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Fatty acid indices of stearoyl-CoA desaturase do not reflect actual stearoyl-CoA desaturase enzyme activities in adipose tissues of beef steers finished with corn-, flaxseed-, or sorghum-based diets¹

S. L. Archibeque^*, D. K. Lunt^*, C. D. Gilbert^*, R. K. Tume and S. B. Smith^*,2

^* Department of Animal Science, Texas A&M University, College Station, 77843; and and Food Science Australia, Brisbane Laboratory, Tingalpa D. C. Queensland 4173, Australia

	Abstract

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We hypothesized that stearoyl-CoA desaturase (SCD) enzyme activity would not correlate with fatty acid indices of SCD activity in steers fed different grains. Forty-five Angus steers (358 ± 26 kg BW) were individually fed for 107 d diets differing in whole cottonseed (WCS) supplementation (0, 5, or 15% of DM) and grain source (rolled corn, flaxseed plus rolled corn, or ground sorghum grain) in a 3 x 3 factorial arrangement. Flaxseed- and corn-fed steers had greater (P < 0.01) G:F (0.119 and 0.108, respectively) than sorghum-fed steers (0.093). Marbling score was decreased by WCS (P = 0.04), and LM area was decreased (P < 0.01) by sorghum. Plasma 14:0, 16:0, 16:1n-7, and 18:2n-6 were greatest in corn-fed steers, whereas plasma 18:3n-3 and 20:5n-3 were greatest in the flax-seed-fed steers (P < 0.01). Plasma 18:1trans-11 was least in sorghum-fed steers, and plasma cis-9,trans-11 CLA was barely detectable, in spite of high intestinal mucosal SCD enzyme activity (118 to 141 nmol•g tissue^–1•7 min^–1). Interfascicular (i.f.) and s.c. cis-9,trans-11 CLA remained unchanged (P 0.25) by treatment, although 18:1trans-11 was increased (P 0.02) in steers fed corn or flaxseed. Steers fed flaxseed also had greater (P < 0.01) i.f. and s.c. concentrations of 18:3n-3 than steers fed the other grain sources. Oleic acid (18:1n-9) was least and total SFA were greatest (P < 0.01) in i.f. adipose tissue of steers fed 15% WCS. Lipogenesis from acetate in s.c. adipose tissue was greater (P < 0.01) in flaxseed-fed steers than in the corn- or sorghum-fed steers. Steers fed flaxseed or corn had larger i.f. mean adipocyte volumes (P < 0.01) than those fed sorghum and tended (P = 0.07) to have larger s.c. adipocyte volumes. Several fatty acid indices of SCD enzyme activity were decreased (P 0.03) by WCS in i.f. adipose tissue, including the 18:2cis-9,trans-11/ 18:1trans-11 ratio. The 18:2cis-9,trans-11/18:1trans-11 ratio also tended to be decreased (P = 0.09) in s.c. adipose tissue by flaxseed; however, SCD enzyme activities in i.f. and s.c. adipose tissue were not affected by dietary WCS (P 0.47) or grain source (P 0.37). Differences in SFA seemed to be independent of SCD enzyme activity in both adipose tissues, suggesting that duodenal concentrations of fatty acids were more important in determining tissue fatty acid concentrations than endogenous desaturation by SCD.

Key Words: Adipose Metabolism • Conjugated Linoleic Acid • Linolenic Acid • Stearoyl Coenzyme A Desaturase • Steers

	Introduction

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The expression of stearoyl-CoA desaturase (SCD) is associated with adipocyte hypertrophy in a number of species (Martin et al., 1999; Smith et al., 1999; Cohen et al., 2002). Therefore, depressing SCD enzyme activity during growth may decrease carcass adiposity. Whole cottonseed (WCS) contains the cyclopropene fatty acid, sterculic acid, which is a potent inhibitor of SCD (Raju and Reiser, 1972; Smith et al., 1998; Yang et al., 1999). Additionally, the cis-9,trans-11 (rumenic acid) and trans-10,cis-12 isomers of CLA inhibit SCD gene expression and enzyme activity posttranslationally (Choi et al., 2001, 2002). Linoleic acid (18:2n-6) and -linolenic acid (18:2n-3) may serve as precursors of rumenic acid as a direct result of isomerization within the rumen (Ward et al., 1964; Wilde and Dawson, 1966). Alternatively, rumenic acid may be derived from desaturation of vaccenic acid (18:1trans-11) postruminally (Santora et al., 2000; Palmquist et al., 2004). Therefore, we provided ruminal and endogenous CLA precursors in a variety of grain sources, in addition to three dietary levels of WCS (0, 5, and 15% of DM), to test the interaction between WCS and CLA on SCD enzyme activity and carcass adiposity in feedlot steers.

The combination of WCS and different grain sources also allowed us to test the validity of calculated fatty acid indices of SCD activity for estimating actual SCD enzyme activity. We hypothesized that, in cattle fed grain sources differing in fatty acid composition, calculated SCD indices would fail to appropriately predict actual SCD enzyme activities in interfascicular (i.f.) and s.c. adipose tissues.

	Materials and Methods

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Animals and Experimental Procedures
Forty-five Angus steers (358 ± 26 kg BW) selected from local livestock auctions were used in this experiment; care, handling and sampling of the steers was approved by the Texas A&M University Institutional Animal Care and Use Committee (AUP No. 2001-75). This study was conducted during spring and summer 2001 at the Texas A&M University Research Center at McGregor. Steers were assigned randomly to pens and treatments on d 1 after arriving at the feedlot. Dietary treatments consisted of a 3 x 3 factorial arrangement of grain source and whole cottonseed inclusion (0, 5, or 15% DM) in the diet. The three grain sources included a cracked corn diet, a cracked corn diet that contained 10% DM as flaxseed, and a ground sorghum diet (Table 1). All diets were formulated to be isonitrogenous and to meet or exceed all nutrient requirements for growing steers (NRC, 1996). Steers were housed in groups of three in partially covered pens equipped with individual Calan gate feeders (American Calan, Northwood, NH). The groups of three animals within a pen consisted of steers on the same grain treatment but with differing WCS concentrations to avoid crossover feeding of the grain sources, which can happen as the steers pull a mouthful of feed from the bunk and drop it on the ground. All diets were fed once daily in the morning in amounts adequate to allow ad libitum access to feed. Dietary composite samples were collected every 28 d for estimates of as-fed fatty acid composition. Steers were switched gradually over a 7-d period to a high-concentrate finishing diet (the corn based, 0% WCS diet). Following this adjustment (d 1 of treatment), steers were adapted over a 7-d period to their respective treatment diets. Steers were weighed and bled via jugular venipuncture on d 0, 28, 56, 84, and 107. Steers were slaughtered on five sequential days and blocked by slaughter date with one steer from each treatment in each block. On the evening of d 109, the block of steers with the heaviest weight was transported to the Rosenthal Meat Science and Technology Center and housed overnight with access to water. The following day, these steers were slaughtered, with the same process repeated on sequential days with the remaining four blocks of steers.

After evisceration, approximately 0.5 m of the duodenum immediately caudal from the pyloric sphincter was removed and emptied of contents. This sample was transported immediately to the laboratory on ice, rinsed gently with 1x Krebs-Henseleit bicarbonate buffer (pH = 7.4), and then scraped lightly to detach the epithelial cells, without incorporating the smooth muscle cells. Samples of liver (n = 23) were taken from the region immediately adjacent to the invagination of the hepatic portal vein. Not all livers were analyzed for SCD activity due to limitations in available resources. Samples were stored at –80°C until further analysis.

Carcass Characteristics
Carcasses were weighed immediately following the slaughter process to determine HCW, and then were chilled at 4°C for 48 h. Carcass measures and grades were measured using standard techniques (USDA, 1997) by trained Texas A&M University personnel. Quality and yield evaluations included determinations of skeletal maturity, lean maturity, marbling, quality grade, fat thickness, LM area, KPH, preliminary yield grade, final yield grade, and dressing percent.

Chemicals
All radiological chemicals were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). All fatty acid standards were purchased from Nu-Chek Prep, Inc. (Elysian, MN). All solvents were analytical reagent grade or greater.

Lipogenesis
Lipogenesis from acetate was conducted as described by Page et al. (1997). Briefly, 100 mg of tissue was incubated in 5 mM sodium acetate, 5 mM glucose, 10 mM HEPES buffer (pH 7.40), and 1 µCi [1-¹⁴C]acetate for 2 h at 37°C. The reaction was stopped by adding 3 mL of 5% trichloroacetic acid. Lipids were extracted and radioactivity counted using a Beckman liquid scintillation spectrometer (Beckman, Palo Alto, CA). Rate of acetate incorporation was calculated and expressed on a cellular basis using the results from the cellularity analyses.

Fatty Acid Composition
Total lipid was extracted from plasma and adipose by the Folch et al. (1957) method. The fatty acids were methylated as described by Morrison and Smith (1964), and the resulting fatty acid methyl esters (FAME) were analyzed with a Varian gas chromatograph (model CP-3800 fixed with a CP-8200 autosampler, Varian Inc., Walnut Creek, CA). Fatty acid methyl esters were separated with a fused silica capillary column CP-Sil88 (100 m x 0.25 mm [i.d.]; Chrompack Inc., Middleburg, The Netherlands), with helium as the carrier gas (flow rate = 2.1 mL/min). The injector temperature and flame ionization detector temperatures were 270 and 300°C, respectively. Total run time was 48 min, with the first 32 min at 180°C, and then increased at 20°C/min to 225°C. Several standards with known fatty acid compositions were used to identify individual peaks. Additionally, these standards were enriched separately with purified sources of specific fatty acids of interest (e.g., trans-18:11) to confirm their identity. Individual FAME were quantified as g/100 g of total FAME analyzed.

Several indices of SCD activity were calculated using a variation of the estimator based on FAME ratios described by Corl et al. (2001). A total index was calculated as (14:1n-5 + 16:1n-7 + 18:1n-9 + 18:2cis-9,trans-11)/ (14:0 + 16:0 + 18:0 + 18:1trans-11). In addition, indices based on 14-carbon (14:1n-5/14:0), 16-carbon (16:1n-7/ 16:0), 18-carbon (18:1n-9/18:0), and CLA (18:2cis-9,trans-11/18:1trans-11) product:precursor ratios also were calculated.

Microsome Extraction
Samples of s.c. adipose, i.f. adipose, liver, and intestinal mucosal tissues (1 to 1.5 g) were homogenized for 60 s at 22°C with a Virtis homogenizer (The Virtis Co., Inc., Gardiner, NY.) in three volumes (wt/vol) of buffer (0.25 M sucrose, 0.01 M potassium phosphate, 1 mM EDTA, and 1 mM dithioerythritol; pH = 7.4). The homogenate was centrifuged for 15 min at 5,000 x g. The infranate was decanted into a separate tube and the pellet and fat cake were discarded. The infranate was centrifuged for 30 min at 17,300 x g, and the supernatant fraction was decanted into a separate tube and centrifuged for 1 h at 104,000 x g. The supernatant fractioin was discarded, and the microsomal fraction was collected and resuspended in 100 mM Tris•HCl buffer (pH = 7.4), snap frozen with liquid N₂, and stored at –80°C until further analysis.

Stearoyl-CoA Desaturase Assay
The SCD activity of microsomal fractions was determined as described by St. John et al. (1991), with modifications as described by Yang et al. (1999). The microsomal fractions were thawed in a 37°C water bath, and 0.5 mL of the microsomal extract was incubated in a total volume of 1.5 mL of a solution containing 100 mM Tris•HCl (pH 7.4), 2 mM NADPH, 0.025 µCi [1-¹⁴C]palmitoyl-CoA, and 50 µM palmitoyl-CoA. There was 0.25, 2.25, and 17.99 mg of protein/incubation vial for the adipose, duodenal, and hepatic incubations, respectively. The incubations were continued for 7 min in a 37°C water bath and terminated by the addition of 3 mL of 10% KOH in methanol. The samples were placed immediately in a 70°C water bath for 30 min and then acidified with 9 mL of 3 N HCl. Fatty acids were extracted by three washes with 6 mL of n-pentane. The pentane phases were evaporated under N₂ and methylated with 14% boron trifluoride in MeOH for 30 min at 70°C. Methyl esters were removed by the addition of 3 mL of distilled deionized water and then extracted by three washes with hexane. The hexane phases were evaporated under N and resuspended in 0.1 mL of hexane and separated by thin layer chromatography on a 5% AgNO₃-impregnated silica gel plate in a petroleum ether:diethylether solvent system (97:3, vol/vol). After separation, the plate was sprayed with 0.2% dichlorofluoroscein in ethanol to visualize the spots. The spots were scraped and counted with a Beckman liquid scintillation spectrometer.

Blanks (containing no microsomes) typically contained measurable disintegrations per minute in the 16:1 spot, although no SCD product can be formed in the absence of microsomes. Therefore, the separation of FAME by thin-layer chromatography was confirmed by developing four preparations of an equal mixture of nonlabeled 16:0 and 16:1 with the same developing system. The methyl esters from the spots were extracted with hexane and analyzed by gas chromatography. This confirmed that there was 37.5% of the 16:0 FAME remaining in the 16:1 FAME spot. Because the spots were discrete with little tailing, we presumed that radioactivity in the 16:1 FAME spot of the blanks represented contamination of the radiolabeled palmitoyl-CoA with a compound that migrated with the 16:1 FAME. Therefore, all reaction 16:1 FAME spots were corrected proportionately.

Cellularity
Adipocyte size, volume, and number were determined by the method of Etherton et al. (1977) with the modifications of Smith et al. (1996). Adipose samples (0.1 g) frozen in liquid N were sliced into 1-mm-thick sections on a chilled-glass surface and placed in 20-mL scintillation vials coated with Sigmacoat (Sigma Chemical Co.), and then processed exactly as described previously (Smith et al., 1996). The fixed cells were filtered through 250-, 62-, and 20-µm nylon mesh screens with 0.01% Triton X-100 in 0.154 M NaCl. Cell fractions from the 62- and 20-µm mesh screens were used to determine mean and peak diameter, mean and peak volume, and adipocytes per gram of tissue with a Coulter counter (model ZM and Coulter Channelyzer 256, Beckman Coulter, Miami, FL).

Statistical Analyses
The Mixed procedure of SAS (SAS Inst., Inc., Cary, NC) was used for statistical analysis of data. The model included the following independent variables: block, grain source, whole cottonseed inclusion, and the grain source x whole cottonseed inclusion rate interaction. Block was considered a random effect. The grain x WCS interaction was not significant and was not included in the model, which did not significantly alter the error sum of squares. The data for plasma fatty acid composition were analyzed as repeated measures, with grain source and WCS inclusion considered to be fixed effects, and days on treatment considered to be a repeated measure using the autoregressive 1 (AR1) covariance structure. This covariance structure assumes that two measurements close in time have a higher correlation than measures farther apart in time. Linear and quadratic estimates of the rate of change in plasma fatty acids were not assessed because most changes occurred within the first 28 d and there were insufficient data points within the first day to make a realistic fit. When treatment effects were significant (P < 0.05), a difference was determined and a tendency for treatment to elicit a response was noted when P < 0.10. Least squares means of significant responses were separated using the PDIFF statement of the Mixed procedure.

	Results

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Animal Production
There was no difference in the formulated protein concentrations of the diets, but there were marked differences in the amounts of dietary fat provided by each of the diets (Table 1). For this reason, dietary energy concentration ranged from 1.74 to 2.07 Mcal of NE_m/kg and 1.17 to 1.46 Mcal of NE_g/kg. The grains altered the dietary fatty acid composition as desired (Table 2), with the corn diet providing approximately 55% of the fatty acids as 18:2n-6, and the flaxseed diet providing approximately 23% of the total fatty acids as 18:3n-3. The flaxseed diet contained the least concentration of 18:2n-6, and both the corn and sorghum diets contained only 1 to 2.4% 18:3n-3.

Plasma Fatty Acids
There was a decrease in plasma 16:0 by d 84 and d 107 (Figure 1), and both the flaxseed- and sorghum-fed steers had a greater decline than did the corn-fed steers (day x grain, P < 0.01; Table 4). Plasma 18:0 (Figure 1) increased over time and was greatest in the sorghum-fed steers at all time points after treatment had begun. There were no apparent differences in 18:0 between the corn- and flaxseed-fed steers. At d 84 and d 107, the sorghum-fed steers had a greater concentration of 18:1n-9 than either the corn- or flaxseed-fed steers (day x grain, P < 0.01; Figure 2). The concentration of plasma 18:2n-6 (Figure 2) was consistently greater in corn-fed steers than in the other treatment groups until d 107, at which time it did not differ from the flaxseed-fed steers (day x grain, P < 0.01). There was a marked increase in plasma 18:2n-6 by d 84 and a sharp decrease by d 107 in all treatment groups.

View larger version (25K): [in this window] [in a new window]   Figure 1. Concentrations (g/100 g of total fatty acids) of 16:0 and 18:0 in plasma from Angus steers fed corn-, flaxseed-, or sorghum-based diets for 107 d. Standard errors are attached to the means.

View larger version (25K): [in this window] [in a new window]   Figure 2. Concentrations (g/100 g of total fatty acids) of 18:1n-9 and 18:2n-6 in plasma from Angus steers fed corn-, flaxseed-, or sorghum-based diets for 107 d. Standard errors are attached to the means. Standard error bars are not apparent if they were smaller in magnitude than the symbols.

View larger version (26K): [in this window] [in a new window]   Figure 3. Concentrations (g/100 g of total fatty acids) of 18:1trans-11 and 18:2cis-9,trans-11 in plasma from Angus steers fed corn-, flaxseed-, or sorghum-based diets for 107 d. Standard errors are attached to the means. Standard error bars are not apparent if they were smaller in magnitude than the symbols.

View larger version (22K): [in this window] [in a new window]   Figure 4. Concentrations (g/100 g of total fatty acids) of 18:3n-3 and 20:5n-3 in plasma from Angus steers fed corn-, flaxseed-, or sorghum-based diets for 107 d. Standard errors are attached to the means. Standard error bars are not apparent if they were smaller in magnitude than the symbols.

Adipose Tissue Fatty Acids
Grain Effects.
The concentration of 18:1n-9 was highest in i.f. (Table 5) and s.c. (Table 6) adipose tissues of sorghum-fed steers and lowest in flaxseed-fed steers, although 18:0 was unaffected by grain type. There was a greater concentration of 18:3n-3 (P < 0.01) in i.f. (Table 5) and s.c. (Table 6) adipose tissues of steers fed flaxseed (approximately 0.8 g/100 g of total fatty acids) than in steers fed corn or sorghum. Adipose tissues of steers fed flaxseed also had the greatest concentration of 18:1trans-11, but cis-9,trans-11 CLA was not affected by treatment in either adipose tissue (P 0.33). The trans-10,cis-12 isomer of CLA was not detectable in any adipose tissue samples. There was a greater percentage of "other" fatty acids in adipose tissues from flaxseed-fed steers than in corn- or sorghum-fed steers. The "other" fatty acids consisted primarily of 14:1n-7, 17:0, 17:1n-8, and 18:1trans-9, and a varied population of eicosanoic fatty acids. The latter were not quantified because of difficulty in assigning identities to many of the fatty acids. However, neither arachidonic (20:4n-6) nor eicosapaentanoic (20:5n-3) were detectable in the adipose tissues.

Whole Cottonseed Effects.
The concentration of 18:1n-9 was higher (P < 0.01) in i.f. adipose tissue of steers fed 5% WCS than in steers fed 15% WCS (Table 5); a similar trend (P = 0.08) was observed for s.c. adipose tissue (Table 6). Total SFA were greatest (P < 0.01) and total MUFA and the SFA:MUFA ratio were lowest (P < 0.01), in i.f. adipose tissue of steers fed 15% WCS. Similar trends were observed for s.c. adipose tissue, but the differences were not significant (P = 0.09 to 0.12).

Lipogenesis and Cellularity
Lipogenesis from acetate in s.c. adipose tissue was greater (P < 0.01) in steers fed flaxseed (5.42 nmol•10⁵ cells^–1•h^–1) than in steers fed corn (3.10 nmol•10⁵ cells^–1•h^–1) or sorghum (1.92 nmol•10⁵ cells^–1•h^–1), whereas lipogenesis was unaffected by treatment (P 0.54) in i.f. adipose tissue (Table 7). Interfascicular adipocyte mean diameter was greatest (P < 0.01) in the steers fed flaxseed (59 µm) or corn (60 µm), and smallest in the steers fed sorghum (49 µm; Table 7). The i.f. adipocyte mean volume tended (P = 0.06) to be smallest in steers fed sorghum (205 pL) and largest in steers fed corn (276 pL) or flaxseed (279 pL). Whole cottonseed had no effect (P 0.21) on mean diameter or volume of i.f. adipose tissue (Table 7).

Stearoyl-CoA Desaturase Activity
Stearoyl-CoA desaturase enzyme activity was not affected (P 0.25) by grain or WCS in i.f. and s.c. adipose tissues, or in duodendal mucosal cells and liver (Table 8). Of the tissues that were analyzed, SCD activity was greatest in s.c. adipose when expressed on a per mg protein basis (32.5 to 44.4 nmol•mg protein^–1•7 min^–1). However, SCD activity of i.f. adipose tissue was more than twice that of s.c. adipose tissue on a per-gram-of-tissue basis (61 to 89 vs. 33 to 38 nmol•g tissue^–1•7 min^–1). Duodenal mucosal cells had by far the greatest SCD activity when rates were expressed on a per-gram-of-tissue basis (118 to 141 nmol•g tissue^–1•7 min^–1). Hepatic SCD enzyme activity was low but detectable.

	Discussion

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Animal Production
The sorghum diet had the lowest lipid content and sorghum-fed cattle had the lowest G:F. We hypothesize that the decrease in feed efficiency with increasing WCS in the sorghum-fed steers may have been caused by a moderate ruminal acidosis brought on by the fine particle size of the ground sorghum in conjunction with impairment of ruminal microbial protein synthesis and postruminal NDF digestibility with the increased WCS (Harvatine et al., 2002); however, this was not measured specifically in this study. Feed efficiency of cattle fed corn-based diets is less responsive to fat supplementation than in cattle fed other fat-supplemented grains, and this may be the result of the higher basal energy from corn than is in other grain sources such as sorghum (Krehbiel et al., 1995a,b; Andrae et al., 2000).

Carcass Quality and Adipocyte Metabolism
Carcass composition of the steers from this study was within the range that currently is being produced by the industry (McKenna et al., 2002). The National Beef Quality Audit (McKenna et al., 2002) reported average carcass weights of 357 kg (SD = 43) and an average yield grade of 3. The carcass quality of the steers from this study would rank in the lower 18% of fed cattle, reflecting the relatively short feeding period and youthful maturity of these steers.

Subcutaneous adipose tissue of steers fed flaxseed had larger adipocytes than sorghum-fed steers, reflecting the higher fat content of the flaxseed diets. However, s.c. adipocytes from flaxseed-fed steers also had the highest rates of de novo fatty acid biosynthesis, which would have contributed to the larger adipocyte volume of this group. The greater s.c. adipocyte volume of the flaxseed-fed steers was not sufficient to cause a significantly greater carcass fat thickness in these steers. Similarly, the greater i.f. diameters and volumes in the flaxseed-fed steers was not sufficient to elicit an increase in marbling scores. Conversely, there was no effect of WCS on i.f. adipocyte volume, but 15% WCS significantly decreased marbling scores. This finding suggests that LM from cattle fed 15% WCS had fewer clusters of marbling adipocytes. Our inability to increase CLA isomers, especially the trans-10,cis-12 isomer, in adipose tissues with the various grain/WCS combinations indicates that these isomers were not responsible for any of the carcass or cellular differences we observed.

Stearoyl-CoA Desaturase
Although we previously reported the effects of oil-seeds on SCD enzyme activity in s.c. adipose tissue (Chang et al., 1992; Page et al., 1997; Yang et al., 1999) and in intestinal mucosal cells and LM (Chang et al., 1992), this is our first report of SCD enzyme activity in i.f. adipose tissue. Desaturase activity was twice as high in i.f. adipose tissue than in s.c. adipose tissue when rates were calculated on a per-gram-of-tissue basis. This apparently had biological significance, because the MUFA:SFA ratio was greater in i.f. adipose tissue (average of 0.74) than in s.c. adipose tissue (average of 0.61).

A primary goal of this study was to decrease carcass adiposity by depressing SCD enzyme activity in adipose tissue. Diets enriched with 18:2n-6 (corn) and 18:3n-3 (flaxseed) were provided to increase the ruminal production of 18:2 cis-9,trans-11 (rumenic acid) and 18:2trans-10,cis-12, both of which have been shown to decrease SCD gene expression and/or enzyme activity (Choi et al., 2001; 2002). -Linolenic acid may serve as a source of 18:1trans-11, bypassing 18:2 cis-9,trans-11 as an intermediate during ruminal biohydrogenation (Ward et al., 1964; Wilde and Dawson, 1966). In addition, Duckett et al. (2002) demonstrated the ruminal production of trans-10,cis-12 CLA in corn-fed steers. Whole cottonseed was added to exacerbate this effect because our previous investigations indicated that WCS markedly increased 18:0 and depressed SCD enzyme activity in Australian cattle (Smith et al., 1998; Yang et al., 1999).

Whole cottonseed (15%) elicited a small but significant increase in SFA, and decrease in MUFA, and a depression of the SFA:MUFA ratio in i.f. adipose tissue, suggesting a depression of SCD enzyme activity. However, WCS was completely ineffective in depressing SCD activity, a result we observed previously in American feedlot cattle (Page et al., 1997). Instead, the increase in total SFA and reduction in MUFA likely was caused by the increase in 16:0 and decrease in 18:1n-9 caused by the inclusion of WCS in the diet. We cannot explain why WCS demonstrably decreased SCD activity and increased 18:0 in Australian cattle, yet was without effect in cattle produced under our production conditions in the United States.

The Endogenous Production of CLA
Although there are studies supporting the endogenous formation of the cis-9,trans-11 CLA from 18:1trans-11 in dairy cattle (Griinari et al., 2000; Corl et al., 2001), these studies provide only estimations of SCD activity, primarily based on milk lipids. Others similarly have used the desaturase products:substrates ratio to estimate SCD activity (Griinari et al., 2000; Duckett et al., 2002). More recently, Palmquist et al. (2004) calculated SCD enzyme activity in ovine adipose tissue based on tissue fatty acid composition. Our data indicate that estimations of SCD may not accurately reflect actual SCD enzyme activity or its contribution to the synthesis of cis-9,trans-11 CLA. For example, sorghum increased the 18:2cis-9,trans-11:18:1trans-11 ratio in i.f. adipose tissue and tended (P = 0.09) to cause the same effect in s.c. adipose tissue, but had no effect on actual SCD enzyme activity in either adipose tissue. The higher 18:2 cis-9,trans-11/18:1trans-11 ratios in adipose tissues of sorghum-fed steers were caused by the dramatically lower concentrations of 18:1trans-11 in plasma and adipose tissue of these steers, especially compared with steers fed flaxseed. We hypothesize that there was more complete ruminal hydrogenation of fatty acids in the sorghum diet, facilitated by the increased surface area of the ground sorghum compared with the other grains.

Unlike our results, Santora et al. (2000) and Corl et al. (2003) demonstrated that increases in tissue concentrations of 18:1trans-11 were associated with proportional increases in 18:2cis-9,trans-11. We clearly obtained different results in adipose tissue, in that cis-9,trans-11 CLA was unchanged by dietary grains, in spite of large increases in adipose tissue 18:1trans-11 in the corn- and flaxseed-fed steers. The fact that cis-9,trans-11 CLA was barely detectable in plasma was especially perplexing considering the high SCD enzyme activity we measured in intestinal mucosal cells. The lack of effect of dietary grains on the concentration of cis-9,trans-11 CLA in plasma or adipose tissues indicates that 18:1trans-11 was not an effective substrate for SCD in these cattle.

The concentration of 18:0 rarely exceeds 15%, and 18:1n-9 usually exceeds 44%, in s.c. adipose tissues (e.g., Huerta-Leidenz et al., 1991; St. John et al., 1991; Chang et al., 1992). In the present study, s.c. adipose tissue contained an average of 22% 18:0 and only 30% 18:1n-9, and the MUFA:SFA ratios for s.c. adipose tissue were only 0.57 to 0.65, the lowest values we have observed. In Chang et al. (1992), we reported that 18:0 did not exceed 13% of total plasma fatty acids, whereas in the current investigation, 18:0 ranged from a low of 30 to a high of 39%. We did not measure duodenal fatty acids in the present investigation, but the plasma concentrations of 18:0 suggest that it was elevated over concentrations we have measured previously (48 to 67% of total duodenal fatty acids; St. John et al., 1991; Ekeren et al., 1992). We also were unable to measure trans-10,cis-12 CLA in adipose tissues and only trace amounts in plasma, although Duckett et al. (2002) reported ruminal production of this CLA isomer in corn-fed steers. Collectively, these data suggest more extensive ruminal biohydrogenation of dietary unsaturated fatty acids in this study than observed previously.

These findings suggest three possibilities: 1) there was functionally less SCD enzyme activity in adipose tissue and intestinal mucosa than we previously observed; 2) the high plasma and adipose tissues concentrations of 18:0 effectively competed with 18:1trans-11 for SCD, so that little of the trans-fatty acid was desaturated in vivo; or 3) adipose tissue and mucosal SCD activities were maximal even at the lowest tissue concentrations of 18:1trans-11. Our measurement of maximal SCD activities would seem to eliminate the first possibility, but this needs to be addressed specifically.

In summary, it was not possible to decrease SCD enzyme activity with the combination of grains and WCS used in this investigation, although some calculated indices of SCD activity were sensitive to grain type or WCS. Flaxseed apparently increased s.c. adipocyte volume by providing more dietary lipid and by increasing de novo fatty acid biosynthesis, and in longer-fed cattle, this may result in greater carcass adiposity. In contrast, feeding WCS at higher levels may decrease carcass quality.

	Implications

Top Abstract Introduction Materials and Methods Results Discussion Implications Literature Cited

 
Indices of stearoyl-CoA desaturase activity based on fatty acid concentrations did not reflect actual enzyme activity. This was especially true when comparing diets that varied markedly in fatty acid composition, which had profound effects on tissue fatty acid composition that were unrelated to desaturase enzyme activity. Ruminally produced vaccenic acid was not converted extensively to rumenic acid in adipose tissue under our production conditions. Dietary polyunsaturated fatty acids of the n-3 and n-6 series also were ineffective in increasing plasma or adipose tissue conjugated linoleic acid isomers. Our data imply that it will be difficult to decrease carcass adiposity of feedlot cattle via a decrease of adipogenesis by endogenously produced conjugated linoleic acid.

	Footnotes

 
¹ Published as a technical article, Texas Agric. Exp. Stn.

² Correspondence: 2471 TAMU (phone: 979-845-3939; fax: 979-458-2702; e-mail: sbsmith{at}tamu.edu).

Received for publication April 5, 2004. Accepted for publication February 9, 2005.

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Top Abstract Introduction Materials and Methods Results Discussion Implications Literature Cited

 


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