) Belinsky, D. L.; Kuhnlein, H. V., Macronutrient, mineral, and fatty acid composition of Canada Goose (Branta canadensis): an important traditional food resource of the Eastern James Bay Cree of Quebec. J Food Compos Anal 2000, 13, 101-15. (28) Herdmann, A.; Martin, J.; Nuernberg, G.; Dannenberger, D.; Nuernberg, K., Effect of dietary n-3 and n-6 PUFA on lipid composition of different tissues of German Holstein bulls and the fate of bioactive fatty acids during processing. J Agr Food Chem 2010, 58, 8314-21.

ACS Paragon Plus Environment

Page 18 of 32

Page 19 of 32

350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390

Journal of Agricultural and Food Chemistry

(29) Folch, J.; Lees, M.; Sloang, G. H., A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957, 226, 497-509. (30) Metcalfe, L. D.; Schmitz, A. A., The rapid preparation of fatty acid esters for gas chromatographic analysis. Anal Chem 1961, 33, 363-64. (31) Sukhija, P. S.; Palmquist, D. L., Rapid method for determination of total fatty acid content and composition of feedstuffs and feces. Journal of Agricultural & Food Chemistry 1988, 36, 1202-06. (32) Li, F.; Duan, Y.; Li, Y.; Tang, Y.; Geng, M.; Oladele, O. A.; Kim, S. W.; Yin, Y., Effects of dietary n-6:n-3 PUFA ratio on fatty acid composition, free amino acid profile and gene expression of transporters in finishing pigs. Brit J Nutr 2015, 113, 739-48. (33) Puthpongsiriporn, U.; Scheideler, S. E., Effects of dietary ratio of linoleic to linolenic acid on performance, antibody production, and in vitro lymphocyte proliferation in two strains of leghorn pullet chicks. Poultry Sci 2005, 84, 846-57. (34) Yongbao W, L. Y. H. Y., Comparative Study of Increasing ω-3 Polyunsaturated Fatty Acids Content in Yolk by Dietary Microalgae and Flaxseed Supplementation. Chinese Journal of Animal Nutrition 2015, 27, 3188-97. (35) Puthpongsiriporn, U.; Scheideler, S. E., Effects of dietary ratio of linoleic to linolenic acid on performance, antibody production, and in vitro lymphocyte proliferation in two strains of leghorn pullet chicks. Poultry Sci 2005, 84, 846-57. (36) Lixiao, L. Study on producing of chicken meat enriched in n-3 polyunsaturated fatty acids. Huazhong Agricultural University, 2007. (37) Lópezferrer, S.; Baucells, M. D.; Barroeta, A. C.; Grashorn, M. A., n-3 enrichment of chicken meat. 1. Use of very long-chain fatty acids in chicken diets and their influence on meat quality: fish oil. Poultry Sci 2001, 80, 741-52. (38) Tu, W. C.; Mühlhäusler, B. S.; Yelland, L. N.; Gibson, R. A., Correlations between blood and tissue omega-3 LCPUFA status following dietary ALA intervention in rats. Prostaglandins, Leukotrienes and Essential Fatty Acids (PLEFA) 2013, 88, 53-60. (39) Indu, M.; Ghafoorunissa, P. J., N-3 fatty acids in Indian diets comparison of the effects of precursor (linlenic acid) vs product (long chain n-3 polyunsaturated fatty acids). Journal of Nutrition Research 1992, 17, 569-82. (40) Jr, A. R.; Coates, W., Effect of dietary alpha-linolenic fatty acid derived from chia when fed as ground seed, whole seed and oil on lipid content and fatty acid composition of rat plasma. Annals of Nutrition & Metabolism 2007, 51, 27-34. (41) Umesha, S. S.; Naidu, K. A., Vegetable oil blends with α-linolenic acid rich Garden cress oil modulate lipid metabolism in experimental rats. Food Chem 2012, 135, 2845-51. (42) Zhao, G.; Etherton, T. D.; Martin, K. R.; West, S. G.; Gillies, P. J.; Kris-Etherton, P. M., Dietary alpha-linolenic acid reduces inflammatory and lipid cardiovascular risk factors in hypercholesterolemic men and women. J Nutr 2004, 134, 2991-97. (43) Tan, B.; Yin, Y.; Liu, Z.; Li, X.; Xu, H.; Kong, X.; Huang, R.; Tang, W.; Shinzato, I.; Smith, S. B., Dietary L-arginine supplementation increases muscle gain and reduces body fat mass in growing-finishing pigs. Amino acids 2009, 37, 169-75.

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427

(44) Liu, W. M.; Lai, S. J.; Lu, L. Z.; Shi, F. X.; Zhang, J.; Liu, Y.; Yu, B.; Tao, Z. R.; Shen, J. D.; Li, G. Q., Effect of dietary fatty acids on serum parameters, fatty acid compositions, and liver histology in Shaoxing laying ducks. Journal of Zhejiang University Science B 2011, 12, 736. (45) Brenna, J. T.; Salem, N.; Sinclair, A. J.; Cunnane, S. C., α-Linolenic acid supplementation and conversion to n-3 long-chain polyunsaturated fatty acids in humans. Prostaglandins, leukotrienes and essential fatty acids 2009, 80, 85-91. (46) Chopra, R.; Sambaiah, K., Effects of rice bran oil enriched with n-3 PUFA on liver and serum lipids in rats. Lipids 2009, 44, 37-46. (47) Talahalli, R. R.; Vallikannan, B.; Sambaiah, K.; Lokesh, B. R., Lower efficacy in the utilization of dietary ALA as compared to preformed EPA+DHA on long chain n-3 PUFA levels in rats. Lipids 2010, 45, 799-808. (48) Huang, F. R.; Zhan, Z. P.; Luo, J.; Liu, Z. X.; Peng, J., Duration of dietary linseed feeding affects the intramuscular fat, muscle mass and fatty acid composition in pig muscle. Livest Sci 2008, 118, 132-39. (49) Opinzar, H.; Kahraman, R.; Abas, I.; Kutay, H. C.; Eseceli, H.; Grashorn, M. A., Effect of dietary fat source on n-3 fatty acid enrichment of broiler meat. Arch Geflugelkd 2003, 67, 57-64. (50) Rymer, C.; Givens, D. I., n-3 fatty acid enrichment of edible tissue of poultry: A review. Lipids 2005, 40, 121-30. (51) Cho, H. P.; Nakamura, M. T.; Clarke, S. D., Cloning, expression, and nutritional regulation of the mammalian ∆-6 desaturase. J Biol Chem 1999, 274, 471-77. (52) Nakamura, M. T.; Nara, T. Y., Structure, function, and dietary regulation of delta6, delta5, and delta9 desaturases. Annu Rev Nutr 2004, 24, 345-76. (53) Al-Hilal, M.; AlSaleh, A.; Maniou, Z.; Lewis, F. J.; Hall, W. L.; Sanders, T. A.; O'Dell, S. D.; Team, M. S., Genetic variation at the FADS1-FADS2 gene locus influences delta-5 desaturase activity and LC-PUFA proportions after fish oil supplement. J Lipid Res 2013, 54, 542-51. (54) Cormier, H.; Rudkowska, I.; Lemieux, S.; Couture, P.; Julien, P.; Vohl, M., Effects of FADS and ELOVL polymorphisms on indexes of desaturase and elongase activities: results from a pre-post fish oil supplementation. Genes & nutrition 2014, 9, 437. (55) Gregory, M. K.; Geier, M. S.; Gibson, R. A.; James, M. J., Functional characterization of the chicken fatty acid elongases. The Journal of nutrition 2013, 143, 12-16. (56) Kartikasari, L. R.; Hughes, R. J.; Geier, M. S.; Makrides, M.; Gibson, R. A., Dietary alpha-linolenic acid enhances omega-3 long chain polyunsaturated fatty acid levels in chicken tissues. Prostaglandins, Leukotrienes and Essential Fatty Acids 2012, 87, 103-09.

ACS Paragon Plus Environment

Page 20 of 32

Page 21 of 32

Journal of Agricultural and Food Chemistry

428

Figure caption

429

Figure 1. Relative hepatic mRNA expression levels of FADS2 (A), ELOVL5 (B), FADS1

430

(C), and ELOVL2 (D) in ducks fed different n-6:n-3 PUFA ratios. The equation represents

431

the quadratic regression curve, and R2 represents the correlation coefficient. Values are

432

presented as mean (n = 6) and standard errors (vertical bars).

433

represent significant differences (P < 0.05).

434

ACS Paragon Plus Environment

a,b,c

Different letters

Journal of Agricultural and Food Chemistry

435

Tables

436

Table 1. Composition and nutrient levels of experimental diets (air-dry basis, %) Dietary n-6 : n-3 PUFA ratio Item 13:1 10:1 8:1 6:1 4:1 Ingredient Corn 54.05 54.05 54.05 54.05 54.05 Flour 13.70 13.70 13.70 13.70 13.70 Soybean meal 23.65 23.65 23.65 23.65 23.65 Rice bran 2.70 2.70 2.70 2.70 2.70 Soybean oil 2.00 1.88 1.76 1.57 1.22 Linseed oil 0.00 0.12 0.24 0.43 0.78 Limestone 1.30 1.30 1.30 1.30 1.30 Dicalcium phosphate 1.20 1.20 1.20 1.20 1.20 Salt 0.40 0.40 0.40 0.40 0.40 1 Premix 1.00 1.00 1.00 1.00 1.00 Total 100 100 100 100 100 Nutrient level 12.51 12.51 12.51 12.51 12.50 ME (MJ/kg)2 3 CP 17.29 17.37 17.44 17.18 17.31 3 CF 4.23 4.52 4.25 3.75 4.13 3 0.87 0.91 0.89 0.90 0.85 Ca 2 Non phosphorous 0.41 0.41 0.41 0.41 0.41 2 0.91 0.91 0.91 0.91 0.91 Lysine 2 Methionine 0.41 0.41 0.41 0.41 0.41 2 Digestible threonine 0.63 0.63 0.63 0.63 0.63 C16:03 7.44 7.68 8.36 7.51 7.60 C18:03 1.02 0.95 0.59 1.15 0.86 3 C18:1 15.48 22.27 17.32 15.73 19.51 3 C18:2n-6 70.66 62.95 65.75 64.92 57.95 3 C18:3n-3 5.40 6.14 7.98 10.69 14.08 2 n-6:n-3 ratio 13.09 10.25 8.23 6.07 4.12

437 438 439 440 441 442

1

Page 22 of 32

2:1 54.05 13.70 23.65 2.70 0.40 1.60 1.30 1.20 0.40 1.00 100 12.49 17.58 3.82 0.93 0.41 0.91 0.41 0.63 8.15 0.82 18.52 48.85 23.59 2.07

Vitamin and mineral premixes supplied per kilogram diet: vitamin A, 9,000 IU; vitamin D3, 3,000 IU; vitamin E, 79 mg; vitamin B2, 8 mg; vitamin K3, 2 mg; pantothenic acid, 3.2 mg; niacin, 11 mg; folic acid, 1.5 mg; biotin, 1 mg; Co, 1 mg; Mn, 49 mg; Cu, 6 mg; Zn, 60 mg; I, 2 mg; Se, 0.18 mg. 2 Calculated values. 3 Analysed based on triplicate determinations.

ACS Paragon Plus Environment

Page 23 of 32

443

Journal of Agricultural and Food Chemistry

Table 2. Primer sequences for real-time PCR amplification of hepatic genes Target gene

Primer sequence (5′–3′)

Size/bp

F: CACAGCCTGTTGAGTATGGC FADS2

186 R: GGATCCCAGAACGCCATAGA F: TCCACAGTGCCTTTCCTCAT

FADS1

191 R: TGCTGGAAGTGGAGGTGATT F: AGGACCAAAGTACATGCGGA

ELOVL5

203 R: ACCACCAGAGGACACGTATG F: CTCAGGGCTCACCTCATTGT

ELOVL2

116 R: AGGTTCTGGCACTGCAAGTT F: ATGTTCGTGATGGGTGTGAA

GAPDH

176 R: CTGTCTTCGTGTGTGGCTGT F: TACGCCAACACGGTGCTG

β-actin

215 R: GATTCATCATACTCCTGCTTG

444

FADS2, fatty acid desaturase 2; FADS1, fatty acid desaturase 1; ELOVL5, elongation of very

445

long-chain fatty acids enzyme 5; ELOVL2, elongation of very long-chain fatty acids enzyme 2.

446

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

447

Page 24 of 32

Table 3. Effects of dietary n-6:n-3 PUFA ratios on growth performance of ducks at 15–42 d of age Dietary n-6:n-3 PUFA ratio Item

P value 13:1

10:1

8:1

6:1

4:1

2:1

BW (kg)

3.51 ± 0.04

3.42 ± 0.05 3.40 ± 0.04 3.43 ± 0.03 3.41 ± 0.06 3.43 ± 0.06

0.52

ADG (g/d)

252 ± 2.76

240 ± 3.26

239 ± 4.77 243 ± 4.48 243 ± 2.91 243 ± 3.92

0.52

ADFI (g/d)

98.3 ± 1.29

96.3 ± 1.92 95.2 ± 1.49 96.6 ± 1.22 95.8 ± 2.02 98.2 ± 1.77

0.53

F/G

2.52 ± 0.05

2.52 ± 0.04 2.58 ± 0.04 2.52 ± 0.08 2.49 ± 0.05 2.53 ± 0.06

0.99

448

ACS Paragon Plus Environment

Page 25 of 32

449

Journal of Agricultural and Food Chemistry

Table 4. Effects of dietary n-6:n-3 PUFA ratios on serum lipid levels of ducks at 42 d of age (µmol/mL) Dietary n-6:n-3 PUFA ratio Item

450

P value 13:1

10:1

TG

0.69 ± 0.05b

0.82 ± 0.04b

TC

4:1

2:1

0.85 ± 0.08b

1.51 ± 0.12a