J. Anim Sci. 2006. 84:3061-3070. doi:10.2527/jas.2005-736
© 2006 American Society of Animal Science
Effects of high-sugar ryegrass silage and mixtures with red clover silage on ruminant digestion. 2. Lipids1
M. R. F. Lee2,
P. L. Connelly,
J. K. S. Tweed,
R. J. Dewhurst,
R. J. Merry and
N. D. Scollan
Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, SY23 3EB, UK
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Abstract
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The experiment investigated the digestion of lipids from different forage silages in beef steers. Six Hereford x Friesian steers prepared with rumen and duodenal cannulas were given ad libitum access to a high-sugar grass silage, control grass silage, red clover silage, or mixtures of the red clover and each of the grass silages (50:50, DM basis). The experiment was conducted as an incomplete 5 x 5 Latin square, with an additional randomly repeated sequence. Total fatty acid and C18:3n-3 concentrations were greater (P < 0.05) for the high-sugar grass silage than the control grass silage or the red clover silage. Dry matter and total fatty acid intake were less (P < 0.05) for steers fed the control grass silage than for steers fed the other diets. Duodenal flow of C18:3n-3 was greater (P < 0.05), and flows of C18:0 and total C18:1 trans were less (P < 0.05), for the red clover silage compared with the 2 grass silage diets, with the mixtures intermediate. These results were supported by a reduction (P < 0.05) in biohydrogenation of C18:3n-3 for the red clover silage, with the mixtures again being intermediate. Flows of total branched- and odd-chain fatty acids were greater (P < 0.05) for the high-sugar grass silage diet, possibly as a result of greater microbial flow, because these fatty acids are associated with bacterial lipid. Duodenal flows of the chlorophyll metabolite, phytanic acid, were greater (P < 0.05) for animals fed the high-sugar grass silage treatments compared with the other treatments. These results confirm the potential for modifying the fatty acid composition of ruminant products by feeding red clover silage.
Key Words: beef steer • biohydrogenation • fatty acid • high-sugar grass silage • phytanic acid • red clover silage
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INTRODUCTION
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Feeding forage diets containing lipids with high proportions of C18:2n-6 and C18:3n-3 to ruminants results in extensive biohydrogenation with the formation of C18:0 as an end point but also numerous intermediary products such as C18:1 and conjugated and nonconjugated C18:2 cis and trans isomers (Jenkins, 1993
). Interest in these intermediary products has grown in recent years with the identification of biologically active isomers of CLA and enzymatic pathways for CLAc9t11 formation from C18:1t11, both in the mammary gland and adipose tissue (Pariza et al., 2001).
Despite this interest, the real challenge to the ruminant nutritionist has been to reduce biohydrogenation to retain PUFA and hence increase the health value of ruminant fat (Tapiero et al., 2002). The few studies that reported on lipid metabolism of complete forage diets indicate extensive biohydrogenation of C18:2n-6 and C18:3n-3 (Outen et al., 1975; Bauchart et al., 1984; Scollan et al., 2003). Lee et al. (2003a) investigated the effect of clover silages (red vs. white and in combination with grass) on ruminal metabolism of forage lipids. Red clover resulted in a greater flow of C18:3n-3 to the duodenum of beef steers per unit DMI than grass silage, which was due to a reduction in the biohydrogenation of C18:3n-3. Dairy cows fed red clover silage had an increase in duodenal flow (Dewhurst et al., 2003a) and milk levels of C18:3n-3 (Dewhurst et al., 2003b).
The major aim of this experiment was to further investigate duodenal flow of fatty acids in beef steers offered red clover silage or grass silage. In addition, the grass silages were prepared with the aim of obtaining different concentrations of residual water-soluble carbohydrate. Previous research (Lee et al., 2003b) has shown that increasing the availability of water-soluble carbohydrate in an in vitro rumen simulation system led to conditions that have been linked to reduced biohydrogenation of dietary unsaturated fatty acids (Jenkins, 1993).
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MATERIALS AND METHODS
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All animal procedures and the care for the animals were carried out under strict regulations described in the Animals (Scientific Procedures) Act 1986 issued by the Home Office of Her Majesty’s Britannic Government.
Experimental Design and Procedures
The experimental design was described in the companion paper (Merry et al., 2006). In brief, 6 Hereford x Friesian steers, initial BW of 163 ± 5.9 kg, prepared with a rumen cannula and a simple T-piece cannula in the proximal duodenum (immediately after the pylorus and before the common bile duct), were offered silage individually at 0900 and 1600 to ensure ad libitum consumption. Feed refusals were removed daily at 0845, and DM content measured to determine actual DMI. The experiment was conducted as a 5 x 5 incomplete Latin square, with an additional randomly repeated sequence. There were 4 experimental periods with 6 animals allocated to 1 of the 5 diets: high-sugar grass silage; control grass silage; high-sugar grass silage and red clover silage (50:50, DM basis); control grass silage and red clover silage (50:50, DM basis); and red clover silage. Each period lasted 24 d, with a 14-d adaptation period to the diets, followed by a 10-d measurement period for collection of rumen fluid and duodenal digesta.
Digesta flow at the duodenum was estimated using a dual-phase marker system with YbAc and Cr-EDTA as the particulate and liquid phase markers, respectively (Faichney, 1975). Ytterbium acetate (244 mg of Yb/d) and Cr-EDTA (1,454 mg of Cr/d) were infused intraruminally via separate lines at a rate of 19 mL/h for 7 d. On d 20 and 21, duodenal digesta (100 mL per sample) was collected every 4 h over a 24-h period, and each daily sample was kept separate for analysis. On d 23 and 24, samples of rumen fluid were collected as described by Merry et al. (2006). All samples were stored at –20°C.
Sample Preparation and Analysis
Separate samples of fresh silage were taken daily during the digestion periods. Subsamples of silage and digesta were stored as sampled at –20°C, or freeze-dried, ground, and retained at –20°C for chemical analysis. Accumulated samples of daily duodenal digesta (n = 7) were thoroughly mixed, and a 200-g subsample of each was freeze-dried to represent whole digesta. Separate 200-g portions of duodenal digesta were centrifuged at 3,000 x g for 25 min to provide the centrifuged solid digesta. These were subsequently freeze-dried, ground, and stored at –20°C for analysis.
Chemical compositions of the silages and digesta were determined as described by Lee et al. (2002b). Chlorophyll was determined spectrophotometrically at 645 and 663 nm using the method of Arnon (1949). Lipid fractions were determined by extracting total lipid from the freeze-dried silage samples using chloroform:methanol (2:1, vol/vol) and fractionating using TLC (Nichols, 1963). The individual fractions were bimethylated using 5 N NaOH in methanol and 5% HCl in methanol (Kramer and Zhou, 2001). The fatty acids in the silages were obtained by direct transmethylation (Sukhija and Palmquist, 1988). Digesta fatty acids were prepared by direct hydrolysis, with an added internal standard (C19:0 methyl ester), in 5 M KOH dissolved in methanol. The resultant potassium carboxylates were transformed into their fatty acids by the addition of 10 N H2SO4, collected in petroleum ether, and methylated using 5% HCl in methanol (Kramer and Zhou, 2001). The samples were analyzed by GLC on a CP-Select column (100 m x 0.25 mm ID, Varian Inc., Palo Alto, CA) that was chemically bonded for fatty acid methyl esters, with split injection (30:1). Peaks were identified from external standards (ME61, Laroden fine chemicals, Malmo, Sweden; S37, Supelco, Poole, Dorset, UK; CLAs, Matreya, Philadelphia, PA) and quantified using the internal standard (C19:0).
Calculations and Statistical Analysis
Digesta flows were calculated after mathematical reconstitution of true digesta (Faichney, 1975). Biohydrogenation of C18 MUFA and PUFA were assessed by the simple difference between feed and duodenal content (g/d). All measurements other than silage composition were subjected to GLM-ANOVA, with DMI as a covariate, according to a 5 x 5 Latin square design, with an extra column (animal) added and 1 row (period) omitted (Cochran and Cox, 1957). Thus, there were 6 animals fed over 4 periods. In each period, 2 animals were fed the same diet. Where the overall treatment effect was significant (P < 0.05), individual treatment differences were determined using the Student-Newman-Keul’s test. Because of the unbalanced nature of the design, the SEM were calculated based on 4 replications. Principal component factor analysis (Massart-Leen and Massart, 1981) was used to describe patterns of fatty acid profiles. All analyses were conducted using Genstat 7 (Lawes Agricultural Trust, 2003).
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RESULTS
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Diet Composition
The major chemical compositions of the silages are described in the companion paper (Merry et al., 2006). Tables 1 and 2 show the chlorophyll, lipid fractions, and fatty acid composition of the silages. The chlorophyll content of the control grass silage was less (P < 0.05) than the other silages. There was a greater proportion of total lipid (P < 0.05) and C18 unsaturated fatty acids in the polar lipid fraction of the red clover silage and the mixtures than the grass silages. Conversely, there was a greater proportion in the triacylglycerol fraction of the grass silages than the silages containing red clover (P < 0.05, for total lipid and C18 unsaturated fatty acids). The proportions of total lipid and C18 unsaturated fatty acids in the diacylglycerol and FFA fractions were not different across treatments. The grass silages had greater concentrations (P < 0.05) of C12:0, C16:1, C18:1n-9, C18:1n-11, and C18:3n-3, whereas the red clover silage had greater concentrations (P < 0.05) of C14:0, C18:0, and C20:0. The mixtures were intermediate between the 2. Concentrations of total fatty acids did not differ among silages and silage mixtures.
Intake and Duodenal Flow of Fatty Acids
Steers fed the control grass silage consumed less (P < 0.05) than the steers fed the other 4 silage treatments (Table 3). Total fatty acid, C14:0, C16:0, C18:0, C18:2n-6, C18:3n-3, and C20:0 intakes were less (P < 0.05) for the steers fed the control grass silage than for steers fed the other treatments. Intake of C18:0 and C18:2n-6 was greater (P < 0.05) with steers fed on red clover silage than steers fed on grass silages but similar among mixtures of grass and red clover silage. Duodenal flows of total fatty acids, C18:0, C18:1 trans, C18:2t9t12, and branched- and odd-chain fatty acids were greater (P < 0.05) for steers fed the high-sugar grass silage than steers fed the red clover silage. In contrast, duodenal flows of C18:2n-6 and C18:3n-3 were greater (P < 0.05) for steers fed the red clover silage compared with steers fed the other treatments. When differences in DMI of the control grass silage were accounted for by calculating flow of fatty acids per kilogram of DMI, the values for fatty acid flow for the mixtures of grass and red clover were intermediate to those for the pure silages.
There were greater (P < 0.05) flows of C15:0 anteiso when grass silages were fed compared with red clover silage (Table 4). Duodenal flows of the chlorophyll metabolite, phytanic acid, were approximately 3 times greater for steers fed the high-sugar grass silage than the red clover silage and 1.5 times greater than the control grass silage.
The isomers C18:1t6/7/8, C18:1t9, C18:1t11, CLAc9t11 were less (P < 0.05) and C18:1c12 and CLAt10c12 greater (P < 0.05) for steers fed the red clover silage compared with the grass silage treatments (Table 5). The results of principal components factor analysis, highlighting potential common pathways, are shown in Figure 1. Fatty acids that appear close together in this analysis behave in the same way across the data set and may share common metabolic pathways. The results of the analysis identified 3 main clusters: cluster 1 = C18:1t6/7/8, C18:1t9, C18:1t11, C18:2t9t12, and CLAc9t11, which tended to increase proportionally for steers fed the grass silage diet; cluster 2 = C18:1c9, C18:1c11, C18:1c13, CLAt9t11, and C18:1t10, which tended to remain constant across diets; and cluster 3 = C18:3, C18:2, C18:1c12, C18:1c15, C18:1t12, C18:1t13, C18:1t15, and CLAc9c11, which tended to increase proportionally for steers fed the red clover silage diet.
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Figure 1. Multivariate analysis plot of the proportions of C18 fatty acids in the duodenal digesta of beef steers fed the experimental silages. Significance of the clusters (P < 0.05).
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Flows of long-chain fatty acids (C20+) were similar among the 5 treatments (Table 6), although C22:0 were less (P < 0.05) for steers fed the red clover silage than for steers fed the high-sugar grass silage, and C20:2 and C23:0 were less (P < 0.05) for steers fed the grass silages than steers fed the other silages.
Ruminal Biohydrogenation
Estimates of the degree of ruminal biohydrogenation of the unsaturated C18 fatty acids are given in Table 7. Proportional biohydrogenation of C18:1c9 was greater (P < 0.05) for steers fed the control grass silage diet than the other diets. There was no significant difference among the diets for C18:2n-6. Biohydrogenation of C18:3n-3 was less (P < 0.05) when red clover silage was fed opposed to grass silages with the mixtures intermediate.
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Table 7. Biohydrogenation (g/g) of unsaturated C18 fatty acids in the rumen of steers fed the experimental silages1
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DISCUSSION
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The lipid fractions of the silages (Table 1
) are different from the profiles of fresh red clover and fresh grass reported by Lee et al. (2003c). Those authors reported values of 73 and 66% polar lipid, 13 and 15% diacylglycerol, 8 and 13% triacylglycerol, and 7 and 7% FFA for grass and red clover, respectively. The decrease in the proportion of polar lipid and the increase in FFA of the silages were likely due to plant-mediated lipolysis of the cell and organelle lipid membrane bilayer of the plant cell, which occurs during periods of stress or during cell death and senescence (Lee et al., 2004). Besides the greater levels of FFA, silage compared with fresh forage has greater levels of triacylglycerol (Fievez et al., 2004). Lee et al. (2002a) reported a doubling of the triacylglycerol in cut grass after 2 h in a rumen-simulated environment without inoculum, suggesting a plant-mediated response. Sakaki et al. (1990) highlighted a mechanism for an increase in triacylglycerol during periods of stress. An enzyme, acylCoA:sn-1,2-diacylglycerol acyltransferase, which is found mainly in the envelope membrane of chloroplasts, upregulates during periods of stress and converts diacylglycerols released by the degradation of the membrane lipid bilayer into triacylglycerol. The preparation of red clover silage required a longer wilt than grass silage in the current study (Merry et al., 2006). Therefore one may expect greater loss of the membrane polar lipid of the red clover because the plant enzymes would have longer to operate before being deactivated as a result of the drop in pH during ensiling. However, the action of the enzyme polyphenol oxidase, within the red clover, may have inhibited plant-mediated lipolysis through the production of highly electrophilic quinones (Lee et al., 2004). These quinones may act by complexing with the polar lipid and so preventing lipolysis or denaturing acyl hydrolase and acylCoA:sn-1,2-diacylglycerol acyltransferase from breaking down the polar lipid and forming triacylglycerol.
The proportions of the 3 main fatty acids, C18:3n-3, C16:0, and C18:2n-6, in silage lipids was similar across silages, averaging 0.51, 0.21, and 0.19, respectively. The feeding of both grass silages resulted in a net synthesis of total fatty acids from mouth to duodenum, whereas red clover silage and the mixtures all resulted in a net loss of total fatty acids, with duodenal flows 1.12, 1.02, 0.94, 0.93, and 0.86 times fatty acid intake for high-sugar grass, control grass, high-sugar grass/red clover mix, control grass/red clover mix, and red clover silages, respectively. This trend has previously been reported with grass and clover silages (Outen et al., 1975; Lee et al., 2003a) and harvested grass (Bauchart et al., 1984; Scollan et al., 2003). This may in part be the result of different microbial populations on the different diets, which through their different microbial lipid content may result in changes in net lipid flow to the duodenum (Merry and MacAllan, 1983).
The changes in fatty acid composition between intake and duodenal flow are characteristic of the processes of lipolysis and biohydrogenation of the dietary unsaturated fatty acids in the rumen. Decreased flow of C18:3n-3 and C18:2n-6 were accompanied by increased flow of C18:0 and the appearance of C18:1trans, C18:2t9t12, and CLA. The apparent proportional biohydrogenations of C18:1c9, C18:2n-6, and C18:2n-3 were 0.23, 0.90, and 0.91, respectively. The low apparent biohydrogenation of C18:1c9 is a common occurrence on forage diets (Wu et al., 1991; Lee et al., 2003a; Scollan et al., 2003) with low concentrations of C18:1c9, reflecting an underestimation of biohydrogenation because of the contributions made by endogenous C18:1c9 from sloughed digestive tract epithelial cells, incomplete biohydrogenation, and de novo synthesis by rumen microorganisms (Merry and MacAllan, 1983). The biohydrogenation values for C18:2n-6 and C18:3n-3 are in the range reported by Doreau and Ferlay (1994) of between 0.70 to 0.95 and 0.85 to 1.0, respectively. There was no difference in the biohydrogenation for C18:2n-6 or C18:3n-3 between the 2 grass silages. It was originally speculated that feeding high-sugar grass silage might reduce ruminal pH (Lee et al., 2003b) and consequently reduce lipolysis and biohydrogenation. Indeed, Van Nevel and Demeyer (1996a,b) showed that in in vitro systems a reduction in pH below 6 resulted in a 20% reduction in lipolysis and a 5% reduction in biohydrogenation. However, the difference in ruminal pH was numerically very small in the current study; 6.67 vs. 6.76 for high-sugar grass silage and control grass silage, respectively (Merry et al., 2006). Scollan et al. (2003) achieved similar results when feeding the same high-sugar grass and control grass varieties as harvested forage, which they postulated was related to the lack of a significant rumen pH differential among treatments.
The main effect on the duodenal flow of fatty acids, per unit DMI, brought about by the inclusion of red clover silage into the diets was an increase in the flows of C18:2n-6 and C18:3n-3 and a decrease in the flows of C18:0, C18:2t9t12, and C18:1 trans. The increase in the flow of C18:3n-3 and the decrease in the flow of the biohydrogenation intermediary and end products are explained by the decrease in biohydrogenation of C18:3n-3 when red clover silage was fed as the sole feed or mixed with grass silage. Previous studies with red clover have shown similar increases in C18:3n-3 in the duodenal digesta of beef steers (Lee et al., 2003a) and the milk of dairy cows (Dewhurst et al., 2003a). The mechanism of action for this reduction in biohydrogenation on red clover is not fully understood but may be due to the action of polyphenol oxidase, as discussed previously. Red clover silage had a greater proportion of polar lipid than grass silage, which may through quinone binding offer some level of protection in the rumen resulting in a greater escape of C18 unsaturated fatty acids. A change in microbial population may provide an alternative explanation. Increasing the content of red clover in the diet was accompanied by a shift in duodenal fatty acids toward increased levels of CLAt10c12 and C18:1c12 and reduced levels of CLAc9t11 and C18:1t11. These shifts in CLA and C18:1 isomer production are observed when increasing the concentrate content of a diet (Beaulieu et al., 2002; Wang et al., 2002), which also coincides with a reduction in biohydrogenation of C18:3n-3. This response appears to be associated with changes in the population of ruminal microorganisms, with a reduction in cellulolytic bacteria (Butyrivibrio fibrisolvens) and an increase in starch degrading bacteria such as Megasphaera elsdenii. This particular bacterium has been shown to produce CLAt10c12 from pure linoleic acid (Kim et al., 2002). However, in the current study the level of starch supply to the rumen was equal across the diets (Merry et al., 2006), making such a microbial population shift unlikely and the rise in CLAt10c12 on red clover silage difficult to explain. One potential explanation may be the result of an artifact coeluting with CLAt10c12 on the GLC column as previous studies (Lee et al., 2005) have alluded to problems of identification of CLA isomers using GLC technologies. This may also explain why this particular fatty acid does not cluster well in the principal components analysis with other fatty acids, which appear to increase on the red clover silage diet (Cluster 3; Figure 1).
Principal components analysis is often used to reduce the dimensionality of data profiles containing intercorrelated variables. Moreover, it aims to display the maximum amount of variation in a data profile within a few principal components. Hence, pairwise score plots derived from principal components analysis are useful to find similarities and contrasts between samples. The aim of using this method of analysis in this study was to assess how different forage silages altered the production of biohydrogenation intermediates and to allude to common pathways for their production. The principal components factor analysis plot of the proportions of C18 fatty acids in the duodenal digesta (Figure 1) highlights these changes by clustering fatty acids that share common pathways or that fluctuate in similar ways. Three main cluster groups are depicted in the figure: cluster 1 = C18:1t6/7/8, C18:1t9, C18:1t11, C18:2t9t12, and CLAc9t11, which tended to increase proportionally on the grass silage diet; cluster 2 = C18:1c9, C18:1c11, C18:1c13, CLAt9t11, and C18:1t10, which tended to remain constant across diets; and cluster 3 = C18:3, C18:2, C18:1c12, C18:1c15, C18:1t12, C18:1t13, C18:1t15, and CLAc9c12, which tended to increase proportionally on the red clover silage diet. The CLAt10c12 isomer appeared to have a weaker association with this cluster, which has been discussed previously.
In addition to the appearance of fatty acids associated with biohydrogenation at the duodenum, branched- and odd-chain and long-chain (C20+) fatty acids were also detected. Kim et al. (2005) identified 3 branched- and odd-chain fatty acids: C15:0 anteiso, C17:0 iso, and C17:0 anteiso, as potential markers to study the microbial colonization of freshly ingested grass. In our study, duodenal flow of C15 and C17 branched- and odd-chain fatty acids and microbial N flow (Merry et al., 2006) were highly correlated (r = 0.77, 0.77, 0.72, 0.52, 0.53, and 0.75; P < 0.05; for C15:0 iso, C15:0 anteiso C15:0, C17:0 iso, C17:0 anteiso, and C17:0, respectively), suggesting potential for the use of these fatty acids as microbial markers. These fatty acids are also of interest because of potential anticancer effects (Yang et al., 2000). The feeding of forages in this study resulted in a greater flow of these beneficial branched- and odd-chain fatty acids to the duodenum per unit DMI compared with the study of Ferlay et al. (1993) when feeding corn silage with concentrate: 1.82 vs. 0.90 g 
C15 and C17/kg of DMI, respectively. Phytanic acid is another branched chain fatty acid produced in the rumen, which appears to be biologically active in animal models. Phytanic acid is a chlorophyll metabolite produced by the bacterial hydrogenation of phytol (Patton and Benson, 1966). It is associated with retinal changes of the eye (Yamamoto et al., 1995) and Refsum disease (Klenk and Kahlke, 1963) in humans with a gene mutation resulting in a deficiency of the enzyme phytanoyl-CoA hydroxylase, which is required for the 
-oxidation of phytanic acid (Milhalik et al., 1995). However, in healthy individuals without this gene mutation, phytanic acid has been implicated in the induction of white and brown adipocyte differentiation (Schulter et al., 2002a, b), the prevention of vitamin A teratogenesis (Arnhold et al., 2002), and the ability to activate peroxisome proliferators-activated receptor-
agonists in a similar way to CLAc9t11 as a potential treatment and prevention of diabetes (McCarthy, 2001). Because it is a by-product of chlorophyll breakdown in the rumen, the main dietary intake in humans is through ruminant products from forage-based systems. Greater duodenal flow of phytanic acid in steers fed high-sugar grass silage was due to the greater chlorophyll content of high-sugar grass silage compared with the control grass silage. Lower duodenal flows of phytanic acid with red clover silage are more difficult to explain and may be due to polyphenol oxidase induced inhibition of chlorophyll or phytol hydrolysis in the rumen in line with the reduced biohydrogenation of C18:3n-3.
Long-chain PUFA (C20+) play a major role in animal nutrition because they are important structural components of the cell membrane and are involved in the formation of eicosanoids (Tapiero et al., 2002). These fatty acids are produced from a series of desaturation and elongation reactions by liver enzymes from the base n-6 and n-3 fatty acids, C18:2n-6 and C18:3n-3 (Allam, 2003). The major source of long-chain PUFA (C20+) in ruminant diets would be from supplements containing marine algae or fish oil (Wachira et al., 2000; Scollan et al., 2001; and Shingfield et al., 2003). Because the desaturase and elongase enzymes are absent from the rumen, it was thought that the measurement of these fatty acids in animals fed all forage diets was of little value. However, the flows of these fatty acids recorded in this study (0.97% of duodenal fatty acids) are within previously reported ranges (Wachira et al., 2000; Lee et al. 2003a) when feeding silage and concentrate (without fish oil) and forage silage alone and may be as a consequence of endogenous lipid from sloughed epithelial digestive tract cells.
The feeding of high water-soluble carbohydrate grass silages to beef steers had no effect on C18 PUFA biohydrogenation. The addition of red clover to the diet reduced C18:3n-3 biohydrogenation, with a resultant increase in its flow to the duodenum. These results confirm that there is potential for modifying the fatty acid composition of ruminant products by feeding grass and red clover silages.
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Footnotes
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1 The authors would like to acknowledge and thank Roger T. Evans and his staff at IGER Trawsgoed for the care of the animals, M. S. Dhanoa for his statistical advice, and Delma Jones and Alison Kingston-Smith for their help with the analysis of the samples. This work was carried out as part of a European Commission Key Action 1 project, HealthyBeef QLRT-CT-2000-31423, and was partly funded by the United Kingdom’s Department for Environment, Food, and Rural Affairs. 
2 Corresponding author: michael.lee{at}bbsrc.ac.uk
Received for publication December 18, 2005.
Accepted for publication May 11, 2006.
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Abstract
INTRODUCTION
