Effects of dietary sesame oil on growth performance and fatty acid composition of muscle and tail fat in fattening Chaal lambs

Accepted Manuscript Title: Effects of dietary sesame oil on growth performance and fatty acid composition of muscle and tail fat in fattening Chaal lambs Author: H. Ghafari M. Rezaeian S.D. Sharifi A.A. Khadem A. Afzalzadeh PII: DOI: Reference:

S0377-8401(16)30469-2 http://dx.doi.org/doi:10.1016/j.anifeedsci.2016.08.006 ANIFEE 13604

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Please cite this article as: Ghafari, H., Rezaeian, M., Sharifi, S.D., Khadem, A.A., Afzalzadeh, A., Effects of dietary sesame oil on growth performance and fatty acid composition of muscle and tail fat in fattening Chaal lambs.Animal Feed Science and Technology http://dx.doi.org/10.1016/j.anifeedsci.2016.08.006 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.

Effects of dietary sesame oil on growth performance and fatty acid composition of muscle and tail fat in fattening Chaal lambs H. Ghafaria, M. Rezaeianb, S.D. Sharifia, A.A. Khadema, A. Afzalzadeha

a

Department of Animal and Poultry Science, Agricultural Faculty of Aburaihan,

University of Tehran, Tehran, Iran b

Department of Animal Health and Nutrition, Faculty of Veterinary Medicine, University

of Tehran, Tehran, Iran

Corresponding

author:

Hadi

Ghafari

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982923040746,

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Highlights:

SO feeding did not affect lamb performance, but kidney fat increased by SO feeding. SO feeding increased the serum total cholesterol and high density lipoproteins. SO feeding decreased the molar ratio of ruminal propionate. SO feeding improved the concentration of CLA cis-9, trans-11 in meat and tail fat.

ABSTRACT This experiment was carried out to study the effect of sesame oil (SO) supplementation on performance and fatty acid composition of meat and tail fat in Iranian Chaal lambs. Eighteen lambs were fed one of the three isocaloric and isonitrogenous diets containing 0 (control), 25 and 50 g SO per kilogram diet in a completely randomized design for 84 days. There were no substantial effects on animal performance and their carcass and non-carcass 1

measurements, except for kidney fat weight which linearly increased (P = 0.05) by increasing level of SO. Supplementation of SO was resulted a decrease in the molar ratio of ruminal propionate (P = 0.03), whereas the ruminal acetate:propionate ratio (P = 0.04), serum total cholesterol (P = 0.01) and high density lipoproteins (HDL) were increased linearly (P < 0.01). The inclusion of SO up to 50 g/kg in diet linearly decreased concentrations of C15:0 (P = 0.02, P < 0.01), C16:0 (P = 0.04, P = 0.05), C16:1 (P = 0.02, P = 0.02), C17:0 (P = 0.03, P < 0.01) and C17:1 (P = 0.02, P < 0.01), and increased concentrations of C18:1 trans (P < 0.01) and conjugated linoleic acid (CLA) C18:2 cis-9, trans-11 (P ≤ 0.01) in both intramuscular and tail fat. Increasing level of SO in the diets had quadratic (P = 0.05) effect on C18:0 and linearly increased (P = 0.05) polyunsaturated fatty acids (PUFA), and decreased saturated fatty acids (SFA) (P=0.03) as well as atherogenicity index (P = 0.05) in tail fat. Our results indicated that increasing level of SO up to 50 g per kg diet may improves tail fat and intramuscular CLA cis-9, trans-11 in young fattening Chaal lambs without affecting animal performance and with little effect on fat deposition.

Abbreviations: ADG, average daily gain; BW, body weight; C, control diet; CLA, conjugated linoleic acid; DM, dry matter; DMI, dry matter intake; FA, fatty acid; FAME, fatty acid methyl esters; FCR, feed conversion ratio; HDL, high density lipoproteins; LDL, low density lipoproteins; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; SO, sesame oil; TC, total cholesterol; TMR, total mixed ration; TG, triglycerides; UFA, unsaturated fatty acids; VFA, volatile fatty acid; VLDL, very low density lipoproteins.

Key words: Conjugated linoleic acid; Sesame oil; Performance; Chaal lambs 2

1. Introduction

The nutritional modulation of the fatty acid (FA) profile of ruminant edible fats is an important research topic, and modification of tissue and milk fat composition could improve the health characteristics of the fat produced by ruminants (Beaulieu et al., 2002; Bessa, et al., 2015). Ruminant edible fats are the primary sources of conjugated linoleic acid (CLA) for humans, which has been associated with a wide range of positive health benefits (Bessa, et al., 2005; Boles et al., 2005). The biological role of conjugated isomers of linoleic acid such as rumenic acid (C18:2 cis-9, trans-11) and C18:2 trans-10, cis-12 have been extensively studied (Castro et al., 2005; Bessa, et al., 2015). Among several biological effects, rumenic acid known as an anticarcinogenic isomer of CLA and has been shown a beneficial effect on cardiovascular disease in several animal and experimental models as reviewed by Gebauer et al. (2011). One of the options of enhancing the beneficial effects of animal products is through dietary manipulation, such as the use of finishing diets supplemented with vegetable oils to improve the concentration of CLA and thus their health benefits (Bolte et al., 2002). Sesame oil is one of the available vegetable oils in some part of the world. In some region, this oil, is obtained by using a traditional pressing method with high levels of impurities and low cost which is not suitable for human consumption and is available for use in animal diets. There have been several studies on the effect of different fats of vegetable origin in diets of beef cattle (Beaulieu et al., 2002; Duckett et al.,2002) and fattening lambs (Haddad and Younis, 2004; Castro et al., 2005; Mansoet al., 2009) but to our knowledge, the influence of SO supplementation in lamb diets has not been studied and information on the effect of fats of vegetable origin in diets on FA profile of tail fat in fat-tailed sheep are also rare. In a 3

recent study by Maleki et al. (2015) it was also reported that there was minimal variability among breeds in their FA profiles and CLA cis-9, trans-11 of tail fat in order to genetically manipulate FA profile in meat and tail fat. Hence, investigation on dietary manipulation to improve the FA profile of tail fat and meat is necessary. Many vegetable oils such as soybean, sunflower and linseed oil have been studied to enrich the beneficial FA in meat of ruminants (Kitessa et al., 2009; Roy et al., 2013). The potential of SO as a new supplemental oil in modifying FA composition of meat and tail fat of lambs has not been studied. Therefore, the objective of this study was to investigate the effects of SO (containing 397 g/kg linoleic and 428 g/kg oleic acids of total FA) supplementation on performance, carcass characteristics, some blood metabolites, rumen fermentation, and meat and tail fat FA composition (especially CLA) in fattening Chaal (an Iranian fat-tailed breed) lambs.

2. Material and methods

2.1. Animals, diets, and experimental procedure

The experiment was carried out at research institute of Aminabad which located at south East of Tehran. All of the animal procedures and protocols used in this study were approved by the college of Aburaihan animal care and use committee. Eighteen male Chaal lambs with similar weight (23.7 ± 0.73 kg) and age (139 ± 6 d) from a pure flock under the uniform rearing condition were used in this experiment. Animals were assigned into three groups (n=6/group) to evaluate three isocaloric and isonitrogenous total mixed rations (TMR) containing 0 (control), 25 and 50 g SO per kg diet (Table 1). Lambs were housed in individual pens with concrete floor. Before initiation of the trial, lambs were gradually 4

adapted over a 14 days period to the experimental condition and control diet. The feeding trial lasted 84 d and the diets were offered in two equal meals at 09:00 and 18:00 h daily with free access to fresh water. Chopped alfalfa hay and barley straw were used in the forage part of TMR. SO was included by partially replacing (isocaloric replacement) barley in diets. The oil was mixed with the concentrate portion of the TMR before being mixed with the forage component. Diets were weighed individually for each lamb. Refusals from each lamb were collected before 09:00 h and weighed. Random grab samples of the diets were taken weekly intervals, combined throughout the trial period, and stored frozen at -20°C until analysis. Feeders were managed to allow up to 5% of diet per animal to be in excess one hour prior to the next feeding. During the experiment, lambs were observed for health problems and their body weights (BW) were recorded at 2-wk intervals before the morning feedings. Feed was withheld, 12 h before initial and final weighing. Average daily gain (ADG) and dry matter intake (DMI) of lambs were measured and feed conversion ratios (FCR) were also calculated.

Feed samples were analyzed for dry matter (DM; method No. 934.01), ether extract (EE; method No. 920.39) and crude protein (CP; method No. 981.10) following the procedures of AOAC (1990). Determination of neutral detergent fiber (NDFom) was performed by using Na sulfite, without heat stable amylase and expressed exclusive of residual ash according to Van Soest et al. (1991), and ash-free acid detergent fiber (ADFom) was also determined and expressed exclusive of residual ash. The SO purchased from a commercial source and concentrations of fatty acids in the oil (g/100 g FA) were: C16:0, 8.26; C16:1, 0.15; C18:0, 5.28; C18:1, 42.79; C18:2, 39.69; C18:3, 0.78; C20:0, 0.92 and other fatty acids, 2.13.

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2.2. Carcass and non-carcass measurements

At the end of the fattening period, all lambs were slaughtered according to the Muslim (Halal) tradition. Immediately, the non-carcass parts of body (i.e., head, feet, skin, lung and trachea, heart, liver, kidneys, spleen, kidneys and mesenteric fats) were removed and weighed and hot carcass weight of lambs including the fat tail was also weighed. The fat tails of lambs were removed from the hot carcasses and weighed separately. The ratios of fat tail and non-carcass parts were calculated as a ratio of hot carcass weights.

2.3. Blood metabolites and ruminal liquid measurements

Individual blood samples were taken before the morning feedings from the jugular vein into simple vacuum tubes on d 40 and 80 of the experiment, and centrifuged at 3000 × g for 20 min. Plasma was stored at -20°C for subsequent analysis. Finally, glucose, total cholesterol (TC), high density lipoproteins (HDL) and the triglycerides (TG) concentrations (mg/dL) were measured using a commercially assay kits (Pars Azmun, Iran). Very low density lipoproteins (VLDL) and low density lipoproteins (LDL) were calculated using the Friedewald et al. (1972) equations: VLDL = (TG/5) and LDL = [TC – (VLDL + HDL)]. Samples of ruminal fluid (about 250 mL) from each sheep were taken after slaughter (in pre-feeding condition). Rumen pH was then immediately measured using a digital pH meter (model WTW pH 330, WTW, Weinheim, Germany). Rumen fluid samples were then strained through 4 layers of cheesecloth. Subsamples of 4 mL were transferred to test tubes containing 80 µL of MgCl2 (saturated solution) and immediately stored at -20°C for the analysis of volatile fatty acids (VFA). For the estimation of protozoal population, 6

subsamples of 4 mL rumen fluid were transferred to test tubes containing 4 mL of formaldehyde and stored at 4°C until enumeration procedures. Total protozoal population was counted using an optical microscope and a Neubauer improved counting chamber after adding 100µL of brilliant green dye to the preserved rumen fluid and allowed to stand for one hour. The concentration of VFAs (mmol/L) was determined by a gas chromatograph (Philips PU 4410) equipped with a flame ionization detector, and a semi-capillary, TRFFAP, 30m × 0.53mm × 1µm column (Supelco, USA), and auto-sampler. Temperatures were 140°C in the column and 250°C both in the injector and the detector, and carrier gas (He) flux was 13 mL/min. Each sample (1µL) was injected automatically with split ratio of 1/3. Chromatograms were integrated using software Star Chromatography Workstation 6.2 (Varian Inc., USA). The ammonia-N of ruminal liquid was also measured according to the method of Conway (1962).

2.4. Determination of tissues fatty acid composition

After slaughter, samples from the longissimus dorsi muscle (6th to 8th ribs) and tail fat were rapidly removed from the left side of the carcass, vacuum-packaged, and stored at 20°C before subsequent analysis. According to the method of Folch et al. (1957), lipid from intramuscular fat and tail fat were extracted using a 2:1 chloroform:methanol solution and stored in chloroform at -20°C. For muscle, fatty acid methyl esters (FAME) were prepared using a sodium methoxide 0.5 M solution in methanol followed by hydrochloric acid in methanol (1:1). For adipose tissue (tail fat) which contains essentially only esterified fatty acids, a base methylation (sodium methoxide 0.5M solution in methanol) was only used (Raeset al., 2001). The analysis of FAME were performed by a gas chromatograph 7

(Shimadzu GC-14 A) equipped with a flame ionization detector, and a 100 m × 0.25 mm i.d. capillary column with a 0.2 µm film thickness (RT-2560, Restek). Nitrogen was used as the carrier gas and the injector split ratio was 1:50. The initial oven temperature was held at 100°C for 4 min and then increased at 3°C/min to 240°C, and held for 20 min. The injector and detector temperatures were 225 and 250°C, respectively. Tridecanoic acid (C13:0) was used as internal standard and identification of FA was accomplished by comparison of sample peak retention times with those of FAME standard mixtures (Sigma, St. Louis, MO, USA). Also, CLAcis-9, trans-11 was identified with reference to methyl esters of CLA (O5507, Sigma-Aldrich, St. Louis, MO, USA). Atherogenicity index was calculated based on the equation described by Ulbricht and Southgate (1991) as follows: Atherogenicity index = (C12:0 + 4 × C14:0 + C16:0)/(MUFA + n-3PUFA + n-6PUFA).

2.5. Statistical analysis

All of the measured variables were presented as means taken from six sheep replicates. The data were analyzed as a completely randomized design using the General Linear Model (GLM) procedure of SAS (SAS Institute Inc., 2002), and linear and quadratic polynomial contrasts were used to examine the effect of different levels of SO. The level of significance was set at P≤0.05.

3. Results

3.1. Sheep performance, and their carcass and non-carcass measurements

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Adding SO to lamb diets had no significant effect on ADG, DMI, FCR and carcass measurements of lambs (Table 2). The weight and proportions of non-carcass components (i.e., head and feet, lung and trachea, heart, kidneys, spleen and mesenteric fat) were also not showed any significant difference among the treatments, except for kidney fat weight, which linearly increased (P = 0.03) by increasing level of SO in the diets (Table 2).

3.2. Ruminal fermentation and blood metabolites

Inclusion of SO to lamb diets had no significant effect on the ammonia concentration, protozoal population and the ruminal pH (Table 3). The concentration of VFAs did not differ among lambs fed either control or SO diets. But, the ruminal propionate proportion decreased (P = 0.01) by feeding SO diets. Increasing level of SO caused a linear increases (P = 0.03) in acetate:propionate ratio (Table 3). Results from blood metabolites showed that TC and HDL were also elevated (P ≤ 0.01) by SO supplementation. No significant differences were found in blood level of glucose, TG, LDL and VLDL between the treatment groups (Table 3).

3.3. Tail fat and meat fatty acid compositions

Fatty acid profile of tail fat of lambs are presented in Table 4. With the exception of C10:0, C12:0 and C14:0, inclusion of SO in the diet up to 50 g/kg resulted a significant linear decreases in the concentration of fatty acids containing less than 18 carbons (C15:0, C16:0, C16:1, C17:0, C17:1) in tail fat (P < 0.01, P = 0.05, P = 0.02, P < 0.01 and P < 0.01, respectively). Additionally, the C18:0 content of tail fat showed a quadratic response (P = 9

0.05) with an increase in C18:0 as SO level was increased from 0 to 25 g/kg diet followed by a decrease in C18:0 when 50 g SO per kg diet was fed. The proportions of C18:1 trans, CLA cis-9, trans-11, C20:0 and PUFA was linearly increased (P < 0.01, P < 0.01, P = 0.03 and P = 0.05, respectively), whereas saturated fatty acids (SFA) and atherogenicity index were decreased (P = 0.03 and P = 0.05) by SO feeding. However, total C18:1, C18:2n-6, C18:3n-3, C20:4n-6, C22:5n-3, unsaturated (UFA) and monounsaturated fatty acids (MUFA) as well as n-6/n-3 ratio were not affected by SO feeding. Fatty acid composition of intramuscular fat of lambs are shown in Table 5. Similar with tail fat, increasing level of SO in the diets decreased the concentrations of C15:0, C16:0, C16:1, C17:0 and C17:1 (P = 0.02, P = 0.04, P = 0.02, P = 0.03 and P = 0.02, respectively), and increased the concentrations of C18:1 trans and CLA cis-9, trans-11 (P < 0.01 and P = 0.01) in muscle. Total C18:1, C18:2n-6, C18:3n-3, C20:3n-9, C20:3n-6, C20:3n-3, C20:4n6, C20:5n-3, C22:4n-6, C22:5n-3, C22:6n-3, UFA, MUFA, PUFA and other fatty acids as well as n-6/n-3 ratio in intramuscular fat were not affected by SO feeding.

4. Discusion

4.1. Lambs performance, and their carcass and non-carcass measurements

The effects of added dietary fat on performance of ruminants are reported to be varied (Haddad and Younis, 2004; Boles et al., 2005; Manso et al., 2009). Such variability could be associated with differences between experiments in terms of composition of the basal diet (i.e., energy density and level of grain), level of fat inclusion, fat type and composition (i.e.,

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contents of free and saturated fatty acids), and whether diets were formulated to be isoenergetic. Our results agree with previous works that have shown adding fats to the diet of fattening lambs does not cause modifications in animal performance or carcass characteristics (Ivan et al., 2001; Boles et al., 2005; Castro et al., 2005). Level of fat used in our study (≤50 g/kg diet on DM basis) and the fact that the rations with fat supplements were isoenergetic and isonitrogenous may explain the absence of significant differences in animal performance and carcass measurements. In general, dietary fat depresses DM intake of ruminants due to chemostatic regulation of voluntary intake by increasing energy density of diets (Boggs et al., 1987). In the current study, diets were calculated to be isocaloric and thereby, had no effect on DMI of lambs which is consistent with other studies (Ivan et al., 2001; Demirel et al., 2004; Boles et al., 2005) who reported no reduction in feed intake when various vegetable fats were included in lamb rations. In contrast, Haddad and Younis (2004) reported a decrease in feed intake of Awassi lambs when 25 and 50 g saturated fatty acids were added to each kg of their diets. Lough et al. (1993) also observed a decrease in dry matter intake of ram and ewe lambs when palm oil was included in their rations (100 g/kg of diet). However, the diets used in both Haddad and Younis (2004) and Lough et al. (1993) studies were not isoenergetic, and the lambs reached the same energy intake levels with reduced DM intake. Under the conditions of this experiment, SO supplementation in isocaloric diets had little effect on fat deposition in different parts of lambs' body (i.e., kidney fat, mesenteric fat and

tail fat), so that only kidneys fat weight was significantly increased. The observation of little differences in fat deposition could be due to the low amount of fat deposition in all lambs. It should be noted that lambs in the present study were slaughtered at the early stage of maturity (8 months of age) and if they had been slaughtered later, the treatment effects could 11

have been different since fatness occurs largely during the later stages of growth. Salinas et al. (2004) reported an apparent effect of diet on the amount of fat deposition at a greater finishing body weight, but lambs in the present study were slaughtered at low finishing body weights. Also, Lough et al. (1993) found that supplementation of 100 g palm oil per kg diet resulted in a greater fat deposition and lower carcass muscle proportions in sheep. These could have been due to the amount of oil used. Although, SO supplementation in our study had little effect on total fat deposition, the increase of kidney fat weight indicates that

substituting fat for carbohydrate calories had its primary impact on internal fat tissues and not on lean tissue mass.

4.2. Ruminal fermentation and blood metabolites

Doreau and Ferlay (1995) reported that linseed and coconut oils have a defaunating effect on rumen environment. Varadyova et al. (2007) also reported that linseed oil at a dose of 50 g/kg in the diet of sheep decreased rumen protozoal population but sunflower (50 g/kg diet) and rapeseed oils had no effect on total rumen protozoal count which is similar to the results of our research. In our study, inclusion of SO up to 50 g/kg of diet was apparently low in modifying rumen protozoal population, and it can be said that the rumen protozoa had no uniform response to oil supplements. Patra (2014) indicated that fat addition generally caused increases in percentage of propionate and decreases in acetate to proportionate proportion with no change in total VFA and acetate. But, in the present study the increasing level of SO in lamb diets caused decreases in ruminal propionate and increases in acetate:propionate ratio. The decreases in proportion of ruminal propionate in our study could be related to a reduced barley grain concentration in the diet since the type and amount 12

of carbohydrate digested by ruminants is a decisive factor in determining the proportions of the resultant rumen VFA. Overall, we replaced SO with barley, but to create the isocaloric and isonitrogenous diets, we reduced barley while slightly increased alfalfa, barley straw, wheat bran and canola meal. Therefore, our feed replacement with oil slightly increased fiber but decreased starch, which could affect ruminal fermentation in this manner. Similarly, according to Rigout et al. (2003), glucose level did not reflect the quantity and proportion of VFA.

Plasma concentration of TC and HDL were increased by SO feeding. Mean concentrations of glucose, LDL, VLDL and TG were similar among treatments. Fat supplementation remarkably increases lipoprotein cholesterols export by the intestine, the major site of de novo cholesterol synthesis in ruminants (Noble, 1981). Several researchers were also reported that cottonseed oil, soybean oil and palm oil have the ability to increase blood cholesterol and TG contents (Hernandezet al., 1978; Garciaet al., 2003). HDL concentration was increased in SO supplemented groups of lambs to approximately the same extent as TC. Hence, the elevation of TC in SO supplemented lambs was due primarily to an increase in HDL. TC represents the summation of all forms of cholesterol presented in the serum.

4.3. Fat tail and meat fatty acid compositions

In agreement with Maleki et al. (2015) and Momen et al. (2016), oleic acid (C18:1) is the most abundant FA in all the depots studied, followed by palmitic acid (C16:0) and stearic acid. Oil supplementation in the present study caused a decrease in the proportion of the saturated fatty acid C16:0 in both intramuscular and tail fat. Lowering the C16:0 content of meat and adipose tissue is desirable for human health as this FA has been identified as a 13

hypercholesterolemic FA (Givens, 2005). In addition, increased dietary oil reduced the concentrations of odd chain fatty acids. These fatty acids probably originate mainly from de novo FA synthesis in intramuscular fat and adipose tissues because feedstuffs contain no significant amounts of these fatty acids (Aurousseau et al., 2004). In this study, all diets were isoenergetic, and so diet including the low level of fat (control diet) supplied more fermentable organic matter than SO-containing diets. Thus, the control diet that led to a greater production of propionic acid, from which odd-chain fatty acids are synthesized. Tissues from lambs fed 25 g SO per kg diet contained a greater proportion of C18:0 as compared with lambs fed 50 g SO per kg diet (a quadratic effect), indicates that low level of oil (25 g SO) undergo greater complete biohydrogenation than high levels of SO (50 g SO). In the current study, the concentration of CLA cis-9, trans-11 as well as C18:1 trans increased in intramuscular and tail fats of lambs receiving SO diets. Similarly, supplementation of sheep diets with safflower oil (rich in C18:2) up to 60 g per kg in diet caused an increase of CLA cis-9, trans-11 in muscle (Boles et al., 2005). The C18:1 trans11 is the major transoctadecenoate of rumen and ruminant products in most situations except for high concentrate or very low fiber diets supplemented with unsaturated oils (Duckett et al., 2002; Sackmann et al., 2003; Bessa et al., 2015). High concentrate diets are also associated with high levels of C18:1 trans-10 and CLA trans-10, cis-12 by inducing rumen biohydrogenation pathways of unsaturated fatty acids that favors the production of C18:1 trans-10 instead of C18:1 trans-11 (Bessa et al., 2005; Sackmann et al., 2003; Bessa et al., 2015). Therefore, CLA cis-9, trans-11 in muscle and adipose tissue is generally low when ruminants are fed diets rich in concentrate and the supplementation of these diets with unsaturated vegetable oil seems to be inadequate to obtain an expressive response (Duckett et al., 2002; Sackmann et al., 2003; Bessa et al., 2005). In a study on the effect of sunflower 14

oil inclusion in steer diets, Sackmann et al. (2003) supported the idea that forage can alter CLA production. They showed that when dietary forage ratio increase from 12% to 36%, the flow of C18:1 trans-10 to the duodenum decreased 63% while that of C18:1 trans-11 increased 237%. In the current experiment, 25 and 50 g SO supplemented diets contained 34 and 35% forage, respectively. Hence, these amounts of forage in the diets could have been promoted the C18:1 trans-11 producing pathway. Furthermore, the direct relationship between C18:1 trans-11 and CLA cis-9, trans-11 is well known (Bessa et al., 2005; Bessa et al., 2015), and about 87% of CLA cis-9, trans-11 present in tissues results from endogenous desaturation by stearoyl-CoA desaturase (SCD) of C18:1 trans-11 (Palmquist et al., 2004). It has also been suggested that C18:1 trans-11 isomer is a common intermediate in the microbial biohydrogenation of dietary C18:1n-9, C18:2n-6, and C18:3n-3 (Harfoot and Hazelwood, 1997). Thus, increases in the concentrations of C18:1 trans and CLA cis-9, trans-11 in tissues with SO supplementation could be due to the high production of C18:1 trans-11 formed during ruminal biohydrogenation of C18:1n-9, C18:2n-6, and C18:3n-3 supplied by SO. Previous studies (Or-Rashid et al., 2007; Varadyova et al., 2007; Varadyova et al., 2008) showed that most species of protozoa are rich in UFA, especially CLA cis-9, trans-11 and C18:1 trans-11. Varadyova et al. (2007; 2008) also reported that the highest concentrations of C18:1 trans-11 and CLA cis-9, trans-11 in the rumen fluid were occurred with adding 50 g/kg sunflower and rapeseed oils in sheep diets without major changes in the rumen protozoal population. In their studies, dietary addition of sunflower and rapeseed oils (50 g/kg diet) resulted in higher incorporation of C18:1 trans-11 into protozoal fractions which probably positively affected C18:1 trans-11 flow from the rumen. Their results confirm the important role of rumen protozoa in formation of C18:1 trans-11 and CLA cis-9, trans-11 in 15

the rumen and supports our findings, because in our study inclusion of SO up to 50 g/kg in lamb diets increased concentrations of C18:1 trans and CLA cis-9, trans-11 in both intramuscular and tail fat without significant change in the rumen protozoal population. The increase in the concentrations of CLA cis-9, trans-11 and C18:1 trans of tail fat was higher than those of intramuscular fat (64 and 107% in tail fat versus 38 and 55% in intramuscular fat, respectively). Similarly, previous works have shown greater changes in subcutaneous than in intramuscular fat (Castro et al., 2005; Manso et al., 2009). This may be attributable to the low proportion of phospholipid in adipose tissue as well as a predominant incorporation of CLA cis-9, trans-11 into the triacylglycerol fraction in ruminant animals (Raes et al., 2003). The greater changes in tail fat than intramuscular fat could also be due to the fact that during the finishing period of fat tailed sheep, tail fat increases at a greater rate than other adipose depots such as intramuscular. On the other hand, tail fat seems to be more responsive to changes in the dietary FA supply or changes in rumen metabolism than intramuscular fat. Breed is one of the main factors affecting the FA profile and carcass composition. Differences in the FA composition of fat have been observed between Iranian sheep breeds, and this variability among breeds might be useful in genetically improvement of fat depots and derived products (Alipanah and Kashan, 2011; Maleki et al., 2015; Momen et al., 2016). In a recent study by Maleki et al. (2015), differences between the sheep breeds in their FA profiles and CLA cis-9, trans-11 were observed in the adipose tissue, but not in the tail fat (there were minimal differences in FA profiles of tail fat). According to our findings it can be said that, if using the breed effects is insufficient to genetically manipulation of CLA cis9, trans-11 of tail fat, nutritional modification by using SO in lamb diets can be one of the

16

ways to manipulate the FA profile (especially, CLA cis-9, trans-11) of tail fat and meat in fat-tailed breeds. In our study, SO feeding led to decrease in SFA and increase in PUFA proportions of tail fat which could be desirable for human health. The increases of PUFA in tail fat was basically due to increases of CLA cis-9, trans-11. In tail fat the main PUFA were CLA cis-9, trans-11, C18:2n-6 and C18:3n-3, and the CLA cis-9, trans-11 have a greater portion in PUFA of tail fat than of intramuscular fat. Furthermore, inclusion of SO up to 50 g/kg in diet of lambs increased the CLA cis-9, trans-11 of tail fat more than of intramuscular fat (64% in tail fat versus 38% in intramuscular fat, respectively). Nutritional advisers recommended an increase of n-3 fatty acids in human diet, and thus a reduction of n-6/n-3 ratio to values below 4 (Department of Health, 1994). In the present trial, SO supplementation up to 50 g/kg diet did not substantially impact on n-6/n-3 ratio in lamb meat and tail fat, and the values for n-6/n-3 ratio of tail fat were lower than 4, and the values for n-6/n-3 ratio of muscle were slightly higher than 4. However, a negative effect was observed on n-6/n-3 ratio when diets were supplemented with soybean oil (Bessa et al., 2005) and sunflower oil (Manso et al., 2009). Fat with high atherogenicity index value is assumed to be more detrimental to the human health (Ulbricht and Southgate, 1991). In the present study, a significant linear decrease in the atherogenicity index of tail fat was found in response to SO supplementation, which could be desirable for human health. These results are in agreement with observation in subcutaneous and intramuscular fats from lambs fed 4% sunflower oil (Manso et al., 2009).

5. Conclusions

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It can be concluded that the inclusion of SO up to 50 g/kg in the diet of young fattening Chaal lambs, as a high value energy source and meat fatty acid modifier, may improves tail fat and intramuscular fatty acid composition without affecting animal performance, and with little effect on fat deposition.

Conflict of interest

The authors declared that there is no conflict of interests.

Acknowledgments

The authors are grateful to the University of Tehran, for providing the funds for this research. The authors would also like to thank the members of laboratory staff from the College of Aburaihan. The help of the staff of Aminabad research institute are kindly acknowledged.

References

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Table 1: Ingredients and chemical composition of 0 (control, C), 25 and 50 g sesame oil (SO) per kg diets. Items Ingredients, DM basis (g/kg) Alfalfa hay Barley straw Barley Wheat bran Canola meal Sesame oil Vit. -min. premix a Salt Sodium bicarbonate Limestone

0 (C)

Diets (g SO per kg) 25

50

250 60 465 130 80 0.0 4.0 4.0 4.0 3.0

270 70 390 140 90 25 4.0 4.0 4.0 3.0

280 70 350 140 95 50 4.0 4.0 4.0 3.0

Composition (g/kg) Dry matter c 887 890 893 b ME, MJ/kg 10.7 10.9 11.2 Crude protein c 147 147 146 c Neutral detergent fiber (NDFom) 347 359 357 Acid detergent fiber (ADFom)c 190 201 203 c Ether extract 23.9 48.8 73.2 d Calcium 5.8 6.1 6.3 Phosphorus d 5.0 5.0 5.0 e Sodium 3.2 3.2 3.2 a Each kg contained: vitamin A, 400,000 IU; vitamin D3, 100,000 IU; vitamin E, 200mg; Ca, 180 g; P, 70 g; Mg, 30 g; Na, 50 g; Mn, 5,000 mg; Zn, 3,000 mg; I, 100 mg; Fe, 3,000 mg; Cu, 300 mg; Co, 100 mg; andSe, 20 mg plus 400 mg antioxidant. b The metabolizable energy of diets was estimated based on tabular values recommended by National Research Council (2007). For sesame oil, the ME content was also estimated using the energy conversions of NRC (2007): 1 kg TDN = 3.6 Mcal ME and 1 Mcal ME = 4.18 MJ ME. Based on tabular values recommended by National Research Council (2001) for vegetable oils, TDN value for sesame oil was considered to be as 1840 g/kg. c Based on laboratory analysis. d Estimated based on National Research Council (2007). e Estimated based on National Research Council (2001).

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Table 2: Animal performance and the carcass and non-carcass measurements of Chaal lambs fed on 0 (control, C), 25 and 50 g sesame oil (SO) per kg diets. Diets (g SO per kg) P value Variables 0 (C) 25 50 SEM linear quad Animal performance Average initial weight, kg 23.7 23.7 23.7 0.32 1.00 1.00 Average final weight, kg 38.4 38.0 39.2 0.50 0.26 0.25 Dry matter intake, kg 1.15 1.14 1.18 0.03 0.38 0.50 Average daily gain, kg 0.18 0.17 0.18 0.01 0.23 0.22 Feed conversion ratio 6.59 6.71 6.43 0.19 0.55 0.43 Carcass measurements Hot carcass weight, kg Dressing carcass yield, kg/100 kg Fat tail, kg Untailed carcass weight, kg

18.6 48.5 2.49 16.1

18.4 48.3 2.50 15.9

19.2 49.0 2.78 16.4

0.35 0.52 0.17 0.24

0.24 0.51 0.27 0.38

0.25 0.54 0.52 0.24

Non-carcass measurements, kg liver Lungs and trachea Spleen Heart Kidney Kidney fat Mesenteric fat Head Four feet Skin Total fata

0.59 0.45 0.08 0.23 0.10 0.08 0.26 2.24 0.87 3.64 2.84

0.60 0.46 0.08 0.23 0.10 0.09 0.25 2.22 0.87 3.71 2.84

0.61 0.45 0.08 0.25 0.11 0.11 0.29 2.27 0.87 3.86 3.17

0.02 0.01 0.01 0.01 0.01 0.01 0.02 0.07 0.02 0.18 0.17

0.51 0.96 0.94 0.14 0.13 0.05 0.30 0.73 0.88 0.40 0.18

0.97 0.66 0.90 0.30 0.63 0.40 0.43 0.75 0.78 0.86 0.43

Proportions, kg/100 kg of hot carcass weight liver 3.19 3.28 Lungs and trachea 2.42 2.48 Spleen 0.43 0.44 Heart 1.25 1.25 Kidneys 0.52 0.54 Fat tail 13.4 13.5 Kidney fat 0.48 0.50 Mesenteric fat 1.37 1.36 Head 12.0 12.1 Four feet 4.68 4.71 Skin 19.6 20.2 a Total fat 15.3 15.4 a Total fat = (Kidney fat + mesenteric fat + fat tail).

3.17 2.34 0.42 1.30 0.56 14.4 0.55 1.50 11.8 4.55 20.1 16.5

0.08 0.05 0.04 0.04 0.03 0.76 0.03 0.11 0.38 0.08 0.93 0.68

0.85 0.27 0.93 0.31 0.31 0.36 0.11 0.43 0.69 0.27 0.74 0.22

0.36 0.13 0.74 0.57 0.94 0.69 0.74 0.60 0.69 0.37 0.77 0.58

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Table 3: Rumen fermentation parameters and blood metabolites of Chaal lambs fed on 0 (control, C), 25 and 50 g sesame oil (SO) per kg diets. Diets (g SO per kg) P value Variables 0 (C) 25 50 SEM linear quad Rumen fermentation Parameters Rumen pH Ammonia-N, mg/L Protozoal numbers, (n/ml)(105)

6.96 128 2.93

7.10 133 3.40

7.06 131 3.13

0.04 3.51 0.66

0.14 0.64 0.83

0.16 0.43 0.66

VFAconcentration, mmol/L Acetate Propionate Butyrate Isovalerate Valerate Total VFA

37.0 12.0 7.17 0.71 0.85 57.7

35.6 9.95 6.89 0.57 0.82 53.8

39.9 10.5 7.45 0.68 0.99 59.5

2.46 0.50 0.38 0.07 0.11 2.89

0.41 0.11 0.61 0.78 0.38 0.66

0.36 0.07 0.38 0.17 0.47 0.20

65.9 18.6 12.8 1.05 1.57 3.57

66.9 17.7 12.6 1.16 1.69 3.84

1.29 0.81 0.55 0.14 0.25 0.21

0.11 0.01 0.92 0.66 0.65 0.03

0.73 0.52 0.68 0.37 0.89 0.70

0.01 <.01 0.35 0.15 0.15 0.60 low

0.92 0.77 0.65 0.73 0.73 0.60

Molar proportions, mol/100 mol of total VFA Acetate 63.8 Propionate 20.9 Butyrate 12.5 Isovalerate 1.25 Valerate 1.53 Acetate: propionate ratio 3.10

Blood metabolites, mg/dL TC 45.8 51.5 56.5 2.70 HDL 24.3 27.8 32.5 1.62 LDL 17.3 19.1 19.3 1.44 VLDL 4.20 4.57 4.73 0.23 TG 21.0 22.8 23.7 1.17 Glucose 66.3 68.2 67.7 1.78 VFA, volatile fatty acid; TC, total cholesterol; HDL, high density lipoproteins; LDL, density lipoproteins; VLDL, very low density lipoproteins; TG, triglycerides.

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Table 4: Fatty acid composition of tail fat (g/100 g FA) of Chaal lambs fed on 0 (control, C), 25 and 50 g sesame oil (SO) per kg diets. Diets (g SO per kg) P value Fatty acids 0 (C) 25 50 SEM linear quad C10:0 0.24 0.22 0.23 0.02 0.75 C12:0 0.20 0.20 0.19 0.01 0.55 C14:0 4.08 4.10 4.14 0.20 0.84 C15:0 1.30 1.07 0.99 0.07 <.01 C16:0 23.2 22.3 21.4 0.62 0.05 C16:1 cis-9 2.52 2.23 2.15 0.10 0.02 C17:0 2.48 2.16 1.83 0.14 <.01 C17:1 cis-9 1.80 1.34 1.26 0.09 <.01 C18:0 9.64 10.6 9.19 0.43 0.48 a C18:1 trans 3.66 5.57 7.59 0.35 <.01 Total C18:1 43.2 44.1 45.9 1.07 0.10 C18:2n-6 2.95 2.96 3.02 0.15 0.77 C18:2 cis-9, trans-11(CLA) 1.06 1.45 1.74 0.09 <.01 C18:3n-3 0.78 0.77 0.73 0.05 0.49 C20:0 0.16 0.21 0.22 0.02 0.03 C20:4n-6 0.13 0.12 0.13 0.01 1.00 C22:5n-3 0.13 0.11 0.12 0.01 0.61 Others b 6.10 6.10 6.83 0.46 0.28 c SFA 41.3 40.8 38.2 0.90 0.03 c UFA 52.6 53.1 55.0 1.18 0.17 MUFA c 47.5 47.7 49.3 1.10 0.28 c PUFA 4.93 5.31 5.61 0.22 0.05 n-6/n-3 3.43 3.51 3.71 0.19 0.33 d Atherogenicity index 0.77 0.75 0.72 0.02 0.05 a Sum of C18:1 trans isomers eluted between C18:0 and C18:1cis-9. b Include unidentified peaks. c SFA = C10:0 + C12:0 + C14:0 + C15:0 + C16:0 + C17:0 + C18:0 + C20:0; UFA = MUFA + PUFA; MUFA = C16:1 + C17:1 + C18:1; PUFA = C18:2n-6 + C18:2 cis-9, trans-11 + C18:3n-3 + C20:4n-6 + C22:5n-3. d Ulbricht and Southgate (1991).

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0.58 0.63 0.96 0.43 0.98 0.42 0.95 0.10 0.05 0.91 0.75 0.89 0.67 0.82 0.43 0.70 0.27 0.52 0.35 0.63 0.60 0.90 0.82 0.78

Table 5: Intramuscular fatty acid composition (g/100 g FA) of Chaal lambs fed on 0 (control, C), 25 and 50 g sesame oil (SO) per kg diets. Diets (g SO per kg) P value Fatty acids 0 (C) 25 50 SEM linear quad C10:0 0.14 0.14 0.15 0.03 0.84 C12:0 0.14 0.12 0.13 0.01 0.85 C14:0 2.49 2.51 2.54 0.15 0.82 C15:0 0.38 0.30 0.24 0.04 0.02 C16:0 19.4 18.5 18.0 0.46 0.04 C16:1 cis-9 1.34 1.20 0.97 0.10 0.02 C17:0 1.01 0.81 0.71 0.08 0.03 C17:1 cis-9 0.67 0.49 0.39 0.08 0.02 C18:0 14.3 14.9 14.0 0.35 0.54 a C18:1 trans 3.46 4.30 5.35 0.20 <.01 Total C18:1 35.9 36.3 37.0 0.59 0.22 C18:2n-6 7.51 7.66 7.85 0.31 0.44 C18:2 cis-9, trans-11(CLA) 0.78 0.91 1.08 0.07 0.01 C18:3n-3 0.94 0.95 0.88 0.05 0.40 C20:3n-9 0.56 0.57 0.60 0.07 0.72 C20:3n-6 0.47 0.49 0.51 0.07 0.68 C20:3n-3 0.15 0.15 0.14 0.01 0.28 C20:4n-6 3.45 3.52 3.63 0.34 0.72 C20:5n-3 0.51 0.51 0.53 0.05 0.69 C22:4n-6 0.31 0.37 0.36 0.03 0.24 C22:5n-3 1.03 0.99 1.01 0.11 0.90 C22:6n-3 0.29 0.28 0.31 0.03 0.76 Others b 8.26 8.24 9.05 0.58 0.34 c SFA 37.8 37.3 35.7 0.79 0.08 UFA c 53.9 54.4 55.2 0.72 0.21 c MUFA 37.9 38.0 38.3 0.70 0.67 c PUFA 16.0 16.4 16.9 0.76 0.41 n-6/n-3 4.12 4.24 4.31 0.27 0.62 d Atherogenicity index 0.56 0.54 0.53 0.01 0.13 a Sum of C18:1 trans isomers eluted between C18:0 and C18:1cis-9. b Include unidentified peaks. c SFA = C10:0 + C12:0 + C14:0 + C15:0 + C16:0 + C17:0 + C18:0; UFA = MUFA + PUFA; MUFA = C16:1 + C17:1 + C18:1; PUFA = C18:2n-6 + C18:2 cis-9, trans-11 + C18:3n-3 + C20:3n-9 + C20:3n-6 + C20:3n-3 + C20:4n-6 + C20:5n-3 + C22:4n-6 + C22:5n3 + C22:6n-3. d Ulbricht and Southgate (1991).

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0.83 0.25 0.96 0.89 0.78 0.73 0.65 0.72 0.08 0.68 0.87 0.97 0.81 0.49 0.94 0.95 0.78 0.96 0.92 0.49 0.82 0.63 0.57 0.56 0.86 0.90 0.96 0.95 0.87