| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
ANIMAL NUTRITION |
Division of Nutritional Biochemistry, School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire, LE12 5RD, U.K.
Abstract
Feeding sheep concentrate-based diets increases the oleic acid content of their tissues, whereas the cis-9, trans-11 conjugated linoleic acid (CLA) content is increased by feeding forage diets. Both these metabolic transformations could be attributable to increased activity of stearoyl-CoA desaturase (SCD). Therefore, the effect of forage or concentrate feeding regimens on the fatty acid composition of sheep tissues were investigated to determine whether any changes are related to an alteration of SCD mRNA levels. Twenty-four ewe lambs were randomly allotted to one of three dietary treatment groups: 1) dehydrated grass pellets, 2) concentrate diet fed to achieve a growth rate similar to that of the dehydrated grass pellets, and 3) the same concentrate diet approaching ad libitum intake. As expected, animals fed ad libitum concentrates grew at a greater (P = 0.001) rate (280 g/d) than those fed either of the other two diets (180 g/d), which were similar. In samples of liver and the three adipose tissue depots studied, the concentration of oleic acid from sheep fed either level of the concentrate diet was greater (P < 0.001) than from animals fed forage. This was associated with an increase (P < 0.05) in the ratio of SCD to acetyl-CoA carboxylase mRNA in adipose tissue and liver. Compared with concentrate-fed, the forage-fed lambs had increased (P < 0.05) levels of the cis-9, trans-11 isomer of CLA and C18:1, trans-11 in all their tissues, although the levels of SCD mRNA were lower. It therefore seems that the increased oleic acid content of sheep tissues in response to concentrate-rich diets is associated with an increase in SCD gene expression. By contrast, the increased concentration of CLA in animals fed forage-based diets is associated with an increase in substrate (C18:1 trans-11) availability.
Key Words: Conjugated Linoleic Acid • Diet • Oleic Acid • Sheep • Stearoyl-CoA Desaturase
Introduction
Lamb meat is high in saturated fatty acids (Christie, 1981
), in particular, stearic acid, which can be desaturated to oleic acid by the enzyme stearoyl-CoA desaturase (SCD; Enoch et al., 1976). Sheep have only one SCD gene (Ward et al., 1998), whose level of expression correlates well with the oleic acid content of the specific adipose tissue depot (Barber et al., 2000). Conjugated linoleic acids (CLA) are produced by the incomplete biohydrogenation of linoleic acid in the rumen and have been implicated with numerous health-promoting properties, as reviewed by Belury (2002). The cis-9, trans-11 CLA isomer may also be made endogenously from vaccenic acid (C18:1 trans-11) through the action of SCD, as speculated by Griinari et al. (2000). Thus, increasing SCD gene expression may significantly improve the nutritional quality of lamb by decreasing the saturated fatty acid content while increasing both the oleic acid, and CLA cis-9, trans-11 content of the tissue.
It is well documented that, compared with forage, feeding concentrate-based diets increases the level of oleic acid in the tissues of cattle (Hidiroglou et al., 1987; Mitchell et al., 1991; Melton et al., 1982) and sheep (Rowe et al., 1999), and we hypothesized that this is at least in part due to increased activity of SCD. Recent studies have also shown that feeding grass-based diets increases the cis-9, trans-11 CLA content of ruminant milk (Stanton et al., 1997; Kelly et al., 1998; Dhiman et al., 1999) and fat (French et al., 2000), and from the evidence reviewed by Griinari and Bauman (1999), we suggest that this may be the result of increased desaturation of C18:1 trans-11 by SCD. Both of these metabolic transformations can therefore be attributed to increased activity of SCD but are clearly in conflict with each other. In this study, sheep were fed either forage or concentrate diets and SCD mRNA levels and fatty acid composition were determined in order to investigate this paradox.
Materials and Methods
Animals
Twenty-four female Mule x Charolais lambs (78 ± 1 d, 28.7 ± 2.3 kg), which had been with their mothers at grass, were individually housed in an environmentally controlled metabolism unit with continuous access to water and a mineral block. During a 4-wk adaptation period, all animals were randomly assigned to one of three dietary treatments and fed a pelleted diet containing (as-fed basis) 485 g/kg barley, 270 g/kg soybean meal, 150 g/kg nutritionally improved straw (ground straw treated with sodium hydroxide and then pelleted at high temperature and pressure to improve its energy value; Nutrition Trading Int. Ltd., Studley, Warwickshire, U.K.), 25 g/kg vitamin and mineral mix (Frank Wright Limited, Ashbourne, Derbyshire, U.K.), 50 g/kg molassed feed meal, 10 g/kg limestone, 5 g/kg salt, and 5 g/kg fish oil before being gradually introduced to their respective trial diets. The treatment diets were 1) dehydrated grass pellets (GP), 2) concentrate diet to give a growth rate similar to that of GP (LO), and 3) the same concentrate diet approaching ad libitum intake (HI). The dehydrated grass pellets were formed from a mixture of Perennial and Italian ryegrass with a pellet diameter of 13 mm (Whatton Estates, Loughborough, Leicestershire, U.K.). The concentrate diet was a pelleted diet containing (as-fed basis) 550 g/kg barley, 350 g/kg oats, 50 g/kg molassed feed meal, 25 g/kg extracted soybean meal, and 25 g/kg mineral and vitamin mix (Frank Wright Limited). The chemical composition of the two treatment diets is shown in Table 1. The fatty acid composition of the less-abundant fatty acids (C16:0, C16:1, and C18:0) was similar between the two treatment diets. However, the dehydrated grass pellets contained a very high proportion of linolenic acid and only low proportions of oleic and linoleic acid, whereas the concentrate diet consisted mainly of oleic and linoleic acids and had a very low linolenic acid content.
|
to achieve similar growth rates for GP and LO animals and adjusted twice weekly according to the mean weight of the dietary treatment group. All animals were fed once a day at 0800, and feed refusals were noted. Seven days before the end of the experiment, all lambs were fitted with an indwelling jugular cannula. Three days later, 10-mL blood samples were collected from each animal every 2 h for 48 h, with a replacement volume of 2 mL of saline (containing 3.8 g/L of trisodium citrate) being administered after each sample was taken. The blood samples were placed in tubes containing EDTA (1.6 mg of EDTA per milliliter of blood) and centrifuged at 1,500 x g for 15 min, and the resulting plasma analyzed for insulin concentrations. Following the bleed, all the animals were slaughtered within 36 h by stunning and exsanguination. Samples of subcutaneous (tail fat), perirenal, and omental adipose tissue; liver; and longissimus dorsi muscle were rapidly removed from the left side of the carcass, frozen quickly in liquid nitrogen, and stored at -80°C. Samples of abomasal content were also taken at slaughter, and the weight of the whole perirenal depot was recorded. The carcass was skinned, and the weight of the cold carcass was recorded before splitting it in half and cutting vertically through the flank to the anterior edge of the last rib, thus dividing it into four portions. The curve of the ribs was followed to the vertebral column, which was severed at the junction of the 12th and 13th thoracic vertebrae. The following measurements were recorded from the anterior surface of this cross section: the width of the longissimus dorsi muscle (maximum distance from the end adjacent to the spinal process, outward along the rib), depth of longissimus dorsi muscle (longest distance, perpendicular, on same surface), and thickness of backfat over the deepest part of the longissimus dorsi muscle.
Determination of mRNA Levels
A 392-nucleotide NcoI-EcoR V fragment corresponding to nucleotides 622 to 1,014 of the ovine SCD cDNA was subcloned into pGEM-7zf+ (Promega, Madison, WI) and used to generate an antisense transcript. Similarly, a 438-nucleotide EcoR I-BamH I fragment of ovine acetyl co-enzyme A carboxylase (ACC) gene (nucleotides 3,854 to 4,292) was used to produce an RNA probe for ACC. Using the Riboprobe In Vitro Transcription System of Promega, with SP6 RNA polymerase and 
-32P UTP for both, ovine SCD and ACC riboprobes were generated and purified through a Sephadex G50 column. Total RNA was isolated using the acidified phenol-chloroform-guanidine thiocyanate method (Chomczynski and Sacchi, 1987), and yield and purity were determined by measurement of absorbance at 260 and 280 nm using the LKB Ultrospec (Amersham Biosciences, Piscataway, NJ). To determine the levels of SCD and ACC mRNA, aliquots of total RNA (10 µg) were used in the Ambion RPAII kit (Ambion Inc., Austin, TX) according to manufacturer instructions, and protected fragments resolved on a 6% polyacrylamide 7 M urea sequencing gel with Tris/borate/EDTA buffer. For each riboprobe, all samples from each tissue were run on the same gel and a standard sample was run on each gel to which all samples were normalized, allowing comparison between samples run on different gels. Gels were exposed to a phosphor screen (Kodak, Rochester, NY) for 2 h, and the resulting images were scanned using the Molecular Imager FX Pro Plus MultiImager System (Biorad, Hercules, CA). The intensity of individual bands was determined using Quantity One image analysis software (Biorad). Figure 1 is representative of images interpreted for the SCD and ACC mRNA data.
|
, lipid was extracted using a 2:1 chloroform:methanol solution and stored in chloroform at -20°C. To minimize isomerization of cis–trans bonds and to ensure that all fatty acids were methylated, fatty acid methyl esters (FAME) were prepared using a combined acid-base methylation based on the methods of Christie et al. (1999) and Kramer et al. (1997). For adipose tissue, which contains essentially only esterified fatty acids, a base methylation only was used (Christie et al., 1999). Separation of FAME was accomplished using a Perkin Elmer Autosystem gas chromatograph (Perkin Elmer, Norwalk, CT) equipped with a flame ionization detector, and a 100 m x 0.25 mm i.d. capillary column with a 0.2-µm film thickness (CP-Sil 88; Varian Inc., Walnut Creek, CA) and helium as a carrier gas. The initial oven temperature was held at 170°C for 50 min and then increased at 25°C/min to 240°C, and held for 10 min. Injector and detector temperatures were both 255°C with a split ratio of 50:1. Peaks in the chromatograms were identified using pure methyl ester standards (Sigma, St. Louis, MO). Data are expressed as moles/100 moles FAME of all fatty acids identified, and the molecular weight of methyl oleate was used for all unidentified fatty acids as this was the region in which most were found.
Determination of Adipocyte Volume and Adipocyte Number
Mass of lipid per gram of adipose tissue was calculated by oven-drying the extract from a chloroform/methanol extraction (Folch et al., 1957) of a known amount of tissue (approximately 200 mg). Adipocyte volume was estimated using the method of Pond et al. (1984), in which five thin slices (<0.25g) of adipose tissue were placed in a drop of PBS on a microscope slide and the coverslip pressed down lightly to compress the tissue. The tissue was then examined under a microscope (Nikon, Melville, NY/Nikon U.K. Ltd., Kingston upon Thames, Surrey, U.K.) and camera (JVC, Yokohama, Japan) that was linked to a computer with an image capture and measurement software package (Optimas-6, Bothell, WA). The software was used to measure the diameters of 50 individual intact adipocytes, and, as adipocytes were assumed to be spherical, the diameter was used to determine the volume of the cells. The number of adipocytes per gram of adipose tissue was calculated from the mean adipocyte volume, total lipid per gram, and an assumed lipid density of 0.915 g/mL (Taylor et al., 1973; Dawson et al., 1993).
Insulin Analysis
The insulin concentrations of the plasma samples taken during the 48-h bleed were analyzed using the Access Ultrasensitive Insulin one-step immunoenzymatic assay (Beckman Coulter Inc., Fullerton, CA).
Statistical Analysis
Results were expressed as means using the combined data from each dietary treatment group, and data was analyzed by ANOVA using Genstat release 6.1 for Windows (Lawes Agricultural Trust, Hertfordshire, U.K.). Data were considered significant when P < 0.05. To test whether any effect of dietary treatment differed among adipose tissue depots, data were analyzed as a two-way split-plot ANOVA with diet as the main plot factor and depots within the same animal as the subplot factor. If dietary treatment or interaction effects were significant, mean separation was conducted using a Bonferroni multiple comparison test, with an -level of 5% used to detect significant differences. When a significant dietary treatment x adipose tissue depot (diet x depot) interaction was determined, mean comparisons were only made between dietary treatments within an adipose tissue depot. The relationship between C18:1, trans-11 and CLA, cis-9, trans-11 was determined by fitting a line through the data and using general linear regression, including diet and depot in the model, to make comparisons between regressions for different diets and depots. Effects of general linear regression were tested by ANOVA. The insulin concentrations from the blood samples collected during the 48-h bleed were analyzed using REML to compare the effects of diet over time.
Results
Animal Performance and Carcass Characteristics
As expected, growth rates of the animals fed GP or LO were not different, but animals fed HI grew at a greater (P = 0.001) rate (Table 2). Carcass weight differed (P < 0.001) between animals fed different diets; animals fed GP or LO had lighter carcasses than animals fed HI. Perirenal adipose depot weight was greater (P < 0.001) in carcasses of animals fed HI than GP or LO, which were similar. Measurement of backfat depth differed with dietary treatment (P < 0.001) with animals fed GP having the lowest backfat depth, and animals fed HI having the greatest backfat depth, which did not differ from those fed LO. However, there were no effects of diet in the width (P = 0.29) or depth (P = 0.24) of the longissimus dorsi muscle.
|
). This resulted in animals fed HI having fewer cells per gram of tissue for all three adipose depots (P < 0.001). Effects of depot were also observed in adipocyte volume and cells per gram (P < 0.001 for both), with subcutaneous adipose tissue having larger adipocyte volumes and therefore fewer cells per gram than the two internal depots.
|
. Diet altered the concentrations of palmitic, oleic, linoleic, and linolenic acids (P < 0.001 for all); animals fed GP had lower concentrations of palmitic, oleic, and linoleic acid, and greater concentrations of linolenic acid, than detected in samples from animals fed both concentrate diets, which did not differ. Lambs fed GP also had lower (P < 0.001) concentrations of C18:1 trans-10 and greater (P < 0.001) concentrations of C18:1 trans-11 than concentrate-fed lambs. The concentrations of cis-9, trans-11 and trans-10, cis-12 CLA were unaffected by dietary treatment.
|
. A diet x depot interaction (P < 0.001) for palmitic acid content was detected. In both the omental and perirenal depots, lambs fed GP had less palmitic acid in their samples than those fed either concentrate diet, although the differences were not as pronounced in the subcutaneous depot. Palmitoleic acid concentrations differed (P < 0.001) between depots, as subcutaneous contained the most and perirenal, the least. Dietary differences (P < 0.001) were also detected in the concentrations of palmitoleic acid as from lambs fed LO contained greater concentrations than those fed either GP or HI, which differed only in the perirenal depot. Statistical analysis showed that the effect of diet on stearate concentrations differed with depot (P = 0.02) because only in the subcutaneous depot did tissue from lambs fed HI contain less stearic acid than from lambs fed GP, and the subcutaneous depot contained the least stearic acid. An effect of diet and depot (P < 0.001 for both) was also seen in the concentrations of oleic acid. Tissue from animals fed concentrate-rich diets had greater amounts of oleic acid than from animals fed GP, and the subcutaneous depot had more oleic acid than the two internal depots. For both linoleic and linolenic acid, there were diet x depot interactions (P < 0.001 for both). Animals fed LO had greater concentrations of linoleic than those fed GP or HI, except for in the omental depot, where the concentrations in animals fed HI were also greater than those in animals fed GP. Compared with animals fed both concentrate diets, animals fed GP had greater concentrations of linolenic acid in all adipose tissue depots. The subcutaneous depot had the lowest (P < 0.001) concentrations of linoleic acid. Lambs fed GP had greater concentrations of linolenic acid than animals fed concentrate, and the concentration of linolenic acid was greatest in perirenal tissue in lambs fed GP, and in omental tissue in lambs fed concentrate (diet x depot interaction, P < 0.001).
|
Liver and Muscle Fatty Acid Composition
The fatty acid composition of liver and longissimus dorsi muscle is shown in Table 6. The total lipid amounts for longissimus dorsi muscle samples from animals on the three dietary treatments were 48.6, 51.8, and 56.7 mg lipid/g of muscle (wet weight) for GP, LO, and HI respectively (P = 0.403, the standard error of the differences of the means = 5.93). There was an effect of diet on the concentrations of both palmitic (P < 0.001) and palmitoleic (P < 0.01) acid concentration of liver samples. Animals fed GP and HI had lower concentrations of palmitic acids compared with animals fed LO, whereas the concentrations of palmitoleic acid were greater in animals fed both concentrate diets compared with those fed GP. No differences in palmitic or palmitoleic acid concentrations of the longissimus dorsi muscle between animals on the different diets were observed. For samples of liver, stearic acid concentrations were higher (P < 0.001) in animals fed GP compared with LO or HI, which did not differ. In the longissimus dorsi muscle, stearic acid concentrations were greatest (P < 0.01) for LO and lowest for HI, neither of which were different from animals fed GP. There was an effect of diet on liver oleic acid concentration (P < 0.001); liver from lambs fed GP had lower concentrations of oleic acid than from lambs fed either concentrate diet, which did not differ. Although there was an effect (P < 0.001) of diet in longissimus dorsi muscle, concentrations of oleic acid were greater in samples from animals fed HI, as the concentration of oleic acid in samples from animals fed GP and LO did not differ. In both liver (P < 0.001) and longissimus dorsi muscle (P = 0.04), there were effects of diet on concentrations of linoleic and linolenic acid; samples from animals fed GP had lower concentrations of linoleic acid and greater concentrations of linolenic acid than samples from animals fed concentrate diets.
|
For liver and longissimus dorsi muscle, there was an effect (P < 0.001) of diet on the concentration of CLA cis-9, trans-11. Lambs fed GP had more liver CLA cis-9, trans-11 than animals fed either concentrate diet, which did not differ, and lambs fed GP contained more CLA cis-9, trans-11 in their longissimus dorsi samples than those fed HI. The CLA cis-9, trans-11 concentration of liver from animals fed LO was also lower than animals fed GP. There were, however, no significant effects of diet on the concentrations of CLA trans-10, cis-12 in samples from either longissimus dorsi muscle or liver.
Correlation Between C18:1 trans-11 and Conjugated Linoleic Acid cis-9 trans-11
The relationships between the concentrations of C18:1 trans-11 and conjugated linoleic acid cis-9, trans-11 in liver and the three adipose tissue depots studied are shown in Figure 2. There was no statistical evidence that the correlation between these two fatty acids differed between diets or that any effects of diet differed with depot. However, there was linear trend (P < 0.001) between the concentration of C18:1 trans-11 and conjugated linoleic acid cis-9, trans-11 and this correlation was shown to differ between tissue. Although there were similar concentrations of C18:1 trans-11 in all four tissues, the accumulation of conjugated linoleic acid cis-9, trans-11 appears to be far greater in the liver.
|
). There was a diet x depot interaction (P = 0.02) on SCD mRNA levels. In both subcutaneous and omental adipose tissue, samples from lambs fed HI had greater levels of SCD mRNA than from those fed GP or LO, which did not differ. However, in the perirenal depot, although samples from animals fed HI were greater than samples from animals fed GP, neither were different from LO. The abundance of SCD mRNA was greatest in the subcutaneous adipose tissue depot, whereas the levels in omental and perirenal adipose tissue did not differ. There were also effects of dietary treatment (P < 0.01) and depot (P < 0.01) on the levels of ACC mRNA, with samples from animals fed HI having the greatest abundance of ACC mRNA and the perirenal depot having the lowest levels. Because the adipose tissue and liver mRNA levels are expressed in different units, it is not possible to compare differences in mRNA levels between the tissues. However, in the liver, there was an effect of diet (P = 0.04), with samples from animals fed LO containing greater levels of SCD mRNA than from animals fed GP (Table 8). However, there were no effects of diet on liver ACC mRNA levels.
|
|
Insulin Profile
We have previously shown that insulin significantly increases the expression of ovine SCD and the synthesis of monounsaturated fatty acids from acetate in cultured ovine adipose tissue explants (Daniel et al., 2004). Insulin concentrations were therefore measured in samples from this trial to determine whether any dietary effects were related to changes in the animals’ insulin levels. Insulin concentrations in plasma samples from animals fed the three dietary treatments were different over time (P < 0.001), as shown in Figure 3. Lambs fed GP and LO had similar insulin profiles—with a peak in the insulin concentration approximately 2 h after feeding, followed by a rapid decrease—and returned to the concentration observed before feeding for the remainder of the day. However, the pattern of insulin concentrations in the plasma of sheep fed HI appeared to be constantly high throughout the day.
|
The present study confirms that feeding a concentrate-rich diet increases the oleic acid content of ruminant adipose tissue, liver, and muscle, whereas a forage-based diet increases the relative concentrations of cis-9, trans-11 CLA in these tissues. The aim of this study was to investigate how oleic acid and cis-9, trans-11 CLA, both potential products of the enzyme SCD, can apparently have divergent responses to diet.
Most studies examining the effect of forage or concentrate feeding on the fatty acid profiles of ruminant fat have compared grass with ad libitum concentrate feeding (Hidiroglou et al., 1987; Mitchell et al., 1991). Cattle that had consumed only grass have been compared with those fed grass and then fattened on a concentrate-based diet for up to 140 d after the grass-only animals had been slaughtered (Westerling and Hedrick, 1979; Melton et al., 1982; Duckett et al., 1993), and, in such studies, differences in tissue fatty acid composition were attributed to type of diet. However, because of the greater energy intake or age differences associated with the concentrate rations, concentrate-fed animals were heavier with fatter carcasses, and so changes in age and fatness may have confounded the effects of ration on the fatty acid composition of tissues. In this study, two groups of lambs were fed forage or concentrate diets with the same level of energy intake, and a third group was fed concentrates ad libitum. At the beginning of the trial, lambs were matched for age and body weight, and all animals were fed the treatment diets for the same length of time before slaughter.
In addition to being produced by the action of SCD, oleic acid deposited in the tissues of the animals could also be derived from the diet. Indeed, the concentrate diets contained considerably more oleic acid than the forage diet (Table 1
). Although a considerable proportion of this will be hydrogenated in the rumen, the portion of oleic acid found in the abomasal fluid of concentrate-fed animals was still considerably greater that those fed the forage-based diet. However, three lines of evidence suggest that differences in dietary fatty acid composition are not primarily responsible for the changes we see in oleic acid content of the tissues. Firstly, the animals fed the HI diet were taking in approximately 85% more dietary fat, and hence oleic acid, than those fed the LO diet. However, no significant difference was seen in the effect of these two diets on the proportion of oleic acid in the tissue. Secondly, the amount of linoleic acid in the abomasal fluid of concentrate-fed animals is comparable to that of oleic acid, but the changes in adipose tissue concentration are much less (Table 5). Finally, the abomasal concentration of oleic acid (as a percentage of other fatty acids) is far below that seen in tissues and the percentage of a dietary fatty acid reaching the small intestine must exceed adipose tissue levels before it can affect concentrations within the tissue. Thus, although dietary intake may make some contribution to the greater oleic acid content of tissues from concentrate-fed animals, we do not believe it is the major factor.
The alternative explanation is that there is an increase in the de novo production of oleic acid in the tissues of the animals. Stearoyl-CoA desaturase catalyses the formation of oleic acid from stearic acid. The latter can be synthesized de novo in the tissues or can be derived from the diet. The conversion of acetyl-CoA to malonyl-CoA, which is catalyzed by the ACC, is the first committed step in the process of fatty acid production and thought to be the major control point in the synthesis of palmitic acid (C16:0; Vernon, 1992). This can then be elongated to produce stearic acid (C18:0) before finally being desaturated to form oleic acid by SCD. It is this final step that is thought to be rate limiting in the production of oleic acid (Enoch et al., 1976; St. John et al., 1991). Previous work from our laboratories indicates that SCD steady-state mRNA concentrations correlate well with the oleic acid content of tissues and that subcutaneous adipose tissue has greater SCD mRNA levels than internal depots (Barber et al., 2000). Data from this study are consistent with these findings because the subcutaneous depot had the highest levels of both SCD mRNA and oleic acid, compared with the two internal depots studied. Furthermore, we have previously shown that when ovine adipose tissue explants are treated with insulin there are increases in both SCD and ACC mRNA concentrations but that the effect on the former is greatest (Daniel et al., 2004). As a result, although there was an increase in overall lipogenesis, the proportion of oleic acid produced increased. We therefore believe that the ratio of SCD to ACC mRNA is a good index of the ability of tissues to produce oleic acid. In the present study, although adipose tissue SCD and ACC mRNA levels were increased by concentrate feeding, the ratio of SCD to ACC mRNA increased. In liver, only SCD mRNA was increased, resulting in a dramatic increase in this ratio. Although it is assumed that most lipogenesis will be occurring in the adipose tissue (Vernon, 1992), it is still possible that the liver plays an important role in the remodeling of fatty acids. Thus, our data are consistent with the suggestion that increased oleic acid content of ruminant tissues in response to concentrate-rich diets is, at least in part, the result of increased SCD activity.
To date, few studies have focused on the control of SCD in ruminants, and, although Martin et al. (1999) measured SCD gene expression, most of the work studying the effect of diet has been in cattle and has looked at enzyme activity (Page et al., 1997; Chang et al., 1992; Yang et al., 1999). As mentioned above, we have previously shown that insulin specifically increases SCD mRNA concentrations and monounsaturated fatty acid synthesis in sheep adipose tissue. However, this does not seem to be the mechanism by which concentrate-rich diets are acting, as the insulin profiles were essentially identical between the GP and LO groups whereas SCD mRNA concentrations were greater in the latter. It is possible that SCD mRNA levels are increased with concentrate feeding in response to some component of the concentrate diet that is not present in the forage diet. Work in rodents has shown that polyunsaturated fatty acids inhibit liver SCD gene expression (Ntambi, 1992; Landschulz et al., 1994). More specifically, in mouse embryo 3T3-L1 adipocytes, Sessler et al. (1996) showed that both linoleic and linolenic acid inhibit SCD1 expression, and the level of repression by linolenic acid was greater than that of linoleic acid. In both the dehydrated grass pellets themselves and the tissues of animals fed them, linolenic acid predominated over linoleic acid, whereas, in the concentrate diet, and in the tissue of animals fed both levels, linoleic acid was more abundant. The data indicate that the differences in SCD/ACC ratios between the forage and concentrate diets could also be a result of a greater inhibition of SCD expression by the linolenic acid-rich dehydrated grass pellets diet compared with the concentrate diet.
Ruminant fats are among the richest natural sources of CLA isomers, in particular the cis-9, trans-11 isomer (Chin et al., 1992), and data from this trial show that animals fed forage had more cis-9, trans-11 CLA in their tissues than animals fed either level of the concentrate diet. This supports findings, in both dairy and beef cattle, that feeding grass-based diets increases the levels of the cis-9, trans-11 isomer of CLA in milk (Stanton et al., 1997; Kelly et al., 1998; Dhiman et al., 1999) and intermuscular fat (French et al., 2000). trans-10, cis-12 Conjugated linoleic acid was detected, but the levels were far lower than that of the cis-9, trans-11 isomer, again supporting findings in beef (Chin et al., 1992; French et al., 2000).
Whereas tissue concentrations of cis-9, trans-11 CLA differed between diets, no such difference was apparent in the abomasal contents. However, significant differences were seen in the nature of the trans monounsaturated fatty acids leaving the rumen, with animals fed forage producing predominantly C18:1 trans-11 whereas animals fed concentrates produced C18:1 trans-10. It is now clear that a major proportion of the cis-9, trans-11 CLA found in ruminant tissues is formed through the action of SCD on trans-11 C18:1 (Griinari et al., 2000). Paradoxically, however, in the present study, the diet that produced the highest level of CLA also suppressed the expression of SCD. We suggest that the high substrate concentration (trans-11 C18:1) associated with the forage-based diet is responsible for the increased cis-9, trans-11 CLA production and more than compensates for the reduction in enzyme expression. The correlation between C18:1 trans-11 and CLA cis-9 trans-11 (Figure 2) is consistent with this suggestion.
Thus, the present study confirms that concentrate-rich diets increase oleic acid content, whereas forage-based diets increase the cis-9, trans-11 content of ruminant tissues. It also suggests mechanisms whereby both these findings may be mediated through the action of SCD. In the case of oleic acid, the concentrate diet increases expression of the enzyme, whereas, in the case of CLA, the grass-based diet results in the formation of more substrate for conversion to CLA.
Implications
Increasing the conjugated linoleic acid and unsaturated fat content of ruminant meat could improve its nutritional quality. It has been speculated that increasing stearoyl-CoA desaturase levels could achieve both these objectives. From the data obtained, it is suggested that in sheep tissues the oleic acid content depends on stearoyl-CoA desaturase mRNA levels, whereas conjugated linoleic acid production is substrate driven. This has implications in development of strategies to manipulate the conjugated linoleic acid and oleic acid content of ruminant meat.
Footnotes
1 Z. C. T. R. Daniel was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) studentship, and R. J. Wynn was funded by a BBSRC CASE studentship with Pfizer. 
2 The authors wish to acknowledge M. C. Barber, M. T. Travers, and R. G. Vernon (Hannah Research Institute, Ayr, KA6 5HL, U.K.) for kindly donating the SCD and ACC expression vectors and J. Craigon (University of Nottingham) for statistical advice.
3 Correspondence—phone: 44 115 9516121; fax: 44 115 9516122; e-mail: peter.buttery{at}nottingham.ac.uk.
Received for publication August 14, 2003. Accepted for publication November 6, 2003.
Literature Cited
Barber, M. C., R. J. Ward, S. E. Richards, A. M. Salter, P. J. Buttery, R. G Vernon, and M. T. Travers. 2000. Ovine adipose tissue monounsaturated fat content is correlated to depot-specific expression of the stearoyl-CoA desaturase gene. J. Anim. Sci. 78:62–68.
Belury, M. A. 2002. Dietary conjugated linoleic acid in health: Physiological effects and mechanism of action. Annu. Rev. Nutr. 22:505–531.[Medline]
Chang, J. H. P., D. K. Lunt, and S. B. Smith. 1992. Fatty acid composition and fatty acid elongase and stearoyl CoA desaturase activities in tissues of steers fed high oleate sunflower seed. J. Nutr. 122:2074–2080.
Chin, S. F., W. Liu, J. M. Storkson, Y. L. Ha, and W. M. Pariza. 1992. Dietary sources of conjugated dienoic isomers of linoleic acid, a newly recognized class of anticarcinogens. J. Food Compos. Anal. 5:185–197.
Chomczynski, P., and N. Sacchi. 1987. Single step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Anal. Biochem. 162:156–159.[Medline]
Christie, W. W. 1981. The composition, structure and function of lipids in the tissues of ruminant animals. Pages 95–191 in Lipid Metabolism in Ruminant Animals. W. W. Christie, ed. Pergamon Press, Oxford, U.K.
Christie, W. W., J. L. Sebedio, and P. Juaneda. 1999. Methodology test for total CLA and CLA isomers. Pages 1–6 in CLA: What’s going on? No. 3. Centre de Recherche et d’Information Nutritionnelles, Paris.
Daniel, Z. C. T. R., S. E. Richards, A. M. Salter, and P. J. Buttery. 2004. Insulin and dexamethasone regulate stearoyl-CoA desaturase mRNA levels and fatty acid synthesis in ovine adipose tissue. J. Anim. Sci. 82:231–237.
Dawson, J. M., C. P. Essex, A. Walsh, D. E. Beever, M. Gill, and P. J. Buttery. 1993. Effect of fishmeal supplementation and beta-agonist administration on adipose tissue metabolism in steers given silage. Anim. Prod. 57:397–406.
Dhiman, T. R., G. R Anand, L. D. Satter, and M. W. Pariza. 1999. Conjugated linoleic acid content of milk from cows fed different diets. J. Dairy Sci. 82:2146–2156.[Abstract]
Duckett, S. K., D. G. Wagner, L. D. Yates, H. G. Dolezal, and S. G. May. 1993. Effects of time on feed on beef nutrient composition. J. Anim. Sci. 71:2079–2088.[Abstract]
Enoch, H. G., A. Catala, and P. Strittmater. 1976. Mechanism of rat liver microsomal stearoyl-CoA desaturase. J. Biol. Chem. 251:5095–5103.
Folch, J., M. Lee, and G. H. Sloan Stanley. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497–509.
French, P., C. Stanton, F. Lawless, E. G. O’Riordan, F. J. Monahan, P. J. Caffrey, and A. P. Moloney. 2000. Fatty acid composition, including conjugated linoleic acid, of intramuscular fat from steers offered grazed grass, grass silage or concentrate-based diets. J. Anim. Sci. 78:2849–2855.
Griinari, J. M., and D. E. Bauman. 1999. Biosynthesis of conjugated linoleic acid and its incorporation into meat and milk in ruminants. Pages 180–200 in Advances in Conjugated Linoleic Acid Research. Vol. 1. M. P. Yurawecz, M. M. Mossoba, J. K. G. Kramer, M. W. Pariza, and G. J. Nelson, ed. AOCS Press, Champaign, IL.
Griinari, J. M., B. A. Corl, S. H. Lacy, P. Y. Chouinard, K. V. V. Nurmela, and D. E. Bauman. 2000. Conjugated linoleic acid is synthesized endogenously in lactating dairy cows by 
9-desaturase. J. Nutr. 130:2285–2291.
Hidiroglou, N., L. R. McDowell, and D. D. Johnson. 1987. Effect of diet on animal performance, lipid composition of subcutaneous adipose tissue and liver tissue of beef cattle. Meat Sci. 20:195–210.
Kelly, M. L., E. S. Kolver, D. E. Bauman, M. E. Van Amburgh, and L. D. Muller. 1998. Effect of intake of pasture on concentrations of conjugated linoleic acid in milk of lactating cows. J. Dairy Sci. 81:1630–1636.[Abstract]
Kramer, J. K. G., V. Fellner, M. E. R. Dugan, F. D. Sauer, M. M. Mossoba, and M. P. Yurawecz. 1997. Evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special emphasis on conjugated dienes and total trans fatty acids. Lipids 32:1219–1228.[Medline]
Landschulz, K. T., D. B. Jump, O. A. Macdougald, and M. D. Lane. 1994. Transcriptional control of the stearoyl CoA desaturase 1 gene by polyunsaturated fatty acids. Biochem. Biophys. Res. Commun. 200:763–768.[Medline]
Martin, G. S., D. K. Lunt, K. G. Britain, and S. B. Smith. 1999. Postnatal development of stearoyl coenzyme A desaturase gene expression and adiposity in bovine subcutaneous adipose tissue. J. Anim. Sci. 77:630–636.
Melton, S. L., J. M. Black, G. W. Davis, and W. R. Backus. 1982. Flavor and selected chemical components of ground beef from steers backgrounded on pasture and fed corn up to 140 days. J. Food Sci. 47:699–704.
Ministry of Agriculture, Fisheries, and Food. 1975. Energy Allowances and Feeding Systems for Ruminants. Tech. Bull. 33. Her Majesty’s Stationery Office, London.
Mitchell, G. E., A. W. Reed, and S. A. Rogers. 1991. Influence of feeding regime on the sensory qualities and fatty acid contents of beef steaks. J. Food Sci. 56:1102–1103.
Ntambi, J. M. 1992. Dietary regulation of stearoyl-CoA desaturase 1 gene expression in mouse liver. J. Biol. Chem. 267:10925–10930.
Page, A. M., C. A. Sturdivant, D. K. Lunt, and S. B. Smith. 1997. Dietary whole cottonseed depresses lipogenesis but has no effect on stearoyl coenzyme desaturase activity in bovine subcutaneous adipose tissue. Comp. Biochem. Physiol. B 118:79–84.[Medline]
Pond, C. M., C. A. Mattacks, and D. Sadler. 1984. The effects of food restriction and exercise on site-specific differences in adipocyte volume and adipose tissue cellularity in the guinea pig. 1. Superficial and intra abdominal sites. Br. J. Nutr. 51:415–424.[Medline]
Rowe, A., F. A. F. Macedo, J. V. Visentainer, N. E. Souza, and M. Matsushita. 1999. Muscle composition and fatty acid profile in lambs fattened in dry lot or pasture. Meat Sci. 51:283–288.
Sessler, A. M., N. Kaur, J. P. Palta, and J. M. Ntambi. 1996. Regulation of stearoyl-CoA desaturase 1 mRNA stability by polyunsaturated fatty acids in 3T3-L1 adipocytes. J. Biol. Chem. 271:29854–29858.
Stanton, C., F. Lawless, G. Kjellmer, D. Harrington, R. Devery, J. F. Connolly, and J. Murphy. 1997. Dietary influences on bovine milk cis-9, trans-11-conjugated linoleic acid content. J. Food Sci. 62:1083–1086.
St. John, L. C., D. K. Lunt, and S. B. Smith. 1991. Fatty acid elongation and desaturation enzyme activities of bovine liver and subcutaneous adipose tissue microsomes. J. Anim. Sci. 69:1064–1073.[Abstract]
Taylor, A. W., J. Garrod, M. E. McNulty, and D. C. Secord. 1973. Regenerating epididymal fat pad size and number after exercise training and three different feeding patterns. Growth 37:345–354.[Medline]
Vernon, R. G. 1992. Control of lipogensis. Pages 59–81 in the Control of Fat and Lean Deposition. P. J. Buttery, K. N. Boorman, and D. B. Lindsay, ed. Butterworth Heineman, Oxford, U.K.
Ward, R. J., M. T. Travers, S. E. Richards, R. G. Vernon, A. M. Salter, P. J. Buttery, and M. C. Barber. 1998. Stearoyl-CoA desaturase mRNA is transcribed from a single gene in the ovine genome. Biochim. Biophys. Acta 1391:145–156.[Medline]
Westerling, D. B., and H. B. Hedrick. 1979. Fatty acid composition of bovine lipids as influenced by diet, sex and anatomical location and relationship to sensory characteristics. J. Anim. Sci. 48:1343–1348.
Yang, A., T. W. Larsen, S. B. Smith, and R. K. Tume. 1999. Delta(9) desaturase activity in ovine subcutaneous adipose tissue of different fatty acid composition. Lipids 34:971–978.[Medline]
This article has been cited by other articles:
|
|
 |
|
  L. Faucitano, P. Y. Chouinard, J. Fortin, I. B. Mandell, C. Lafreniere, C. L. Girard, and R. Berthiaume Comparison of alternative beef production systems based on forage finishing or grain-forage diets with or without growth promotants: 2. Meat quality, fatty acid composition, and overall palatability J Anim Sci, July 1, 2008; 86(7): 1678 - 1689. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Gomez-Cortes, P. Frutos, A. R. Mantecon, M. Juarez, M. A. de la Fuente, and G. Hervas Milk Production, Conjugated Linoleic Acid Content, and In Vitro Ruminal Fermentation in Response to High Levels of Soybean Oil in Dairy Ewe Diet J Dairy Sci, April 1, 2008; 91(4): 1560 - 1569. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. W. Hess, G. E. Moss, and D. C. Rule A decade of developments in the area of fat supplementation research with beef cattle and sheep J Anim Sci, April 1, 2008; 86(14_suppl): E188 - E204. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Cruz-Hernandez, J. K. G. Kramer, J. J. Kennelly, D. R. Glimm, B. M. Sorensen, E. K. Okine, L. A. Goonewardene, and R. J. Weselake Evaluating the Conjugated Linoleic Acid and Trans 18:1 Isomers in Milk Fat of Dairy Cows Fed Increasing Amounts of Sunflower Oil and a Constant Level of Fish Oil J Dairy Sci, August 1, 2007; 90(8): 3786 - 3801. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Pavan and S. K. Duckett Corn oil supplementation to steers grazing endophyte-free tall fescue. II. Effects on longissimus muscle and subcutaneous adipose fatty acid composition and stearoyl-CoA desaturase activity and expression J Anim Sci, July 1, 2007; 85(7): 1731 - 1740. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Fukuda, Y. Suzuki, M. Murai, N. Asanuma, and T. Hino Augmentation of vaccenate production and suppression of vaccenate biohydrogenation in cultures of mixed ruminal microbes. J Dairy Sci, March 1, 2006; 89(3): 1043 - 1051. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. J. Shingfield, C. K. Reynolds, G. Hervas, J. M. Griinari, A. S. Grandison, and D. E. Beever Examination of the Persistency of Milk Fatty Acid Composition Responses to Fish Oil and Sunflower Oil in the Diet of Dairy Cows J Dairy Sci, February 1, 2006; 89(2): 714 - 732. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. L. Palmquist, N. St-Pierre, and K. E. McClure Tissue Fatty Acid Profiles Can Be Used to Quantify Endogenous Rumenic Acid Synthesis in Lambs J. Nutr., September 1, 2004; 134(9): 2407 - 2414. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
