Biology Medicine
AgriculturalAndFoodSciences,1970-2015-Agricultural And Food Sciences-2013.jsonl.gz.gz
Agricultural And Food Sciences
e63a1bd68db77985a8d05616f8b5bdc8b79dcaee
Effect of Linseed Oil Dietary Supplementation on Fatty Acid Composition and Gene Expression in Adipose Tissue of Growing Goats

This study was conducted to determine the effects of feeding oil palm frond silage based diets with added linseed oil (LO) containing high α-linolenic acid (C18:3n-3), namely, high LO (HLO), low LO (LLO), and without LO as the control group (CON) on the fatty acid (FA) composition of subcutaneous adipose tissue and the gene expression of peroxisome proliferator-activated receptor (PPAR)α, PPAR-γ, and stearoyl-CoA desaturase (SCD) in Boer goats. The proportion of C18:3n-3 in subcutaneous adipose tissue was increased (P < 0.01) by increasing the LO in the diet, suggesting that the FA from HLO might have escaped ruminal biohydrogenation. Animals fed HLO diets had lower proportions of C18:1 trans-11, C18:2n-6, CLA cis-9 trans-11, and C20:4n-6 and higher proportions of C18:3n-3, C22:5n-3, and C22:6n-3 in the subcutaneous adipose tissue than animals fed the CON diets, resulting in a decreased n-6:n-3 fatty acid ratio (FAR) in the tissue. In addition, feeding the HLO diet upregulated the expression of PPAR-γ (P < 0.05) but downregulated the expression of SCD (P < 0.05) in the adipose tissue. The results of the present study show that LO can be safely incorporated in the diets of goats to enrich goat meat with potential health beneficial FA (i.e., n-3 FA).


Introduction
e usually high content of saturated fatty acids (SFA) in ruminant meat can increase the risk of cardiovascular diseases [1]. However, ruminant meat may also be a good dietary source of some nutrients with health bene�ts including some FA such as long chain (C20) polyunsaturated fatty acids (LC-PUFA) and conjugated linoleic acid (CLA) isomers [2]. e decrease of SFA and the increase of health-bene�cial FA have been an important topic in ruminant meat research. e inclusion of sources of C18:3n-3 in lamb diets, such as forages [3][4][5], pastures [6], linseed [7,8], or linseed oil [9,10], had increased the concentration of n-3 PUFA in the meat. Ruminant fats are among the richest natural sources of CLA isomers, particularly of rumenic acid (CLA cis-9 trans-11), and are the main sources of these isomers in the human diet [11]. e CLA cis-9 trans-11 is produced during ruminal biohydrogenation of C18:2n-6 to stearic acid [12] and by endogenous conversion of C18:1 trans-11 by Δ9desaturase in tissues [13]. Feeding animals with diets rich in linoleic acid (C18:2n-6) and -linolenic acid (C18:3n-3) acids increased the CLA cis-9 trans-11 content of ruminant meat [3,6,9,14,15]. However, feeding linseed oil (rich in C18:3n-3) seems to be less effective in the increase of CLA cis-9 trans-11 in intramuscular fat than sun�ower oil (rich in C18:2n-6) [9,16]. Bessa et al. [9] observed that a blend of sun�ower and linseed oil might be a good approach to obtain simultaneously an enrichment in n-3 PUFA and CLA in lamb intramuscular fat.
Both n-3 and n-6 PUFA appear to suppress the genes that encode for several enzymes, which are involved in carbohydrate and lipid metabolism, whereas saturated, trans-, and monounsaturated fatty acids (MUFA) fail to suppress [17,18]. e PPAR are activated by FA and regulate FA uptake and oxidation in the liver [19]. e PUFA activates PPAR and stimulates peroxisomal -oxidation and peroxisome proliferation [20]. e expression of the SCD gene in the ruminants is regulated by the transcription factors SREBP1 [21,22], PPAR- [22], and PPAR- [21,22]. Furthermore, unsaturated FA are important because they play a role in the cellular activities, metabolism, and nuclear events that govern gene transcription [23]. In this sense, the dietary n-6 and n-3 PUFA, especially arachidonic acid (C20:4n-6), have been shown to repress SCD gene expression [23]. In most cases these studies have been carried out in the rodents, where lipid metabolism is different from that in ruminants.
is study was conducted to determine the effects of feeding oil palm frond (OPF) silage-based diets containing different levels of linseed oil on the FA composition of subcutaneous adipose tissue and the PPAR-, PPAR-, and SCD gene expression in Boer goats.

Animals, Diets, and Management.
Twenty-one �vemonth-old male Boer goats weighing 13.66 ± 1.07 Kg (mean initial body weight ± standard error) were initially drenched against parasites and randomly assigned to different dietary treatment groups. Goats were housed individually in wooden pens measuring 1.2 m × 1 m each, built inside a shed with slatted �ooring 0.5 meter above the ground. e experimental diets were formulated to have a high LO (HLO), low LO (LLO), and no LO as the control group (CON). e feed ingredients and composition of experimental diets are shown in Table 2. Sun�ower oil (SFO) (Lam Soon Edible Oils Sdn. Bhd.) which contained 6.40% of C16:0, 3.66% of C18:0, 28.32% of C18:1n-9, 61.23% of C18:2n-6, and 0.39% of C18:3n-3 expressed as a percentage of total identi�ed FA, palm kernel oil (PKO) (Malaysian Palm Oil Board) which contained 52.55% of C12:0, 16.75% of C14:0, 9.00% of C16:0, 2.36% of C18:0, 16.62% of C18:1n-9, and 18.47% of C18:2n-6 expressed as a percentage of total identi�ed FA, and linseed oil (LO) (Brenntag Canada, Inc., Montreal, QC, Canada) which contained 5.15% of C16:0, 3.17% of C18:0, 16.62% of C18:1n-9, 16.12% of C18:2n-6, and 57.09% of C18:3n-3 expressed as a percentage of total identi�ed FA were used as oil sources to incorporate different levels of C18:3n-3. e linseed oil was used as the main source of -linolenic acid (C18:3n-3) while sun�ower oil was used as the main source of linoleic acid (C18:2n-6). e animals were fed twice daily at 3.7% of BW (DM basis), with adjustments made weekly according to the changing body weight. Fresh OPF were chopped to 2-3 cm length and mixed with cellulase enzyme (Onozuka R-10; Yakult Ltd, Tokyo, Japan; 2 g/Kg of fresh matter) and lactic acid bacteria (Lactobacillus plantarum MTD1, Ecosyl, Stokesley, Yorkshire, UK) calculated to contain at least 1 × 10 6 colonies forming units (CFU) per gram as per manufacturer's instructions and ensiled in 200-liter plastic drums for 12 weeks. e concentrations (70% DM basis) and OPF silage (30% DM basis) were mixed and offered in 2 equal meals at 0800 and 1700. e diets were adjusted to be isonitrogenous and isocaloric and to meet the energy and protein requirements of growing goats (Table 2) [24]. All the goats had free access to drinking water and a mineral block. e feeding trial lasted for 100 days with a three-week adaptation period.

Chemical
Analyses. Samples (500 g) of concentrate and OPF silage were collected every 7 d and stored at 4 ∘ C. Individual goat refusals of feed were weighed daily and stored at 4 ∘ C until analyzed for dry matter (DM). Concentrates and OPF silage samples were dried at 60 ∘ C for 48 h to determine the DM content, ground to pass a 1 mm screen and analyzed for crude protein (CP), ether extract (EE), ash, organic matter (OM), neutral detergent �ber (NDF), and acid detergent �ber (ADF) according to standard methods. Crude protein (CP, total nitrogen × 6.25) was determined by the method (number 990.03) of the [25]. Neutral detergent �ber (NDF) and acid detergent �ber (ADF) were determined according to van Soest et al. [26].
2.3. Measurement of FA. e total FA were extracted from oils, experimental feeds, and subcutaneous adipose tissue based on the method of [27] modi�ed by [28] and described by [29], using chloroform : methanol 2 : 1 (v/v) containing butylated hydroxytoluene to prevent oxidation during sample preparation. e extracted fatty acids were transmethylated to their fatty acid methyl esters (FAME) using 0.66 N KOH in methanol and 14% methanolic boron tri�uoride (BF 3 ) (Sigma Chemical Co., St. Louis, MO, USA) according to the methods by AOAC (1990). e FAME was separated by gas liquid chromatography on an Agilent 7890A GC system (Agilent, Palo Alto, CA, USA) using a 100 m × 0.25 mm ID (0.20 m �lm thickness) Supelco SP-2560 capillary column (Supelco, Inc., Bellefonte, PA, USA). One microliter of FAME was injected by an autosampler into the chromatograph, equipped with a �ame ionization detector (FID). e carrier gas was He, and the split ratio was 10 : 1 aer injection of the FAME. e injector temperature was programmed at 250 ∘ C, and the detector temperature was 300 ∘ C. e column temperature program initiated runs at 120 ∘ C held for 5 min, increased by 2 ∘ C/min up to 170 ∘ C, held at 170 ∘ C for 15 min, increased again by 5 ∘ C/min up to 200 ∘ C, held at 200 ∘ C for 5 min, then increased again by 2 ∘ C/min to a �nal temperature of 235 ∘ C, and held for 10 min. e FA concentrations are expressed as percent of total identi�ed FA. A reference standard (mix C4-C24 methyl esters; Sigma-Aldrich, Inc., St. Louis, MO, USA) and CLA standard mix (CLA cis-9 trans-11 and CLA trans-10, cis-12, Sigma-Aldrich, Inc., St. Louis, MO, USA) were used to determine recoveries and correction factors for the determination of individual FA composition.

2.�. Tiss�e Collection an� RNA Extraction an� P�ri�cation.
Immediately aer slaughtering the animals the subcutaneous adipose tissue was quickly excised and snap-frozen in liquid nitrogen and stored at −80 ∘ C until RNA extraction. Total RNA was extracted from 100 mg of frozen tissue using the RNeasy lipid tissue mini kit (Cat. no. 74804, Qiagen, Hilden, Germany), and DNase digestion was completed during RNA puri�cation using the RNase-Free DNase set (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Total RNA purity was determined by the 260/280 nm ratio of absorbance readings using NanoDrop ND-1000 UV-Vis Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA).

Complementary DNA Synthesis.
Puri�ed total RNA (1 g) was reverse transcribed using a QuantiTect reverse transcription kit (Qiagen, Hilden, Germany) in accordance with the manufacturer's recommended procedure.

Real-Time Polymerase Chain Reaction (PCR).
Real-time PCR was performed with the Bio-Rad CFX96 Touch (Bio-Rad Laboratories, Hercules, CA, USA) using optical grade plates using QuantiFast SYBR green PCR kit (Cat. no. 204054, Qiagen, Hilden, Germany). e sequences of primers are shown in Table 1.
e -actin was used as the reference gene to normalize the tested genes. All primers were purchased through 1st BASE oligonucleotide synthesis (1st Base, Singapore). Each reaction (20 L) contained 8.5 L SYBR green PCR mix, 1 L cDNA, 1 L each of forward and reverse primers, and 8.5 L RNase free water. Target genes were ampli�ed through the following thermocycling program: 95 ∘ C for 10 ′ , 40 PCR cycles at 95 ∘ C for 30 ′′ , 60 ∘ C for 20 ′′ , and 72 ∘ C for 20 ′′ . Fluorescence was measured at every 15 ′′ to construct the melting curve. A real-time PCR was conducted for each primer pair in which cDNA samples were substituted with dH 2 O to verify that exogenous DNA was not present. Additionally, 1 g of RNA isolated by the procedure described above was substituted for cDNA in a real-time PCR reaction to con�rm that there were no genomic DNA contaminants in the RNA samples. Both negative controls showed no ampli�cation aer 40 cycles. E�ciency of ampli�cation was determined for each primer pair using serial dilutions. e cycle numbers at which ampli�ed DNA samples exceeded a computer generated �uorescence threshold level were normalized and compared to determine relative gene expression. Higher cycle number values indicated lower initial concentrations of cDNA and thus lower levels of mRNA expression. Each sample was run in triplicate, and averaged triplicates were used to assign cycle threshold (CT) values. e ΔCT values were generated by subtracting experimental CT values from the CT values for -actin targets ampli�ed with each sample. e group with the highest mean ΔCT value (lowest gene expression) per ampli�ed gene target was set to zero, and the mean ΔCT values of the other groups were set relative to this calibrator (ΔΔCT). e ΔΔCT values were calculated as powers of 2 (−2 ΔΔCT), to account for the exponential doubling of the PCR.

Statistical Analysis.
Results were analyzed using analysis of variance with the different LO content as the main effects. FA data and all the gene expression data were analyzed by one-way ANOVA, using the MIXED procedure of the SAS soware package, version 9.1 (SAS Inst. Inc., Cary, NC). e statistical models used the following equation: where was the overall mean, was the different dietary LO, was the animal effect, and was the residual error. e random effect was the animals. Means were separated using the "PDIFF" option of the "least-squares means (LSMEANS)" statement of the MIXED procedure. Differences of 0.05 were considered to be signi�cant. Linear and quadratic contrasts were used to determine the effect of increasing amounts of LO on the response variables. e data were checked for normality using the UNIVARIATE procedure of SAS soware, and the results in the tables are presented as means ± standard error of the mean.

Composition of Experimental
Diets. e three experimental diets which were isocaloric are shown in Table 2 As expected the FA composition of linseed oil contained the highest amount of -linolenic acid and sun�ower oil contained the highest amount of linoleic acid.

FA Composition of Experimental Diets.
Oleic and linoleic acids were the predominant FA in all treatment diets, whereas the HLO diet showed a more balanced supply of the three main FA of plant origin, namely, oleic, linoleic, andlinolenic, although the latter was quantitatively the most abundant (Table 3). Table 4. e proportion of subcutaneous adipose tissue FA having 18 carbons was quite consistent across the three treatment groups, averaging between 56.77 and 58.23%. Mean concentrations of C18:0, C18:1n-9, C18:2n-6, and C18:3n-3 were 7.58, 39.72, 5.52, and 0.98%, respectively. On the other hand, C18:0 increased in a linear manner with increasing LO. e different levels of dietary LO had signi�cant ( ) effects on some of the polyunsaturated fatty acids (PUFA) in the subcutaneous adipose tissue of especially the HLO treatment group. However, oleic acid and other monounsaturated fatty acid (MUFA) were not affected by the different LO. e C18:1 trans-11 (vaccenic acid) (VA) showed a linear increase in the CON diet compared to the LLO and HLO diets. e CLA cis-9 trans-11 (rumenic acid) also showed a positive linear effect with decreased LO.

Adipose Tissue FA Composition. e FA composition of the goat subcutaneous adipose tissue fed different dietary LO is presented in
Decreasing the LO increased the C20:4n-6 ( ), while the HLO diet increased ( ) the C18:3n-3 concentration in the subcutaneous adipose tissue when compared to the CON group. e total n-3 PUFA content in the HLO diet signi�cantly increased ( ) compared to the CON diet and the n-6 : n-3 fatty acid ratios (FAR) in the subcutaneous adipose tissue of the HLO treatment group were signi�cantly ( ) decreased compared to the CON treatment group (Table 4). Incremental amounts of linseed oil in the diet resulted in dose-dependent increases in most 22-carbon PUFA in the adipose tissue. e concentrations of C22:5n-3 and C22:6n-3 were signi�cantly ( ) increased by increasing the LO in the diet.
e total SFA, MUFA, and PUFA were not affected by different dietary LO. However total n-3 PUFA increased with increasing dietary LO. In contrast, the sum of CLA isomers showed a positive linear decrease to increasing LO in the diet. e major increase in this ratio was observed in the CON diet with the highest level of n-6 : n-3 FAR (Table 4). Increasing the dietary LO by 3.96-fold (from 13.63 to 3.44; Table 2) via oil supplementation changed by almost the same ratio by 3.31fold in the subcutaneous adipose tissue from 1.59 to 0.48 (Table 4).

Adipose Tissue Gene
Expression. e relative expression of the genes in the subcutaneous adipose tissue of the HLO group compared to CON group is shown in Figures 1, 2  all treatment groups ( , Figure 1) indicating that different LO levels in the diet had no effect on the upregulation of the PPAR-gene expression. e current study showed that the LO altered the PPAR-expression in the goat tissues where increasing the LO increased the PPARexpression in the HLO and LLO treatment groups compared to the CON group. e SCD gene expression showed a signi�cant ( , Figure 3) reduction in the HLO group compared to the CON group suggesting that the SCD gene was downregulated by the HLO and LLO treatment. is effect was more pronounced in the LO group.   1 e data are expressed as the percentage of total identi�ed fatty acids. 2 SFA = sum of C10:0 + C12:0 + C14:0 + C15:0 + C16:0 + C17:0 + C18:0. 3 UFA = sum of C14:1 + C16:1 + C17:1 + C18:1n-9 + C18:2 + C18:3 + C20:4, C22:6, C20:5n-3 + C22:5-3 + C22:6n-3. 4 MUFA = sum of C14:1 + C16:1 + C17:1 + C18:1n-9. 5 n-3 PUFA = sum of C18:3n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3. 6 n-6 PUFA = sum of 18:2n-6 + 20:4n-6. 7 Total CLA = sum of CLA cis-9 trans-11 + CLA cis-12 trans-10. 8 n-6 : n-3 FAR = (C18:2n-6 + C20:4n-6) ÷ (C18:3n-3 + C20:5n-3 + C22:5n-3 + C22:6n-3). e linear increase in subcutaneous adipose tissue concentrations of C18:1 trans-11 by decreasing dietary LO may be explained by the ability of ruminal bacteria to synthesis this isomer from C18:2n-6 [12]. Harfoot et al. [31] reported that C18:1 trans-11 was the primary end product in the rumen rather than C18:0 when C18:2n-6 was supplied in higher concentrations. e concentrations of C18:2n-6 that ranged from 4.06 to 6.76% were somewhat similar to the 4.78% reported for the subcutaneous adipose tissue of Hanwoo steers fed linseed [32] but higher than the 1.43% reported in heifers fed different vegetable oils [16]. e concentration of C18:2n-6 tended to decrease ( ) with increasing dietary LO, possibly because of its partial conversion to C18:0 in the rumen. e decrease in C20:4n-6 when the dietary LO was increased to 1.30% which coincided with the major decrease in dietary concentrations of C18:2n-6 was also reported by Igarashi et al. [33] for rat adipose tissue. e quadratic increases in the concentration of n-3 PUFA (C18:3n-3, C22:5n-3, and C22:6n-3) coincided with the major increase in dietary concentration of C18:3n-3 of the HLO diet. ese increases are in agreement with the results of Kim et al. [34] who fed cattle with linseed. Jerónimo et al. [35] also reported that the concentration of C22:5n-3 and C22:6n-3 increased in ruminant intramuscular fat when they were fed high levels of linseed oil rather than sun�ower oil. e capacity of conversion of C18:3n-3 to health promoting n-3 LC-PUFA is limited in humans [36] stressing the importance for its dietary supply. Increasing the LO in the diet resulted in a partial substitution of n-6 PUFA by n-3 PUFA in membranes. A possible reason can be the competition between C18:2n-6 and C18:3n-3 for the same desaturation and elongation enzymes which affect the conversion to LC-PUFA derivatives. Generally, a positive relationship has been reported between the concentrations of dietary C18:2n-6 and adipose tissue CLA cis-9 trans-11 in grazing heifers fed diets supplemented with plant oilenriched concentrates [16]. e C18:1 trans-11 isomer is an intermediate product in the microbial biohydrogenation of dietary C18:1n-9, C18:2n-6, and C18:3n-3 [12]. e C18:1 trans-11 concentration in the subcutaneous adipose tissue, which could be converted to CLA cis-9 trans-11, tended to increase linearly with decreasing dietary LO. Likewise, the concentrations of CLA cis-9 trans-11 increased linearly ( ) as the goats consumed diets of decreasing LO, with the concentration of CLA cis-9 trans-11 increasing dramatically in goats fed the CON diet. is result may be explained by the increase in C18:2n-6 in goats fed the diet without the addition of LO, indicating that more C18:2n-6 was isomerized to CLA cis-9 trans-11 and hydrogenated to C18:1 trans-11 in the rumen of goats fed the higher C18:2n-6 leading to a higher deposition in the adipose tissue. Jerónimo et al. [35] reported that when linseed oil fed to lambs was replaced with soybean oil which contained high amounts of C18:2n-6 as the fat supplement, the concentrations of C18:1 trans-11 as well as CLA cis-9 trans-11 in intramuscular fat were increased. erefore, the synthesize of these isomers from C18:2n-6 may have been more efficient than that from C18:3n-3. At least for forage-based diets, the well-established mainstream pathway for the ruminal biohydrogenation of C18:2n-6 is straightforward with an initial isomerization with the formation of CLA cis-9 trans-11 and its reduction to C18:1 trans-11. e C18:2n-6 supplementation would therefore result in more rumen-derived CLA cis-9 trans-11 and less diverse biohydrogenation-derived FA compared with LO. e content of CLA cis-9 trans-11 in meat decreased with increasing the dietary LO, con�rming the previous results of Bessa et al. [9]. In addition Noci et al. [16] observed that the CLA cis-9 trans-11 content in intramuscular fat was higher in heifers supplemented with sun�ower oil as a source of C18:2n-6 than with LO. e n-6 : n-3 FAR is highly in�uenced by the FA composition of the diet fed to the animals [37]. Lowering the ratio of n-6 to n-3 FA in food products have been recommended to prevent or modulate certain diseases in humans [38]. e n-6 : n-3 FAR in food should range between 1 and 4 [39]. e n-6 : n-3 FAR found in the HLO treatment (2.07) was within this range. In the HLO treatment, only 48.51% of n-3 PUFA were n-3 LC-PUFA. is can be important considering that the health bene�ts of n-3 PUFA are mostly associated with the n-3 LC-PUFA as the metabolism of C18:3n-3 in humans is limited [36]. e PUFA : SFA and n-6 : n-3 FAR are indices used to evaluate the nutritional value of fat for human consumption. Increasing the PUFA content of the diet, by including sources rich in either n-6 or n-3 PUFA, generally improves the PUFA : SFA ratio [40]. is was also observed in the present trial, and in all diets where the PUFA : SFA ratio was always lower than 0.29, which is the minimum value recommended for the human diet.

Adipose Tissue Gene Expression.
Previous studies have also revealed that the PUFA activated the PPAR efficiently, although the very LC-PUFA such as erucic acid and nervonic acid and short-chain FA ( Cl0) cannot activate the PPARand PPAR-because these FA are exclusively metabolized in the peroxisomes [41]. e PPAR-responds to changes in dietary fat by activating the expression of various enzymes involved in fatty acyl CoA formation and hydrolysis, FA elongation and desaturation, and FA oxidation [42]. e signi�cant differences in the mRNA expression of PPAR-in the subcutaneous adipose tissue between the different dietary treatments suggest that the principal pathway through which FA act to modulate the expression of lipogenic genes is through the altered expression of PPAR-. Al-Hasani and Joost [43] also showed that increasing the dietary LO in the rodent diet can increase PPAR-activity in target tissues which is associated with increased insulin sensitivity. An alternative explanation is that the effects of FA on PPAR-signaling are mediated through changes in PPARactivity rather than changes in gene expression, which was not measured in this study, since FA have been shown to act as natural ligands of the PPAR-gene in other studies [44]. is study also showed that the PPAR-contributes to the regulation of subcutaneous adipose tissue gene expression. As PPAR-is related to the expression regulation of several gene-encoding proteins involved in adipocyte metabolism, it could also be a candidate gene affecting the fat deposition, including intramuscular and subcutaneous adipose tissue deposition [45]. e results of the current study clearly support that there is a relationship between gene expression of PPAR-and the intake of the n-3 PUFA. e increase in n-3 PUFA of the adipose tissues in the present study increased the PPAR-gene expression. Increasing the dietary LO might stimulate PPAR-target gene expression such as lipoprotein lipase (LPL), fatty acid transport protein (FTTP), and acyl-CoA synthase (ACS) [46,47].
To the best of our knowledge, this is the �rst study that examined the effects of different dietary LO levels on the PPAR-, PPAR-, and SCD mRNA expression in goat tissues. is study demonstrated for the �rst time that the dietary LO inhibits the expression of the gene that codes for the critical enzyme required to desaturate vaccenic acid to CLA in the goat adipose tissue. Furthermore, there is evidence from the present study that the degree of inhibition of transcription for this gene was related to the dietary LO level. e expression of SCD is known to be strongly modulated by several nutrients such as FA, carbohydrates and hormones [18,22,48], and cholesterol [49]. Similarly, Daniel et al. [50] also showed a reduction in SCD gene expression in the adipose and liver tissues of lambs fed forage compared with a concentrate-based diet. e downregulation in mRNA levels was probably due to the high concentration of C18:3n-3 in the forage compared with the concentrate diet. Alphalinolenic acid (C18:3n-3) also inhibited SCD gene expression in the mouse adipocytes [18,51]. Given that C18:3n-3 is the predominant essential FA in grass [52], these results have implications for strategies to further augment concentrations of CLA in the tissue of goats reared on pasture. Waters et al. [22] and Igarashi et al. [18] found a negative relationship between SCD gene expression and n-3 PUFA in beef cattle and the rat, respectively, which is supported by the present �nding where downregulation of SCD occurred for the HLO dietary treatment group with the highest concentration of -linolenic acid. Bellinger et al. [53] showed that feeding a mixture of n-3 PUFA, -linolenic acid (eicosapentaenoic acid), EPA, and docosahexaenoic acid (DHA) resulted in a 50% suppression of SCD mRNA in the rat liver. Nutrients, especially FA, have been shown to regulate SCD at both the enzyme activity [51] and transcriptional level [54]. In human cell lines, the transcription of SCD is under the control of two transcription factors, namely, PPAR-and PPAR- [54]. e present study also examined the gene expression of these two transcription factors as affected by the different dietary LO, and while there was downregulation of the SCD gene expression by increasing the dietary LO, the PPAR-gene expression was signi�cantly increased in the adipose tissue. Actually, the PPAR is a key regulator of SCD which mediates its transcriptional activation [55].
Nutritionists strongly recommend an increased human consumption of CLA and n-3 PUFA [56][57][58]. e CLA in human tissues may be synthesized through the tissue desaturation of vaccenic acid by SCD [59] and thus may be increased by vaccenic acid in the human diet. Results of the present study have important implications with regards to ingesting n-3 PUFA which may have negative effects on the de novo synthesis of CLA in the human muscle through potential reductions in the SCD gene expression as shown in this and many other studies. However, because a positive relationship of n-6 PUFA and particularly the n-6 : n-3 FAR with SCD gene expression exists, a correct balance of dietary n-3 PUFA appears to be of critical importance to achieve optimal SCD gene expression levels and, in turn, CLA production in the adipose tissue. A ruminant product that naturally contains CLA and n-3 PUFA could be a good regular source of these important FA and a good alternative to more expensive nutritional supplements. e present study has important implications for the establishment of dietary strategies to augment the concentration of both CLA and n-3 PUFA in ruminant tissues. However, further work is required to determine the biochemical and molecular mechanisms controlling the synthesis and deposition of n-3 PUFA and CLA in the goat tissues to optimize the effects of n-3 PUFA on the PPAR-, PPAR-, and SCD gene expression. is will provide better strategies to consistently produce nutritionally enhanced chevon.

Conclusion
Increasing the linseed oil in the goat diet had increased the total n-3 PUFA due to the high levels of C18:3n-3 in linseed oil, resulting in a reduced n-6 : n-3 FAR of the subcutaneous adipose tissue. However, the synthesis of EPA, docosapentaenoic acid (DPA), and DHA from dietary C18:3n-3 seems to be limited, and thus the EPA, DPA, and DHA enriched goat meat would contribute only a small amount compared to the recommended daily intake for human diet. e results indicate that maximum of tissue CLA cis-9 trans-11 concentration was observed with the low C18:3n-3 in the diet which contained low amounts of linseed oil and it decreased linearly by increasing the C18:3n-3.
e present study investigated how changes in the dietary FA affect the mRNA level expression of genes related to fat metabolism in the subcutaneous adipose tissue in Boer goats. e results showed that different dietary fat led to different FA pro�les in the adipose tissues and levels of PPAR-, PPAR-, and SCD gene expression. Goats fed treated OPF-based diets with high n-3 PUFA showed an upregulation of the PPARand downregulation of the SCD gene expression compared to the goats fed with low n-3 PUFA.
us, the data indicate that utilization of diets with the highest -linolenic acid is a valid approach to obtain goat meat enriched with n-3 PUFA. Increasing the n-3 PUFA in the diet using linseed oil decreased the n-6 : n-3 FAR in the subcutaneous adipose tissue of growing Boer goats. ese changes would likely improve the health status of the chevon produced.