Fatty acid composition of beef steers as affected by diet and fat depot

C. Mapiye, P. Vahmani, J.L. Aalhus, D.C. Rolland, V.S. Baron, T.A. McAllister, H.C. Block, B. Uttaro & M.E.R. Dugan 1 Department of Animal Sciences, Faculty of AgriSciences, Stellenbosch University, P. Bag X1, Matieland 7602, South Africa 2 Agriculture and Agri-Food Canada (AAFC), Lacombe Research Centre, 6000 C & E Trail, Lacombe, Alberta, Canada T4L 1W1, 3 AAFC, Lethbridge Research Centre, 1 Avenue South 5403, PO Box 3000, Lethbridge, Alberta, Canada T1J 4B1


Introduction
In the past two decades, links between intake of trans fatty acids (FA) and saturated FA with heart disease have led to recommendations to limit their consumption in foods, including beef (Mapiye et al., 2015;Nantapo et al., 2015;Vahmani et al., 2015).Recently, growing consumer interest in healthy diets has triggered research to enrich beef with polyunsaturated fatty acids (PUFA) and biohydrogenation products (BHP), particularly rumenic acid (cis 9, trans 11-18:2) and its precursor vaccenic acid (trans 11-18:1; Mapiye et al., 2012;2015) which have potential human health benefits (Field et al., 2009;Dilzer et al., 2012).The proportions of these BHP in beef can be increased by feeding forages in combination with high levels of PUFA (Mapiye et al., 2012;2015;Vahmani et al., 2015).However, feeding PUFA in high-forage diets results in lower growth rates, smaller carcasses and an undesirable meat appearance compared with high concentrate diets (Webb, 2006;Mapiye et al., 2013a).Recently, it was demonstrated that these problems could in part be alleviated by replacing forage (red clover silage (RC)) with wheat dried distillers' grains plus solubles (DDGS) as a non-forage fibre source (Mapiye et al., 2014a).
The composition of PUFA-BHP in beef is influenced by fat depot, with external depots (e.g.subcutaneous fat (SCF)) and internal depots (e.g.perineal fat (PRF)) with the greatest propensity to accumulate rumenic and vaccenic acids, respectively (Jiang et al., 2013;Mapiye et al., 2013b;2014b).These differences have important health implications when one examines which depot could be incorporated into meat products such as minced beef.Currently, SCF is used when making minced beef, which is considered by many to be the most popular and most versatile of all beef products (Brewer, 2012).On the other hand, PRF is an underutilized fat depot, but has a higher content of total PUFA-BHP and is more easily accessible during the slaughter process than SCF (Mapiye et al., 2014b).Strategies to enhance the PUFA-BHP composition of beef products should therefore include feeding designer diets and exploiting fat depot heterogeneity.The current study was an extension of the research by Mapiye et al. (2014a).The objective was to compare PUFA-BHP profiles in SCF and PRF fat from the same group of steers fed RC sunflower seed and varying levels of wheat dried distillers' grain with solubles (DDGS).

Material and Methods
Animal management and the ingredients and nutritional composition of experimental diets were previously described by Mapiye et al. (2014a).Briefly, sixty-four 12-month-old British × Continental crossbred steers with an initial mean bodyweight (BW) of 362.7 ± 4.50 kg were randomly allocated to four dietary treatments (control, sunflower seed (SS), DDGS-15 and DDGS-30), with two pens of eight steers per dietary treatment.The control diet was composed of 70% red clover silage, 25.8% barley grain and 4.2% vitaminmineral supplement on a dry matter (DM) basis (Table 1).The SS diet contained 11.4% SS substituted for barley grain, and the DDGS-15 and DDGS-30 diets contained 15% and 30% DDGS, respectively, substituted for red clover silage and SS to maintain a targeted 5% added oil in the diets from SS or DDGS (Table 1).
At slaughter, PRF was collected closest to the geometric centre of the whole PRF, vacuum packed and held in a 2 ºC cooler with a wind speed of 0.5 m/sec pending FA analysis.Carcasses were cooled for 24 h under the same conditions prior to collecting and vacuum packing SCF covering the longissimus thoracis et lumborum.A 50 g subsample of each fat depot was homogenized using a Robot Coupe Blixir BX3 food processor (Robot Coupe USA Inc., Ridgeland, Miss, USA) and frozen at −80 ºC for subsequent FA analysis.
Statistical analyses were conducted using Proc Mixed of statistical analytical systems (SAS, 2009).All the data were analysed as a two-way ANOVA, including fixed effects of diet, fat depot and diet × fat depot interaction, and slaughter day and animal (diet) as random effects.Treatment means were generated and separated using the LSMEANS and PDIFF options, respectively.The significance threshold was set at P <0.05.

Fatty acids of dietary or endogenous origin
Diet and its interaction with fat depot did not (P >0.05) affect total FA content (Table 2).Overall, the proportions of total saturated FA (SFA), 14:0 and 16:0 decreased (P <0.05) with SS addition, but further substitutions with DDGS led to no further changes (P >0.05;Table 2).Conversely, the proportions of 18:0 increased (P <0.05) with the addition of SS to the control diet, but further substitutions with DDGS led to no (P >0.05) further changes.These results resemble patterns of dietary proportions of the individual SFA observed in the current study.Current results may also be partly linked to the influences of both rates of complete biohydrogenation of PUFA to 18:0 and high proportions of 18:2n-6, which were previously reported by Shingfield et al. (2013) to down-regulate Δ-9 desaturase activity in adipose tissues.
The proportions of total cis-monounsaturated FA (c-MUFA) were not (P >0.05) influenced by diet.The proportions of c9-14:1 tended to decline (P = 0.06) with the addition of SS to the control diet.The inclusion of DDGS led to further reductions (P = 0.06).Shingfield et al. (2013) reported similar results and attributed them to the inhibitory effects of high dietary levels of 18:2n-6 on de novo synthesis of FA, including conversion of 14:0 to c9-14:1.Adding SS to the control diet did not (P >0.05) change the proportions of c9-16:1 (Table 2), but further substitutions with DDGS did reduce (P <0.05) its proportions.The results could be linked partly to the high proportions of 18:2n-6 in adipose tissues that were observed when feeding DDGS diets (Table 2).
According to Nakamura et al. (2004), high levels of 18:2n-6 can inhibit Δ-9 desaturase activity, consequently reducing de novo synthesis of c9-16:1 from 16:0.Feeding SS diet had no (P >0.05) effect on the proportions of c9-18:1, but further substitutions with DDGS increased (P <0.05) its proportions (Table 2).In line with current findings, Cruz-Hernandez et al. (2007) demonstrated that increasing 18:2n-6 in the diet is capable of inhibiting biohydrogenation in the rumen, leading to the accumulation of intermediate products in adipose tissues.Overall, variations in c-MUFA are difficult to interpret, because c-MUFA can originate from both diet and endogenous synthesis.Inclusion of SS in the control diet led to reductions (P <0.05) in the proportions of total and major n-3 PUFA, but further substitutions with DDGS led to no (P >0.05) further changes (Table 2).This is consistent with the dietary proportions of 18:3n-3 for the various dietary treatments.Thus, these results could be related to the substitution of barley grain and RC with SS and DDGS, which both have relatively lower concentrations of 18:3n-3.There is also a possibility that high proportions of 18:2n-6 in the adipose tissues of steers fed SS and DDGS might have reduced the elongation of 18:3n-3 to 22:5n-3 due to competition for the Δ-6 desaturase (Johnson et al., 2012).
Fat depot influenced the total FA content, with PRF having a higher (P <0.05) content than SCF (Table 3).This could be linked to differences in water content between these fat depots, with SCF having higher water content than PRF (Anderson et al., 1972).Saturated FA (SFA) and cis-monounsaturated FA (c-MUFA) were the major families of FA of dietary or endogenous origin (Table 3).Fat depot influenced (P <0.05) proportions of total SFA, with PRF having larger (P <0.05) proportions than SCF.These dissimilarities could be linked to the proportions of 18:0, which exhibit a similar pattern.Generally, less mature fat depots situated externally, such as SCF, are less saturated than mature fat depots situated internally, such as PRF (Christie, 1978;Lee et al., 2011;Jiang et al., 2013).This is linked to a greater Δ-9 desaturase activity index in external depots as opposed internal ones, and replacement of 18:0 with c9-18:1 in external fat depots.

Fatty acids of microbial origin
Branched-chain FA (BCFA), trans-monounsaturated FA (t-MUFA), CLA and non-conjugated 18:2 BHP (i.e.atypical dienes (AD)) were the major FA groups of microbial origin (Table 4;Fig 1A).Inclusion of SS in the diet led to reductions (P <0.05) in the proportions of total and major BCFA.Proportions of these FA were further reduced (P <0.05) by substitution with increasing levels of DDGS.Given that several BCFA in animal tissue are synthesised de novo by rumen microbes (Vlaeminck et al., 2006), it may be speculated that high levels of 18:2n-6 in SS and DDGS diets might have inhibited rumen microbes (Liu et al., 2011) leading to a decline in BCFA production.Differences in dietary protein content and interaction between dietary nutrients on rumen ecology and fermentation might also have played a role (Vlaeminck et al., 2006).Subsequent decreases when feeding DDGS could be due to reduced ruminal propionate (a precursor for BCFA biosynthesis) production from readily fermentable starch (Pethick et al., 2004).Total and major BCFA were affected (P <0.05;Table 4) by fat depot and diet, but their interaction was not significant (P >0.05).Perirenal fat as opposed to SCF had greater (P <0.05) proportions of total BCFA and slightly lower (P <0.05) proportions of anteiso-17:0.These findings could be linked to depot-specific differences in incorporation of individual FA (Hood et al., 1976).-15:0, anteiso-15:0, iso-16:0, iso-17:0, anteiso-17:0, iso-18:0.The interaction between fat depot and diet was significant for total and major t-18:1 FA (Figure 1A).Perirenal fat as opposed to SCF had greater (P <0.05) proportions of total and major t-18:1 FA including vaccenic acid (t11-18:1).These proportions were accentuated (P <0.05) by feeding SS, maintained (P > 0.05) by feeding DDGS-15, and attenuated (P <0.05) by feeding the DDGS-30 diet.This could be the result of a combination of factors including lower Δ-9 desaturase activity indices in PRF vs. SCF (Lee et al., 2011;Jiang et al., 2013), higher dietary 18:2n-6 observed for the diets containing SS, greater bypass of 18:2n-6 when feeding DDGS diets, and a decline in dietary fibre with increasing levels of DDGS, which might have reduced ruminal pH and negatively influenced biohydrogenation in the rumen (Hristov et al., 2005;Felix et al., 2012).It may also be due to greater de novo synthesis of FA in SCF and dilution of PUFA-BHP when DDGS were added to the diet.
Total AD and t8,c12-18:2, one of the major AD isomers that is probably derived from 18:2n-6, were affected by fat depot × diet interaction, with SCF having greater proportions than PRF.These AD proportions were increased (P <0.05) by inclusion of SS, but further substitutions with DDGS led to no further changes (P >0.05; Figure 1C).These findings could reflect selective uptake or lower rate of metabolism of these AD in SCF (Kramer et al., 1998) and greater proportions of 18:2n-6 observed for the SS containing diets.

Conclusions
The remarkable differences between fat depots were related to the greater proportions of SFA and lower proportions of c-MUFA in PRF, as opposed to SCF.Feeding SS and DDGS-15 diets led to increases in proportions of vaccenic acid in PRF and rumenic acid in SCF, but feeding DDGS-30 was not so effective.Future strategies to enhance the composition of t-18:1 and CLA in beef fat without compromising animal performance and meat quality may therefore include feeding SS and 15% DDGS in forage-based diets and exploiting fat depot heterogeneity.

Fig 1
Fig 1 Effects of fat depot and diet on trans-18:1 isomers (A), CLAs (B) and atypical dienes (C) from beef steers.

Table 1
Ingredients, nutrients and fatty acid composition of the dietary treatments

Table 2
Effect of diet on adipose tissue fatty acids (% total FA) of endogenous or dietary origin from beef steers

Table 3
Effect of fat depot on fatty acids (% total fatty acids) of endogenous or dietary origin from steers fed red clover silage with sunflower seed and dried distillers' grains plus solubles

Table 4
Effect of fat depot and diet on branched-chain fatty acid profiles (% total FA) of beef steers Means with different superscripts for a particular fatty acid profile are significantly different (P <0.05); SEM: standard error of mean; SS: sunflower seed; DDGS-15: 15% wheat dried distillers' grain with solubles + sunflower seed; DDGS-30: 30% wheat dried distillers' grain with solubles + sunflower seed; ∑ BCFA: branched chain fatty acids: sum of iso