Influence of dietary oils on performance, blood metabolites, purine derivatives, cellulase activity and muscle fatty acid composition in fattening lambs

Accepted Manuscript Title: Influence of dietary oils on performance, blood metabolites, purine derivatives, cellulase activity and muscle fatty acid composition in fattening lambs Author: R. Parvar T. Ghoorchi M. Shams Shargh PII: DOI: Reference:

S0921-4488(17)30062-7 http://dx.doi.org/doi:10.1016/j.smallrumres.2017.03.004 RUMIN 5442

To appear in:

Small Ruminant Research

Received date: Revised date: Accepted date:

2-8-2016 16-1-2017 8-3-2017

Please cite this article as: Parvar, R., Ghoorchi, T., Shargh, M.S.,Influence of dietary oils on performance, blood metabolites, purine derivatives, cellulase activity and muscle fatty acid composition in fattening lambs, Small Ruminant Research (2017), http://dx.doi.org/10.1016/j.smallrumres.2017.03.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Influence of dietary oils on performance, blood metabolites, purine

2

derivatives, cellulase activity and muscle fatty acid composition in fattening

3

lambs

ip t

1

4

R. Parvar,a* T. Ghoorchi,a and M. Shams Shargha a

Dept. of Animal and Poultry Nutrition, Faculty of Animal Science, University of Agricultural Sciences and Natural Resources, P.O. Box: 49189-43464, Gorgan, Iran.

us

6 7 8

cr

5

* Corresponding author. Reza Parvar, Dept. of Animal and Poultry Nutrition, University of Agricultural Sciences and Natural Resources, Gorgan, Iran. P. O. Box: 49189-43464; Tel.: +98-9166633543; Fax: +98-173- 2440093; E-mail: [email protected]

M

10 11 12

an

9

13

17 18 19 20 21

te

16

Ac ce p

15

d

14

22 23 24 25

1 Page 1 of 27

Influence of dietary oils on performance, blood metabolites, purine

27

derivatives, cellulase activity and muscle fatty acid composition in fattening

28

lambs

ip t

26

29

ABSTRACT

31

The aim of this study was to investigate the effects of canola, soybean and fish oils on performance,

32

cellulase enzyme activity, microbial protein synthesis and the fatty acid profile of longissimus muscle in

33

fattening lambs. Thirty-five male lambs with an initial body weight of 27.8 ± 2 kg were used in a

34

completely randomized design for an 84-day feeding period. The experimental treatments included: 1-

35

control diet (without oil), 2- diet with 3% fish oil, 3- diet with 3% canola oil, 4- diet with 3% soybean oil,

36

5- diet with 1.5% fish oil + 1.5% canola oil (FO +CO), 6- diet with 1.5% fish oil + 1.5% soybean oil (FO

37

+ SO), 7- diet with 1.5% canola oil + 1.5% soybean oil (CO + SO). No differences were found among

38

treatment of soybean oil, canola oil, FO + CO and control treatment for Daily weight gains (DWG).

39

However, the diets which contained fish oil, FO + SO and CO + SO had lower DWG. Oil

40

supplementations did not affect dry matter intake, feed conversion ratio and hot carcass weight (P > 0.05).

41

Ruminal pH and ammonia nitrogen did not differ among treatments. Oils had no effect on concentrations

42

of serum glucose, cholesterol, triglyceride and blood urea-N concentrations (P > 0.05). No significant

43

differences were found among treatments for purine derivatives and the microbial N (P > 0.05). The

44

particulate material and total activity of carboxy methyl-cellulase (CMM) were negatively affected by

45

inclusion of oils in feeds (P<0.05). No differences were observed for the microcrystalline-cellulase

46

activity (MCC) among treatments (P > 0.05). The particulate material had the highest enzyme activity in

47

different fractions of CMC and MCC. The most abundant fatty acid was oleic (C18:1 cis-9), followed by

48

palmitic (C16:0) and stearic (C18:0). The lambs fed with fish oil had the highest the C20:5n−3 and C22:5

49

or C22:6n−3. The concentrations of saturated fatty acids significantly reduced with different oil

Ac ce p

te

d

M

an

us

cr

30

2 Page 2 of 27

supplementations (P<0.05). The oil supplementations effectively decreased n-6 to n-3 ratio in the

51

longissimus muscle compared with the control diet (P<0.05). In conclusion, soybean, canola and fish oils

52

up to 3% separately or binary-blend of these oils in equal proportions (also up to 3%) can be used in diet

53

of fattening lambs. In addition, inclusion of oils in ration improved the meat content of polyunsaturated

54

fatty acids, especially the level of long-chain n-3 fatty acids.

ip t

50

Keywords: oil, performance, microbial protein, enzyme activities, fatty acids, lamb

us

56

cr

55

57 1. Introduction

59

Oils enhance the energy density of the diets and provide highly digestible diets for animals with high

60

nutrient requirements. Also, oils reduce ruminal acidosis, and increase the absorption of liposoluble

61

nutrients (Manso et al., 2006). Moreover, the use of oil reduces methane emission in the rumen and

62

increases energy efficiency, and results in a lower harmful environmental impact (Machmüller, 2006).

63

Although the use of oils in the diet has many benefits for animals, their usage is limited since they are

64

highly prone to rotting. The use of high levels of oils due to the negative effects of unsaturated fatty acids

65

on rumen fermentation leads to lower fiber digestibility (up 50%) and feed intake (Jenkins and Palmquist,

66

1984). Fish oil is rich in omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid

67

(DHA), When added as a supplement to the diet of ruminants, these acids transmute into vaccenic acid in

68

the rumen (Toral et al., 2010), which can be used for synthesis of Conjugated linoleic acid (CLA) in

69

different tissues (Piperova et al., 2002). Vegetable oils such as soybean and canola contain

70

polyunsaturated fatty acids linoleic and linolenic (Kwan et al., 1991) and their potential benefits have

71

been proved for human health. Previous work has indicated that a diet supplemented with soybean oil

72

separately and mixed with fish oil did not significantly affect daily weight gain (DWG) but reduced feed

73

intake of lambs (Ferreira et al., 2014). Jaworska et al. (2016) demonstrated that a diet supplemented with

74

fish and rapeseed oils reduced the values of ratio of n-6 to n-3 and saturated fatty acids (SFA) in the

Ac ce p

te

d

M

an

58

3 Page 3 of 27

longissimus muscle of lambs. However, we are unaware of any work done on the effect of feeding oils

76

supplemented diets to lambs on microbial protein synthesis and the activity of enzymes in rumen of

77

animals. This study was carried out to investigate the effects of vegetable oil (canola oil and soybean oil)

78

and fish oil on performance, purine derivatives, cellulase enzyme activity, microbial protein synthesis and

79

fatty acid composition of longissimus muscle in fattening lambs.

cr

80

ip t

75

2. Materials and methods

82

2.1. Animal management and dietary treatments

83

This study was conducted at the experimental farm of Gorgan University of Agricultural Sciences and

84

Natural Resources, Gorgan, Iran. Thirty-five-male Afshari lambs with an initial body weight of 27.8 ± 2

85

kg and 4-5 months were used in a completely randomized design for an 84-day feeding period within a

86

14-day adaptation period. Before starting the experiment, lambs were dewormed by Dieverm

87

(Albendazole 2.5%, Damloran Pharmaceutical Co., Iran) and vaccinated against enterotoxaemia. The

88

lambs were weighed and randomly allocated to one of seven dietary treatments according to live weight.

89

Ingredients and chemical compositions of the diets are presented in Table 1. During the experiment,

90

lambs were housed in individual pens. Diets were fed as a Total Mixed Ration (TMR) and were

91

formulated to meet requirements for growing lambs (NRC, 1985). The diets were offered twice daily ad

92

libitum (08:00 and 16:00 h) and the lambs had free access to fresh water. Diets were weighed individually

93

for each lamb. Refusals from each lamb were collected before 08:00 h and weighed. The lambs were

94

taken care of in accordance with guidelines of the Iranian Council on Animal Care (1995). Experimental

95

treatments included: 1- control diet (without oil), 2- diet supplemented with 3% fish oil, 3- diet

96

supplemented with 3% canola oil, 4- diet supplemented with 3% soybean oil, 5- diet supplemented with

97

1.5% fish oil + 1.5% canola oil (FO +CO), 6- diet supplemented with 1.5 % fish oil + 1.5% soybean oil

98

(FO + SO), 7- diet supplemented with 1.5% canola oil + 1.5% soybean oil (CO + SO). The fatty acid

99

composition of the diets is presented in Table 2. Five lambs were then randomly allocated to each

Ac ce p

te

d

M

an

us

81

4 Page 4 of 27

treatment. During the period lambs were weighed individually every 14 days and average daily gain,

101

average feed intake and feed conversion ratio were determined.

102

2.2. Blood and Rumen sampling

103

Five ml of blood were collected from jugular vein and poured in sterile test tubes from all lambs on day

104

80 just before the morning feeding and 6 h after feeding. The serum was separated from blood by

105

centrifuging at 1500 × g for 15 min, and was stored at −20 ◦C for further analysis. Concentrations of

106

glucose, total cholesterol, triglyceride, blood urea-N (BUN), alkaline phosphatase (ALP), aspartate amino

107

transferase (AST) and alanine amino transferase (ALT) in serum were measured using Pars Azmoon kits

108

and associated procedures (Pars Azmoon Co., Tehran, Iran).

109

On 70th day of the period, sampling of rumen fluid was performed 4 hours after morning feeding by

110

stomach tube. Three lambs from each group were selected to determine rumen fermentation. The samples

111

were filtered through four layers of muslin cloth and were immediately transported to the laboratory by

112

insulating flask. The pH was determined immediately after sampling. Ruminal ammonia N was

113

determined using a phenol-hypochlorite method according to the procedure of Broderick and Kang

114

(1980).

cr

us

an

M

d

te Ac ce p

115

ip t

100

116

2.3. Extraction of enzymes

117

The effects of dietary treatments on the activity of carboxymethyl-cellulase (CMM) enzyme and

118

microcrystalline-cellulase (MCC) were measured in different fractions of Rumen fluid particulate

119

material (PM), extracellular enzymes (EC) and cellular (C). The enzyme activity in three fractions of

120

rumen content was determined according to the procedure described by Agarwal (2000). The extraction

121

of enzymes from the various fractions of rumen contents was performed according to Hristov et al.

122

(1999). Glucose released by the activity of enzymes was estimated by dinitrosalicylic acid (DNS) method

123

as described by Miller (1959). The enzyme activity was expressed as µmole of released sugars produced

124

per minute per ml under assay conditions.

5 Page 5 of 27

125 2.3. Estimation of microbial protein

127

Purine derivatives (PD) in urine include uric acid, allantoin, xanthine + hypoxanthine. Estimation of

128

microbial N was performed based on colorimetric method (Chen and Gomes, 1992). Daily urine samples

129

were collected in a plastic bucket containing 100 ml of 10% (vol/vol) sulphuric acid solution to reduce the

130

ultimate pH below 3. Every morning the total urine produced by the animal was measured individually

131

and to prevent the precipitation (particularly of uric acid) of PD urine samples during storage, a sub-

132

sample of 10 ml of daily amount was diluted with 40 ml distilled water and then stored at −20◦ C for the

133

estimation of PD. The samples collected from each sheep were pooled to give one pooled sample for

134

analysis (Chen and Gomes, 1992).

an

us

cr

ip t

126

M

135 2.4. Fatty acid analysis

137

At the end of the experimental period, lambs were slaughtered after 16 h of starving. Sampling of

138

longissimus muscle was performed between 12th and 13th ribs from the left halves of each carcass. For

139

determination of fatty acid composition, the samples were ground to homogeneity after the removal of the

140

any external fat. The lipid were extracted from meat samples using a 2:1 solution of chloroform:methanol,

141

as described by Folch et al. (1957). The lipid extract was dried under nitrogen, and methyl esters of fatty

142

acids (FAME) derivatives were prepared by methylation of the fatty acids, as described by Metcalfe and

143

Schmitz (1961). The FAMEs were separated using a Unicam 4600 gas-chromatograph (model Unicam

144

4600, UK) equipped with flame ionization detector and a fused silica capillary column (BPX70, 30 m,

145

0.22 mm ID, 0.25 Inc, Australia). Pentadecanoic acid (Sigma Chemical Co., St. Louis, MO) was used as

146

internal standard. The fatty acids identification was performed by matching their retention times with

147

those of their respective standards.

Ac ce p

te

d

136

148 149

2.5. Statistical analysis

6 Page 6 of 27

A completely randomized design was used to determine the effect of the oils contents included in the

151

diets on different parameters. Data were analyzed by general linear models (GLM) procedures of SAS

152

(version 9.1; SAS Institute Inc., Cary, NC, USA), based on the statistical model: Yij = µ + Ti + εij, where

153

Yij: observation for animal i receiving diet j, Ti: the effect of treatment, εij: the residual error. Least-

154

square means were computed and tested for differences by the Tukey's test. Differences among means

155

with P<0.05 were accepted as representing statistically significant differences.

cr

ip t

150

us

156

3. Results

158

3.1. Performance

159

Data on feed intake, daily weight gain and feed conversion ratio are shown in Table 3. The results of

160

current study showed that the experimental diets had significant effect on daily weight gain of lambs

161

(P<0.05). Daily weight gains (DWG) of control and canola oil was higher than diets containing fish oil,

162

blend of fish oil plus soybean oil (FO + SO) and blend of canola oil plus soybean oil (CO + SO).

163

Vegetable and fish oil did not affect dry matter intake (DMI) and feed conversion ratio (FCR) of lamb

164

during total feeding trial (P>0.05). However, lambs fed the diets supplemented with different oils had

165

numerically lower feed intake than lambs fed with the control diet. Oil supplementation had no effect on

166

the hot carcass weight (P>0.05). Dressing percentage was similar between lambs fed with different oil

167

sources.

M

d

te

Ac ce p

168

an

157

169

3.2. Ruminal fermentation and blood metabolites

170

Including oils in the diets did not affect ruminal pH and ammonia nitrogen (NH3-N) concentrations

171

(P>0.05). NH3-N was numerically higher in control group. Concentrations of glucose in serum of lambs

172

were not affected by oils (Table 4). Serum cholesterol and triglyceride concentrations tended to increase

173

for treatments containing oils versus control, but differences were not significant. No differences were

174

found among treatments for BUN concentrations (P >0.05). The concentration of liver enzymes was

7 Page 7 of 27

175

modified by dietary treatment (P<0.05). Lambs fed this treatment had the numerically highest

176

concentration of enzymes (ALP, AST and ALT) compared to control (Table 4).

177 3.3. Microbial N

179

The effects of different fat sources included in the diet on microbial N supply and PD are presented in

180

Table 5. No differences were found among treatments for urinary allantoin, uric acid, xanthine +

181

hypoxanthine and the estimated microbial N (P > 0.05). Inclusion of oil in diets did not affect the total PD

182

absorbed. Microbial protein synthesis was lower in treatment containing oils compared to the control but

183

this difference was not significant.

an

us

cr

ip t

178

184 3.4. Enzyme activities

186

The effect of experimental diets on cellulase activity is shown in table 6. The results of this study showed

187

the highest activity of hydrolytic enzymes occurs in the PM, followed by C and EC fraction for CMC and

188

MCC. Enzyme activity in the PM, C and total CMC was higher in control than other treatments (P<0.05)

189

but no significant differences were observed for EC activity of CMC (Table 6). No differences were

190

observed among treatments for the MCC activity. However, enzyme activity of control was numerically

191

higher than other treatments.

d

te

Ac ce p

192

M

185

193

3.5. Fatty acid analysis

194

The control group had the highest proportion of C16:0, C17:0 and C18:0. Differences were not detected

195

in the quantities of C12:0, C16:1, C18:2, C20:0 and C20:4 fatty acids in the longissimus muscle (Table 7).

196

However, the C18:2 had an increasing trend in lambs fed with different oil except fish oil (P=0.06). There

197

was a significant increase in the proportion of linolenic acid (P<0.05), and it was higher in the muscle

198

from lambs fed the canola and soybean oils diet. The lambs fed with fish oil had the highest C20:5n−3

199

(EPA) and C22:5 (DPA) or C22:6n−3 (DHA). The concentrations of SFA were significantly reduced with

200

different oil supplementation (P<0.05). The highest concentration of monounsaturated fatty acids 8 Page 8 of 27

(MUFA) was observed in lamb fed with diets containing canola oil and soybean oil. All experimental

202

diets contain oils stimulated the accumulation of polyunsaturated fatty acids (PUFA) in the longissimus

203

muscle compared with the control (P<0.05). The n−6 to n−3 ratio was affected by oil supplementation

204

(P<0.05). Lambs that used diets containing fish oil, FO +CO and FO + SO had the lowest n-6: n-3 fatty

205

acid ratio among treatments.

ip t

201

cr

206 4. Discussion

208

4.1. Dry matter intake and growth performance

209

In the current study, the results should be interpreted with caution due to the low number of animals per

210

treatment and high variability of data. Findings of present study showed that the inclusion of fish oil, FO

211

+ SO and CO + SO reduced DWG of animals compared to the control. In contrast to the results of this

212

study, Ferreira et al. (2014) reported that adding fish oil separately, and blend of fish oil and soybean oil

213

did not affect DWG. Also, different oil sources at 2% (Najafi et al., 2012) and 4.5% (Roy et al., 2013) had

214

no significant effect on DWG. One reason for weight loss maybe high consumption of oil which leads to

215

the reduction of digestibility and consequently reduction of feed intake in lambs (Song et al., 2010).

216

Overall results indicate that the DMI of lambs was not affected by treatments. In agreement with our

217

results, Roy et al. (2013) reported that adding soybean oil or sunflower oil (45 g/kg DM) to diet of goat

218

did not affect feed intake. Similarly, Maia et al. (2012) reported that DMI and other nutrients were not

219

affected by oil supplementation (soybean, castor and sunflower oils up to 30 g/kg DM of diet). Other

220

studies showed that the use of fish oil as the sole source of supplemental fat or mixed with other fat

221

sources caused a decrease in feed intake (Toral et al., 2010; Ferreira et al., 2014). Reports on the effects of

222

oil on DMI of lambs are inconsistent. Also, unaffected DMI in this study is probably concerned with

223

amount and type of supplement oil (Doreau and Ferlay, 1994). Oils containing long chain polyunsaturated

224

fatty acids, therefore they increase cholecystokinin and decrease ruminal movements and eventually

225

influence feed intake (Agazzi et al., 2010

Ac ce p

te

d

M

an

us

207

9 Page 9 of 27

226 4.2. Ruminal fermentation and blood metabolites

228

The unchanged rumen pH as a result of oil supplementation is similar to the report of Bhat et al. (2011).

229

Ferreira et al. (2016) demonstrated an increase in rumen pH for animals consuming the diets with oils.

230

However, Kholif et al. (2016) reported that soybean and flaxseed oils supplementation in diet reduced the

231

ruminal pH of goats. Animals fed with diets containing different oils had numerically lower ruminal

232

ammonia concentrations in comparison to the control. Ferreira et al. (2016) observed decreased rumen N-

233

NH3 in lambs when the diets were supplemented with soybean oil and fish oil at 40 g/kg DM. Also, Maia

234

et al. (2012) indicated that the supplementation of different oils at 30 g/kg in diets had no effect on

235

ruminal ammonia concentration. In this study, different oils had no significant effect on the concentration

236

of glucose in serum of lambs and levels were within normal ranges for healthy ruminants (Blood et al.,

237

1983). Consistent with our findings, previous studies showed that feeding oils had no effect on

238

concentrations of serum glucose and cholesterol (Roy et al., 2013; Dai et al., 2011). However, many

239

studies reported increased plasma glucose concentration via oil supplementation (Li et al., 2012; Kholif et

240

al., 2016). The unaffected cholesterol and serum triglyceride in this study were consistent with the report

241

of Li et al. (2012). Many researchers reported an increase in levels of cholesterol in dairy cows fed diet

242

with vegetable oil supplementations while triglyceride levels in blood were not affected (Dai et al., 2011).

243

Liver enzymes of serum are sensitive indicators of hepatocellular injury. Therefore, the elevated levels of

244

these enzymes are indications of liver damage (Yap and Choon, 2010). The major functions of liver are

245

metabolism of fat, protein and carbohydrate. Scarpino et al. (2014) reported that high concentrations of

246

AST in animals fed with oil may indicate liver damage which is attributed to loss of liver weight. Bianchi

247

et al. (2014) also reported that when soybean oil was supplemented to a diet of sheep at 6% of dietary

248

DM, the activity of AST and gamma-glutamyl transferase increase considerably.

Ac ce p

te

d

M

an

us

cr

ip t

227

249

10 Page 10 of 27

4.3. Microbial N

251

Diet protein is degraded to ammonia, peptides and amino acids by rumen microbial enzymes. As a result,

252

these compounds are converted into microbial protein. Various methods are used to determine the

253

microbial protein synthesis, such as DAPA and RNA. The measurement purine derivatives of urine are a

254

simple and inexpensive method for estimation of microbial protein (Chen and Ørskov, 2004). Microbial

255

protein synthesis in ruminants provides most of the amino acids required for growth, maintenance and

256

animal production. Purine derivatives are used for determining microbial protein in ruminant since there

257

is a correlation between the duodenum amino acids and purine derivatives (Chen and Gomez, 1992).

258

Allantoin included about 60 to 80 percent of total purine derivatives and is the most important PD for

259

estimation of the microbial protein in sheeps, uric acid and xanthine + hypoxanthine contain 10-30 and 5-

260

10 percent of purine derivatives (Chen and Gomez, 1992). In this study, PD of lambs that were fed with

261

different oils was lower numerically than the control; as a result, microbial protein synthesis was lower in

262

all treatments compared to control. Ammonia and energy are essential for rumen microorganisms to

263

produce microbial protein. Another issue, the simultaneous availability of these resources is very

264

important (Makkar, 2003). If the rate of degradation of protein is greater than that of carbohydrate, high

265

amount of nitrogen can be converted into ammonia. However, a decrease in microbial protein synthesis

266

may occur in response to excessive fermentation of carbohydrate rather than protein degradation (Bach et

267

al., 2005).

cr

us

an

M

d

te

Ac ce p

268

ip t

250

269

4.4. Enzyme activities

270

High enzymatic activity of PM reflects the population of cloned bacteria on feed particles. The

271

microorganisms mostly cling to the PM part, and a few of them are freely suspended in the rumen liquid

272

which may be due to the lesser enzyme activity in the cellular fraction. The EC fraction had the lowest

273

enzymatic activity among three rumen fraction, because these enzymes are bound to the cell membrane, 11 Page 11 of 27

and only a few of them release into the liquid portion due to mechanical injury or disintegration of the

275

fibre degrading microbes (Minato et al., 1966; Agarwal., 2000). Minato et al. (1966) indicated that 50-70

276

percent of rumen bacteria are attached to the particle feed. High activity of CMC compared to MCC is

277

probably due to their higher substrates which is consistent with the findings of other researchers

278

(Raghuvansi et al., 2007). CMC can be used as an indicator of cloned total population of bacteria on feed

279

particles and is also a proper way to evaluate rumen environment that affects fiber degradation in the

280

rumen (Silva et al., 1987). In contrast with our findings, Durge et al. (2014) reported that the inclusion of

281

Brassica juncea oil in goat diets did not affect CMC. In the current study, the diets containing fish oil had

282

the highest negative effect on enzyme activity, since the high proportion of unsaturated fatty acids are

283

toxic to rumen microbial populations and particularly to cellulolytic bacteria.

284

4.5. Fatty acid profile of longissimus muscle

285

Reduction in palmitic (C16:0) and stearic acids (C18:0) is consistent with other studies using soybean oil

286

or fish oil with ruminants (Scollan et al., 2001; Santos-Silva, et al, 2004., Jaworska et al., 2016).

287

Researchers reported that inclusion of soybean oil reduced stearic and palmitic fatty acid in lamb,

288

probably because the elongation process and de novo fatty acid synthesis are inhibited as a result of

289

higher amount of polyunsaturated fatty acids (Santos-Silva et al., 2004). Conversely, Ponnampalam et al.

290

(2001) and Scollan et al. (2001) showed that supplementing the diet of beef cattle and lambs with fish oil

291

reduced the proportion of C18:0 in muscle. In this study, the most abundant fatty acid was oleic (C18:1

292

cis-9), followed by palmitic (C16:0) and stearic (C18:0). In our study, the main fatty acids were C18:1,

293

C16:0 and C18:0, respectively (Table 7). Oliveira et al., (2016) reported a similar amount when dietary oil

294

concentration was 60 g/kg of sunflower and linseed oil blend in lambs. Lambs fed a ration containing 3%

295

of canola oil had significantly increaced levels of C18:3 acids in the longissimus muscle compared with

296

lambs fed with a control ration (Karami et al., 2013), but this did not alter C12:0, C20:0 and C20:4 (Table

297

7), since canola oil contains a high percentage of polyunsaturated fatty acids mainly linolenic (C18:3n−3)

298

and linoleic (C18:2n−6) fatty acids (Kwan et al., 1991). These results of current study show that oil

Ac ce p

te

d

M

an

us

cr

ip t

274

12 Page 12 of 27

sources with long chain contain polyunsaturated fatty acids can bypass rumen microbial degradation

300

when added to diet of ruminant, and be absorbed in the small intestine and deposited in organs and

301

muscles through the circulatory system. However, arachidonic acid and palmitic acid fatty acids were not

302

affected by the type of oil (Table 7). In contrast with this result, C20:4 decreased in beef cattle fed with

303

fish oil (Morgan et al., 1992; Mandell et al., 1997; Scollan et al., 2001). In current study, lambs

304

supplemented with fish oil had greater concentrations of EPA, DPA and DHA in the longissimus muscle

305

compared with other treatments, which agrees with other studies (Scollan et al., 2001; Ponnampalam et

306

al., 2001; Wistuba et al, 2007; Jaworska et al., 2016). Inclusion of oil in diets increased the concentration

307

of polyunsaturated fatty acids and decreased the proportion of saturated fatty acids in longissimus muscle.

308

The increase in concentration of C18:1 in muscle may be explained by the ability of ruminal bacteria to

309

make this isomer from C18:2n-6 (Harfoot and Hazelwood, 1997). In this study, the lowest ratio n-6/n-3

310

was obtained for lambs fed with fish oil, fish oil plus other oils and canola oil, respectively, and this is

311

consistent with the results of Najafi et al. (2012) in goat and Wistuba et al. (2007) in steers which

312

consumed 3% fish oil. Karimi et al. (2013) documented that n-6/n-3 ratio decreased in goat kids muscle

313

fed with 3% canola oil compared with animals fed with palm oil. No differences were found between

314

treatment of soybean oil and control treatment for n-6/n-3 ratio (Table 7). In agreement with our results,

315

Najafi et al. (2012) reported that Supplementation of 3% soybean oil in the ration of goat did not affect n-

316

6/n-3 ratio. Our results are consistent with finding of Jaworska et al. (2016), who reported that n-6/n-3

317

proportion was decreased in lamb receiving rapeseed oil and fish oil.

318

5. Conclusions

319

The results of the present study showed that feeding lambs with a diet supplemented with different oil

320

sources did not affect DMI and FCR. The diets which contained fish oil, FO + SO and CO + SO had

321

lower DWG. Rumen and blood parameters were not differential among treatments. The addition of oils

322

did not affect microbial protein synthesis, PD and MCC activity of lambs. Although, we observed a

323

declining trend for this parameters in lamb fed different oils compared with the control. The incorporation

Ac ce p

te

d

M

an

us

cr

ip t

299

13 Page 13 of 27

of soybean, canola and fish oils up to 3% effectively decreased n-6 to n-3 ratio in the longissimus muscle

325

and had beneficial effects on fatty acid composition of meat. Therefore, the results suggest that the

326

soybean oil, canola oil and fish oil separately or binary-blend of these oils in equal proportion (at 3%

327

DM) can be used in diets without affecting on performance of lambs. Also, that is a strategy to increase

328

the meat content of PUFA, especially the level of long-chain n-3 fatty acids.

329

Conflict of Interest

330

The authors declare no known conflicts of interest, including competing financial interests, associated

331

with this publication.

an

us

cr

ip t

324

332 References

334

Agarwal, N. 2000. Estimation of fiber degrading enzyme. In: Feed Microbiology (edsChaudhary, L.C.,

335

Agarwal, N., Kamra, D.N., Agarwal, D.K.,), pp. 278–291. CAS Animal Nutrition, IVRI, Izatnagar, India.

336

Agazzi, A., Invernizzi, G., Campagnoli, A., Ferroni, M., Fanelli, A., Cattaneo, D., Galmozzi, A., Crestani,

337

M., Dell’Orto, V., Savoini, G., 2010. Effect of different dietary fats on hepatic gene expression in

338

transition dairy goats. Small Rumin. Res. 93, 31–40.

339

Bach, A., Calsamiglia, S., Stern, M.D., 2005. Nitrogen metabolism in the rumen. J. Dairy Sci. 88, E9–

340

E21.

341

Bhatt, R., Soren, N., Tripathi, M. Karim, S., 2011. Effects of different levels of coconut oil

342

supplementation on performance, digestibility, rumen fermentation and carcass traits of Malpura lambs.

343

Anim. Feed Sci. Technol. 164, 29–37.

344

Bianchi, A.E., Macedo, V.P., Franca, R.T., Lopes, S.T.A., Lopes, L.S., Stefani, L.M., Volpato, A., Lima,

345

H.L., Paiano, D., Machadoe, G., Da Silva, A.S., 2014. Effect of adding palm oil to the diet of dairy sheep

346

on milk production and composition, function of liver and kidney, and the concentration of cholesterol,

347

triglycerides and progesterone in blood serum. Small Rumin. Res. 117, 78–83.

Ac ce p

te

d

M

333

14 Page 14 of 27

Blood, D.C., Radostis, O.M., Handerson, J.A., Arunded, J.H., Gay, C.C., 1983. Normal laboratory values.

349

In: Veterinary Medicine, 6th ed. ELBS and Baillière, Tindall, Eastbourne, UK.

350

Broderick, G., Kang, J.H., 1980. Automated simultaneous determination of ammonia and total amino

351

acids in ruminal fluid and in vitro media. J. Dairy Sci. 54, 1176–1183.

352

Che, X.B., Ørskov, E.R., 2004. Research on urinary excretion of purine derivatives in ruminants: past,

353

present and future. In estimatian of microbial protein supply in ruminants using urinary purine derivative

354

(eb.H.S. Makkar and X.B.Chen), pp.180-210 FAO/IAEA, Kluwer Academic Publishers Dordrecht.

355

Chen, X.B., Gomes, J.M., 1992. Estimation of microbial protein supply to sheep and cattle based on

356

urinary excretion of purine derivatives: An overview of the technical details. Occasional publication of

357

the International Feed Resources Unit, Rowett Res. Inst., Bucksburn, Aberdeen, UK.

358

Dai, X.J., Wang, C., Zhu, Q., 2011. Milk performance of dairy cows supplemented with rapeseed oil,

359

peanut oil and sunflower seed oil. Czech J. Anim. Sci. 56, 181–191.

360

Doreau, M., Ferlay, A,. 1994. Digestion and utilization of fatty acids by ruminants. Anim. Feed. Sci.

361

Tech. 45, 379-396.

362

Durge, S.M., Tripathi, M.K., Tripathi, P., Dutta, N., Rout, P.K., Chaudhary, U.B., 2014. Intake, nutrient

363

utilization, rumen fermentation, microbial hydrolytic enzymes and hemato-biochemical attributes of

364

lactating goats fed concentrate containing Brassica juncea oil meal. Small Rumin. Res. 121, 300-307.

365

Ferreira, E.M., Pires., A.V., Susin, I., Gentil, R.S., Parente, M.O.M., Nolli, C.P., Meneghini, R.C.M.,

366

Mendes, C.Q., Ribeiro, C.V.D.M., 2014. Growth, feed intake, carcass characteristics, and meat fattyacid

367

profile of lambs fed soybean oil partially replaced by fish oil blend. Anim. Feed. Sci. Tech. 187, 9– 18.

368

Ferreira, E.M., Pires., A.V., Susin, I., Gentil, R.S., Parente, M.O.M., Nolli, C.P., Meneghini, R.C.M.,

369

Mendes, C.Q., Ribeiro, C.V.D.M., 2016. Nutrient digestibility and ruminal fatty acid metabolism in

370

lambs supplemented with soybean oil partially replaced by fish oil blend. Anim. Feed. Sci. Tech. 216, 30-

371

39.

372

Folch, J., Lees, M., Sloane Stanley, G.H., 1957. A simple method for the isolation and purification of

373

total lipids from animal tissues. J. Biol. Chem. 226, 497–509.

Ac ce p

te

d

M

an

us

cr

ip t

348

15 Page 15 of 27

Harfoot, C.G., Hazelwood, G.P., 1997. Lipid metabolism in the rumen. Pages 382–426 in The Rumen

375

Microbial Ecosystem. P. N. Hobson and C. S. Stewart, ed. Blackie Academic & Professional, London,

376

UK.

377

Hristov, A.N., McAllister, T.A., Cheng, K.J., 1999. Effect of diet, digesta processing, freezing and

378

extraction procedure on some polysaccharide degrading activities of ruminal contents. Can. J. Anim. Sci.

379

79, 73-81.

380

Jaworska, D., Czauderna, M., Przybylski, W., Rozbicka-Wieczorek., A.J., 2016. Sensory quality and

381

chemical composition of meat from lambs fed diets enriched with fish and rapeseed oils, carnosic acid

382

and seleno-compounds. Meat Sci. 119, 185–192.

383

Jenkins, T.C., Palmquist, D.L., 1984. Effect of fatty acids or calcium soaps on rumen and total nutrient

384

digestibility of dairy rations. J. Dairy Sci. 67, 978–986.

385

Iranian Council of Animal Care., 1995. Guide to the Care and Use of Experimental Animals, vol. 1.

386

Isfahan University of Technology, Isfahan, Iran.

387

Karami, M., Ponnampalam, E.N., Hopkins, D.L., 2013. The effect of palm oil and canola oil (saturated-

388

versus polyunsaturated- fatty acids) on feedlot performance, plasma and tissue fatty acid profiles and

389

meat quality in goats. Meat Sci. 94,165–169.

390

Kholif, A., Morsy, T.A., Abd El Tawab, A.M., Anele, U.Y., Galyean, M.L., 2016. Effect of

391

supplementing diets of anglo-nubian goats with soybean and flaxseed oils on lactational performance. J.

392

Agric. Food Chem. 64, 6163−6170.

393

Kwan, J.S., Snook, J.T., Wardlaw, G.M., Hwang, D.H., 1991. Effects of diets high in saturated fatty

394

acids, canola oil, or safflower oil on platelet function, thromboxane B2 formation, and fatty acid

395

composition of platelet phospholipids. Am. J. Clin. Nutr. 54, 351–358.

396

Li, X.Z., Yen, C.G., Lee, H.G., Choi, C.W., Song, M.K., 2012. Influence of dietary vegetable oils on

397

mammary lipogenic enzymes and the conjugated linoleic acid content of plasma and milk fat of lactating

398

goats. Anim. Feed Sci. Tech. 174, 26–35.

Ac ce p

te

d

M

an

us

cr

ip t

374

16 Page 16 of 27

Machmüller, A., 2006. Medium-chain fatty acids and their potential to reduce methanogenesis in

400

domestic ruminants. Agr. Ecosyst. Environ. J. 112, 107–114.

401

Maia, M.D.O., Susin, I., Ferreira, E.M., Nolli, C.P., 2012. Intake, nutrient apparent digestibility and

402

ruminal constituents of sheep fed diets with canola, sunflower or castor oils1. Braz. J. Anim. Sci. 41,

403

2350-2356.

404

Makkar, H.P.S., 2003. Effects and fate tannin in ruminant animals, adaptation to tannin, and strategies

405

to overcome detrimental effects of feeding tannin-rich feeds. Small Rumin. Res. 49, 241-256.

406

Mandell, I., Buchanan-Smith, J., Holub, B., Campbell, C., 1997. Effects of fish meal in beef cattle diets

407

on growth performance, carcass characteristics, and fatty acid composition of longissimus muscle. J.

408

Anim. Sci. 75, 910-919.

409

Manso, T., Castro, T., Mantecón, A.R., Jimeno, V., 2006. Effects of palm oil and calcium soaps of palm

410

oil fatty acids in fattening diets on digestibility, performance and chemical body composition of lambs.

411

Anim. Feed. Sci. Tech. 127, 175–186.

412

Metcalfe, L., Schmitz, A., 1961. The rapid preparation of fatty acid esters for gas chromatographic

413

analysis. Anal. Chemi. 33, 363–364.

414

Miller, G.L., 1959. Modified DNS method for reducing sugars. Anal. Chem. 31, 426-428.

415

Minato, H., Endo, A., Higuchi, M., Ootomo, Y., Uemura, T., 1966. Ecological treatise on the rumen

416

fermentation. 1. The fractionation of bacteria attached to the rumen digesta solids. J. Gen. Appl.

417

Microbiol. 12, 39-53.

418

Morgan, C.A., Noble, R.C., Cocchi, M., McCartney, R., 1992. Manipulation of the fatty acid composition

419

of pig meat lipids by dietary means. Journal of the Science of Food and Agriculture, 58, 357–368.

420

Najafi, M.H., Zeinoaldini, S., Ganjkhanlou, M., Mohammadi, H., Hopkins, D.L., Ponnampalam, E.N.,

421

2012. Performance, carcass traits, muscle fatty acid composition and meat sensory properties of male

422

Mahabadi goat kids fed palm oil, soybean oil or fish oil. Meat Sci. 92, 848–854.

Ac ce p

te

d

M

an

us

cr

ip t

399

17 Page 17 of 27

NRC., 1985. Nutrient Requirements of Small Ruminants; Sheep, Goats, cervids, and New World

424

camelids. Natl Academy Press.

425

Oliveiraa, M.A., Alves, S.P., Santos-Silva, J.S, Bessaa, R.J.B., 2016. Effects of clays used as oil

426

adsorbents in lamb diets on fatty acid composition of abomasal digesta and meat. Anim. Feed. Sci. Tech.

427

213, 64–73.

428

Piperova, L.S., Sampugna, J., Teter, B.B., Kalscheur, K.F., Yurawecz, M.P., Ku, Y., Morehouse, K.M.,

429

Erdman, R.E., 2002. Duodenal and milk trans octadecenoicacid and conjugated linoleic acid (CLA)

430

isomers indicate that post absorptive synthesis is the predominant source of cis-9 containing CLA in

431

lactating cows. J. Nutr. 132, 1235–1241.

432

Ponnampalam, E.N., Trout, G.R., Sinclair, A.J., Egan, A.R., Leury, B.J., 2001. Comparison of the color

433

stability and lipid oxidative stability of fresh and vacuum packaged lamb muscle containing elevated

434

omega-3 and omega-6 fatty acid levels from dietary manipulation. Meat Sci. 58, 151–161.

435

Raghuvansi, S.K.S., Prasad, R., Tripathi, M.K., Mishra, A.S., Chaturvedi, O.H, Misra, A.K., araswat,

436

B.L.S., Jakhmola, R.C., 2007. Effect of complete feed blocks or grazing and supplementation of lambs on

437

performance, nutrient utilisation, rumen fermentation and rumen microbial enzymes. Animal. 1, 221–226.

438

Roy, A., Mandal, G.P., Patra, A.K., 2013. Evaluating the performance carcass traits and conjugated

439

linoleic acid content in muscle and adipose tissues of black Bengal goats fed soybean oil and sunflower

440

oil. Anim. Feed. Sci. Tech. 185, 43–52.

441

Santos-Silva, J., Mendes, I., Portugal, P., Bessa, R., 2004. Effect of particle size and soybean oil

442

supplementation on growth performance, carcass and meat quality and fatty acid composition of

443

intramuscular lipids of lambs. Livest. Prod Sci. 90, 79–88.

444

Scarpino, F.B.O., Ezequiel, J.M.B., Silva, D.A.V., Van Cleef, E.H.C.B., 2014. Soybean oil and residual

445

soybean oil in diets for feedlot sheep: blood parameters. Arch. Zootec. 63, 207–210.

446

Scollan, N.D., Choi, N.J., Kurt, E., Fisher, A.V., Enser, M., Wood, J.D., 2001. Manipulating the fatty acid

447

composition of muscle and adipose tissue in beef cattle. Brit. J. Nutr. 85, 115–124.

Ac ce p

te

d

M

an

us

cr

ip t

423

18 Page 18 of 27

Silva, A.T., Wallace, R.J., Orskov, E.R., 1987. Use of particle-bound microbial activity to predict the rate

449

and extent of fibre degradation in the rumen. Brit. J. Nutr. 57, 407-415.

450

Song, M.K., Jin, G.L., Ji, B.J., Chang, S.S., Jeong, J., Smith, S.B., 2010. Conjugated linoleic acids

451

content in M. longissimus dorsi of Hanwoo steers fed a concentrate supplemented with soybean oil,

452

sodium bicarbonate-based monensin, fish oil. Meat Sci. 85, 210–214.

453

Toral, P.G., Belenguer, A., Frutos, P., 2010. Effect of the supplementation of a high-concentrate diet with

454

sunflower and fish oils on ruminal fermentation in sheep. Small Rumin. Res. 81,119-125.

455

Wistuba, T.J., Kegley, E.B., Apple, J.K., Rule, D.C., 2007. Feeding feedlot steers fish oil alters the fatty

456

acid composition of adipose and muscle tissue. Meat Sci. 77, 196–203.

457

Yap, C.Y.F., Choon, A.W., 2010. Liver function tests (LFts). Proceedings of Singapoor Healthcare, 19,

458

80-2.

cr

us

an

M

459

464 465 466 467 468 469

te

463

Ac ce p

462

d

460 461

ip t

448

470 471 472 473 474 19 Page 19 of 27

Table 1

Ingredients and chemical composition of experimental diets. Dietsa Soybean oil 15.0 36.0 13.5 15.0 9.0 6.0 0.0 0.0 3.0 1.0 0.5 0.5 0.5

Item

476 477 478 479 480

Ac ce p

475

te

d

M

an

us

cr

ip t

Control Fish oil Canola oil (FO +CO) (FO + SO) (CO + SO) Alfalfa 16.0 15.0 15.0 15.0 15.0 15.0 Barley 51.0 36.0 36.0 36.0 36.0 36.0 Wheat straw 12.5 13.5 13.5 13.5 13.5 13.5 Wheat bran 6.0 15.0 15.0 15.0 15.0 15.0 Corn 7.0 9.0 9.0 9.0 9.0 9.0 Soybean meal 5.0 6.0 6.0 6.0 6.0 6.0 Fish oil 0.0 3.0 0.0 1.5 1.5 0.0 Canola oil 0.0 0.0 3.0 1.5 0.0 1.5 Soybean oil 0.0 0.0 0.0 0.0 1.5 1.5 CaCO3 1.0 1.0 1.0 1.0 1.0 1.0 Salt 0.5 0.5 0.5 0.5 0.5 0.5 Mineral-vitamin premixb 0.5 0.5 0.5 0.5 0.5 0.5 Sodium bicarbonate 0.5 0.5 0.5 0.5 0.5 0.5 Chemical composition 2.6 2.6 2.6 2.6 2.6 2.6 2.6 Metabolism Energyc (Mcal/kg) Crude proteinc (%) 14.6 14.6 14.6 14.6 14.6 14.6 14.6 Ether extractd (%) 2.4 5.4 5.4 5.4 5.4 5.4 5.4 Organic matterc (%) 93.9 93.0 93.0 93.0 93.0 93.0 93.0 Neutral detergent fiberd (%) 38.5 37.5 37.5 37.5 37.5 37.5 37.5 Acid detergent fiberd (%) 18.5 19.0 19.0 19.0 19.0 19.0 19.0 Calciumc (%) 0.36 0.36 0.36 0.36 0.36 0.36 0.36 Phosphorusc (%) 0.24 0.24 0.24 0.24 0.24 0.24 0.24 a FO, Fish oil; CO, Canola oil; SO, Soybean oil. b Each kilogram of vitamin–mineral premix contained: vitamin A (50,000 IU), vitamin D3 (10,000 IU), vitamin E (1000 IU), Ca (196 g), P (96 g), Na (71 g), Mg (19 g), Fe (3 g), Cu (0.3 g), Mn (2 g), Zn (3 g), Co (0.1 g), I (0.1 g) and Se (0.001 g). c Calculated using feed composition tables of all ingredients (NRC, 1985). d Based on laboratory analysis.

481 482 483 484 485 20 Page 20 of 27

486 487

Fatty acid composition of experimental diets (% of total fatty acids). Control

Fish oil

Canola oil

Treatmenta Soybean oil

C12:0

0.12

0.10

0.04

0.06

0.07

C14:0

1.43

4.65

1.23

1.25

2.94

2.95

1.24

C16:0

20.07

22.40

16.21

17.32

19.31

19.86

16.77

C16:1

1.43

5.60

1.14

1.09

3.37

3.34

1.11

C17:0

0.24

2.10

0.11

0.11

1.11

1.11

0.11

C17:1

0.04

1.65

0.09

0.05

0.87

0.85

0.07

C18:0

2.65

4.90

1.50

1.87

3.20

3.39

1.69

C18:1

21.24

25.90

22.60

19.21

24.25

22.56

20.91

C18:2

46.30

3.80

49.10

53.40

26.45

28.60

51.25

C18:3

6.01

1.86

7.48

5.46

4.67

3.66

6.47

C20:0

0.41

1.24

0.04

0.82

0.64

0.22

C20:4

0.08

0.08

0.10

0.14

0.09

0.11

0.12

C20:5

0.00

7.54

0.00

0.00

3.77

3.77

0.00

FO+SO

Co+ SO

0.08

0.05

us

cr

FO+CO

an

M

d 0.40

te

Ac ce p

Itemb

C22:5

0.00

0.86

0.00

0.00

0.43

0.43

0.00

C22:6

0.00

17.32

0.00

0.00

8.66

8.66

0.00

SFA

24.91

35.39

19.49

20.65

27.44

28.02

20.07

PUFA

52.39

31.46

56.68

59.00

44.07

45.23

57.84

N6

46.38

3.88

49.20

53.54

26.54

28.71

51.37

6.01

27.58

7.48

5.46

17.53

16.52

6.47

N3 a

ip t

Table 2

FO, Fish oil; CO, Canola oil; SO, Soybean oil. SFA, saturated fatty acids; PUFA, polyunsaturated fatty acids; N6, omega 6; N3, omega 3.

b

488 489 490 491 21 Page 21 of 27

492 493 494

ip t

495 496

Fish oil

Canola Soybean (FO +CO) (FO + SO) oil oil Daily weight gain (g/day) 224a 184b 218a 207ab 205ab 184b Dry matter intake (g/day) 1573 1439 1457 1490 1481 1439 Feed conversation ratio 7.02 7.89 6.69 7.28 7.14 8.13 Final body weight (kg) 45.98a 44.33ab 46.33a 46.41a 45.54a 41.72b Hot Carcass weight (kg) 22.57 21.72 22.44 22.41 22.51 20.40 Dressing percentage (%) 49.64 47.38 48.18 48.04 47.72 47.62 Superscripts: means within a raw without a common superscript differ significantly (P<0.05). A FO, Fish oil; CO, Canola oil; SO, Soybean oil.

501 502 503 504 505

183b 1471 8.22 41.84b 20.73 48.59

8.165 43.03 0.382 0.973 0.331 0.385

0.0038 0.4542 0.0629 0.0018 0.4878 0.8142

te

500

P-Value

Ac ce p

499

SEM

d

497 498

(CO + SO)

M

an

Control

us

Item

cr

Table 3 Dry matter intake and growth performance of fattening lambs fed diets with different oil type. TreatmentA

506 507 508 509 510 22 Page 22 of 27

511 512 513 514

ip t

515 Table 4

Control

6.42 pH 12.01 NH 3-N (mg/dl) Blood metabolites (mg/dl)B 66.0 Glucose 32.0 Blood urea-N 24.5 Triglycerides 57.0 Cholesterol 565.67d ALP (IU/lit) 109.73c AST (IU/lit) 20.5d ALT (IU/lit)

Fish oil 6.43 10.48

Canola oil 6.46 11.23

(FO +CO) 6.60 11.24

(FO + SO)

(CO + SO)

SEM

P-Value

6.46 10.83

6.61 11.32

0.154 0.865

0.8278 0.9216

66.5 41.9 37.5 59.0 755.43b 154.83a 50.53a

7.585 3.215 11.440 5.778 41.707 4.744 2.481

0.9448 0.3044 0.4215 0.1414 0.0001 0.0035 0.0001

us

Item

TreatmentA Soybean oil 6.68 11.46

cr

Ruminal pH, NH3-N concentration and blood metabolites of fattening lambs fed diets with different oil type.

518 519 520 521 522

M

d te

517

Ac ce p

516

an

64.0 68.0 56.5 65.5 66.0 40.0 43.9 36.0 40.0 39.3 28.5 55.0 26.5 24.8 47.5 46.5 69.5 42.5 52.0 51.0 a b dc bc 1116.17 766.8 620.13 697.00 696.43bc 153.93a 124.27bc 112.67c 140.37ab 116.07bc dc a ab bc 29.43 49.73 41.20 39.0 33.56bc Superscripts: means within a row without a common superscript differ significantly (P<0.05). A FO, Fish oil; CO, Canola oil; SO, Soybean oil. B ALP, alkaline phosphatase; AST, aspartate amino transferase; ALT, alanine amino transferase .

523 524 525 526 527 23 Page 23 of 27

528 529 530

ip t

531

cr

532

Treatmenta Control

Fish oil

Canola oil

Soybean oil

Urinary purine derivatives, (mmol/d)b

9.20 3.07 1.36 15.52 13.64 11.28 70.52

535 536 537 538 539

8.90 2.39 1.25 14.26 12.56 10.36 64.79

8.81 2.92 1.30 14.85 13.03 10.79 67.48

SEM

P-Value

1.507 0.7474 0.2125 2.526 2.125 1.836 11.474

0.9023 0.9917 0.9227 0.9231 0.9227 0.9229 0.9231

te

534

(CO + SO)

Ac ce p

533

9.05 2.90 1.32 15.12 13.29 10.99 68.72

(FO + SO)

d

M

Allantoin 10.93 8.04 9.49 Uric acid 3.34 2.98 3.16 X +h 1.58 1.22 1.40 Total PD absorbed 18.17 13.87 16.03 Total PD excreted 15.86 12.24 14.07 Microbial N(gr/d) 13.21 10.08 11.66 MPS (gr/d) 82.59 63.03 72.87 a FO, Fish oil; CO, Canola oil; SO, Soybean oil. b X +h, Xanthine +hypoxanthine; MPS, microbial protein supply.

(FO +CO)

an

Item

us

Table 5 Effects of different oil type supplementation on urinary purine derivatives (PD) and microbial protein supply (MPS) in lambs fed experimental diets.

540 541 542 543 544 24 Page 24 of 27

545 546 547

ip t

548 549

cr

550

us

551

an

Table 6 Effect of dietary oil type on changes in activities of enzymes in various fractions of rumen contents (µmol glucose released / min/ml) TreatmentA Item

Control

Fish oil

Canola oil

Soybean oil

(FO +CO)

(FO + SO)

552 553 554

Ac ce p

te

d

M

Carboxymethyl cellulaseB C 122.83 82.03 102.09 87.75 88.45 EC 80.41 82.15 71.29 81.95 81.28 PM 132.84a 85.92d 116.43b 107.48bc 106.54bc a b ab b Total 336.09 250.09 289.82 277.19 276.27b Microcrystalline cellulase C 55.08 45.41 46.77 46.04 45.2 EC 45.51 40.92 43.34 42.66 40.2 PM 58.24 47.06 51.02 48.71 46.24 Total 158.85 133.42 141.14 137.42 131.66 Superscripts: means within a raw without a common superscript differ significantly (P<0.05). A FO, Fish oil; CO, Canola oil; SO, Soybean oil. B C, Cellular fraction; EC, Extracellular fraction; PM, Particulate material.

(CO + SO)

SEM

P-Value

72.35 86.52 84.26d 243.14b

82.75 66.43 98.86b 248.14b

11.556 7.53 4.832 16.625

0.1246 0.5369 0.0001 0.0184

42.54 36.85 48.49 127.89

45.2 38.22 49.7 133.13

4.560 5.391 4.740 7.570

0.6173 0.9211 0.6477 0.1637

555 556

25 Page 25 of 27

0.52 2.1a 26.98a 1.72 2.38a 0.34c 18.77a 35.62c 7.26c

0.46 0.47c 24.24ab 1.54 1.28bc 0.41c 15.84dc 37.91bc 9.22c

0.49 1.44b 23.06bc 1.51 1.60b 0.64b 14.50d 41.11a 9.53abc

0.40 1.40ab 23.24bc 1.67 1.48bc 0.70b 14.39d 41.16a 9.94abc

0.46 0.78c 21.46bc 1.50 1.33bc 0.97d 16.99bc 36.98bc 12.03ab

Co+ SO

SEM

p-value

0.49 0.55c 20.18c 1.54 1.47bc 0.97a 18.67ab 39.02bc 10.52ab

0.48 0.70c 22.09bc 1.32 1.06c 0.36d 17.53ab 38.58b 12.41a

0.040 0.180 1.280 0.069 0.311 0.080 0.489 0.558 0.520

0.9968 0.0008 0.0264 0.8898 0.0027 <.0001 0.0016 0.0056 0.0645

ip t

C12:0 C14:0 C16:0 C16:1 C17:0 C17:1 C18:0 C18:1 C18:2

FO+ SO

cr

Table 7 Fatty acid composition of longissimus muscle in lambs (g/100 g fat) TreatmentA B Item Control Fish oil Canola oil Soybean oil FO+ CO

557 558 559 560 561

Ac ce p

te

d

M

an

us

0.82b 1.09a 0.86ab 0.84b 0.82b 0.66bc 0.055 0.0095 C18:3 0.45d C20:0 1.43 1.18 1.36 1.38 1.17 1.43 0.77 0.087 0.5346 C20:4 1.67 1.70 1.51 1.38 2.03 1.64 1.94 0.080 0.3876 C20:5 (EPA) 0.48dc 1.94a 0.68bc 0.28d 1.80a 0.72bc 0.80b 0.168 <.0001 C22:5 (DPA) 0.29d 0.90a 0.44dc 0.53bc 0.81a 0.80a 0.72ab 0.061 0.0018 e a ec de b bc C22:6 (DHA) 0.32 1.78 0.44d 0.36 1.12 0.78 0.75bcd 0.138 0.0007 SFA 51.83a 43.75b 42.47b 43.06b 42.20b 42.81b 42.74b 0.923 0.0022 MUFA 37.68c 39.86bc 43.26a 43.53a 38.73bc 41.54ab 39.96bc 0.613 0.0095 PUFA 10.48c 16.37ab 13.70bc 13.36bc 18.64a 15.27ab 17.78a 0.760 0.0101 c ab b b a ab P/S 0.20 0.37 0.32 0.31 0.44 0.36 0.40ab 0.021 0.0072 N6 8.94d 10.91dc 11.04bdc 11. 32abcd 14.07ab 12.16abc 14.85a 0.552 0.0446 d a c d b c N3 1.54 5.54 2.65 2.03 4.57 3.11 2.93c 0.357 <.0001 N6/N3 5.77a 2.03d 4.14b 5.47ab 3.07c 3.91bc 4.91ab 0.361 0.0005 Superscripts: means within a row without a common superscript differ significantly (P<0.05). A FO, Fish oil; CO, Canola oil; SO, Soybean oil. B EPA, eicosapentaenoic acid; DPA, docosapentaenoic acid; DHA, docosahexaenoic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; P/S, polyunsaturated fatty acids /saturated fatty acids; N6/N3, omega6/omega 3.

562 563 564 565 566

Highlights 26 Page 26 of 27

•

No significant differences were found among treatments for purine derivatives and the microbial N

569 570

•

The particulate material and total activity of carboxy methyl-cellulase (CMM) were negatively affected by inclusion of oils in feeds

571 572

•

incorporation of soybean oil, canola oil and fish oil separately or binary-blend of these oils in equal proportion can be used in diet of lambs

573

•

The oil supplementations effectively decreased n-6 to n-3 ratio in the longissimus muscle.

574 575

•

Incorporation of oil sources had beneficial effects on fatty acid composition of meat and can be used as a strategy to increase the meat content of polyunsaturated fatty acids.

us

cr

ip t

567 568

576

Ac ce p

te

d

M

an

577

27 Page 27 of 27