The effects of breed , grazing system and concentrate supplementation on the fatty acid profile of the musculus longissimus dorsi and the kidney fat of steers

The study is aimed at determining the effect of breed (German Simmental vs. German Holstein), grazing system (continuous grazing system [CGS] vs. rotational grazing system [RGS]) and concentrate supplementation (level and type of concentrate) on the fatty acid profile of longissimus muscle and kidney fat of steers. The trial involved four phases: in P1 all animals remained indoors; in P2 and P4 they were allocated on CGS or RGS; during P3 one group remained outdoors, the other indoors. In P1 and P3 the steers were offered grass silage and concentrate. In P3 the indoors group received a supplement with a medium or low-concentrate level. For the last 28 days of P4 the steers were offered a concentrate type with 4.9 % linseed oil or with 1.0 % rapeseed oil. CGS-pasture resulted in higher fatty acid values than RGS-pasture; linseed-oil concentrate resulted in higher ALA, ∑n-3 and lower LA, ∑n-6 than rapeseed-oil concentrate. German Simmental breed had lower IMF-content and higher ∑n-3, ∑n-6, n-6/n-3 ratio and PUFA/SFA ratio in longissimus dorsi and kidney fat than German Holstein breed. The proportion of CLA was higher in German Holstein breed than in German Simmental breed (0.56 vs. 0.50 g/100 g FAME). RGS group showed lower ALA and higher n-6/n-3 in longissimus dorsi and kidney fat than CGS-group. Neither the level nor the type of concentrate affected the LD and kidney fat fatty acids. Healthy fatty acids levels were higher in the German Holstein breed meat. The CGS-group meat had higher contents of ALA and EPA. However, the legal requirements for human nutrition and other health related claims could not be met.


Introduction
The increasing demands of consumers for meat with a healthy fatty acid profile and low fat content offering additional health benefits are promoting the extensification of the beef produc tion system.
The consumption of ruminant's meat and milk rich in saturated fatty acids (SFA) has been linked with coronary heart disease, hypertension, inflammation, mammary cancer and high cholesterol concentration in blood (de Deckere et al. 1998, Tapiero et al. 2002).Nevertheless ruminant meat and milk are also good sources of n-3 PUFA in the human diet (Scollan et al. 2001).
However, it is known that fatty acids of adiposities in ruminants derive from de novo synthesis or from certain diets, suggesting that enzymes involved in lipogenesis are sen sitive to dietary energy levels and possibly to the energy source itself.Studies on breed type and cattle feed strategies have demonstrated that it is possible to manipulate the profile of fatty acids including conjugated linoleic acid (CLA) isomers (Dannenberger et al. 2009).Some studies have indicated the presence of a breed related pattern of fat deposition and fatty acid synthesis.However, there are conflicting results, as either certain authors referred to a higher content of n-3 fatty acids in meat breeds than in dairy breeds (Nuernberg et al. 2005) or observed the opposite effect (Choi et al. 2000, Moreno et al. 2008).Pasture-finished cattle resulted in meat with a lower proportion of SFA, greater n-3 and less n-6 PUFA and higher CLA compared to high-grain-finished cattle (French et al. 2003, Nuernberg et al. 2002, Realini et al. 2004, Noci et al. 2007, Fincham et al. 2009).Additionally, the inclusion of PUFA-rich plant-oil or seeds in ruminant rations (soya oil, linseed oil) increased the concentration of n-3 PUFA, especially ALA and CLA , despite the extensive biohydrogenation of dietary lipids within the rumen.Also restricted grazing plus plant-oil-enriched rations improved the content of some healthy fatty acids in the meat of heifers (Noci et al. 2007).However, the aim to achieve the values of n-3 fatty acids required to use the label »source of n-3 fatty acids« was not achieved.According to the legal claims on nutritional value and health benefits of food (Regulation [EC] 1924[EC] /2006[EC] and 432/2012) ) only food with at least 0.3 g/100 g of ALA or 40 mg/100 g asthe sum of EPA and DHA at a maximum energy content of 100 kcal are allowed to be considered »source of n-3 fatty acids«.Is it possible to achieve these levels with grazing animals supplemented with a con cen trate rich of n-3 fatty acids?Therefore, the purpose of the present study was to test the effect of two grazing systems supplemented with two plant oil-enriched rations during the last 28 days of the final grazing period on the fatty acid profiles of meat and kidney fat of the two most important breeds in Germany.

Experimental design and animal management
The study was carried out in the research station »Zurnhause« at the University of Applied Sciences Weihenstephan-Triesdorf, Germany.
A group of 96 steers (German Simmental [GS] and German Holstein [GH]) were fattened from January 2011 to October 2012.The initial weight of the animals: GS=171 kg; GH=157 kg.Details of the fattening process and feeding regimen used are extensively described in Schmutz et al. (2013).
As is observed in Table 1, the experiment was divided in four phases (from P1 to P4): During P1 all animals remained indoors, subsequently in P2 they were moved to two different grazing systems (continuous grazing system [CGS] or rotational grazing system [RGS]).In P3 a group of 24 animals stayed outdoors and the rest of the group was kept indoors.During P4 the animals were raised as in P2.Animals were fed according to Table 1.In P4, 28 days prior to slaughter, two kinds of concentrate were offered to the steers (a concentrate mixture with 4.9 % linseed oil or a concentrate mixture with 1.0 % rapeseed oil).After reaching the final weight, 94 steers were slaughtered during the period running from the end of August until mid-October 2012.

Slaughter procedure, sampling and analyses
Steers were slaughtered at two-week intervals when they reached a live weight of 625 kg (average).They were transported to the experimental abattoir belonging to the Bavarian State Research Center for Agriculture (LfL) in Poing-Grub, which is located 48 km away from the farm.The transport was carried out according to the EU regulations on animal welfare rules.After a fasting period of 24 h, the steers were slaughtered according to EU laws.Pro cedures were conducted according to the guidelines of the Council Directive 2010/63/EU on the protection of animals used for experimental and other scientific purposes.
Samples of kidney fat were taken from the left side of the carcasses.These were sub sequently vacuum-sealed in plastic bags and deep frozen at −20 °C.After chilling the carcasses at −4 °C for 24 h, a muscle sample was taken from the musculus longissimus dorsi (LD) at the 9th rib of the left side.Each sample was cleared of adipose tissue, cut into small cubes and mashed in a knife mill to form a homogenous paste.Then they were vacuum-sealed in plastic bags and kept frozen at −20 °C until they were analyzed.Intramuscular fat (IMF) of LD was determined with the near-infrared spectroscopy (NIRS) method (Schmutz et al. 2013).
The fatty acid profile of pasture (P4), concentrate (P4), LD and kidney fat were determined according to Firl et al. (2014) in the Bioanalytik laboratory Weihenstephan of the Technical University Munich (TUM).
The fat extraction of the samples was carried out according to Bligh & Dyer (1959) modified by Hallermayer (1976).One gram of homogenate sample was mixed with chloroform/metha nol (1:1, v/v) and the internal Standard Trinonanoate (Sigma, Taufkirchen, Germany) for 90 s (Ultra Turrax, 8 000 rpm).Next the samples were centrifuged for 5 min at 4 °C (4 000 rpm).The overlap was decanted into a separating funnel and the pellet was extracted twice more and the overlaps combined in the separating funnel.After adding 21 ml of physiological solution of

RS-oil
Level of offered concentrate: medium (M)=1.5 kg/animal/d, low (L)=0.75kg/animal/d, LS-oil: linseed-oil concentrate, RS-oil: rapeseed-oil concentrate, total consumption of concen trate in the whole trial: M=275 kg/animal, L=191 kg/animal sodium chloride the mixture was then shaken for one minute.After the phase separation (1 h) the chloroform was evaporated to dryness under vacuum conditions at 37 °C by use of a rotary evaporator (Rotavapor R 210, Büchl Labortechnik GmbH, Essen, Germany).The residue was dried with nitrogen for 15 min and then dissolved in one milliliter tertiary butyl-methylether (t-BME).Of this solution 100 µl together with 50 µl trimethylsulfonium-hydroxide (TMSH) were pipetted into a microvial, shaken and injected on the gas chromatograph.The fatty acid methyl ester (FAME) was broken down with a Hewlett Packard 6890 GC, equipped with an Agilent 7683 Autosampler (Agilent Technologies, Böblingen, Germany).A CP 7420 separation column was used (coating select FAME 100 % bonded cyano-propyl-phase, 100 m × 0.25 mm, Chrompack, Varian, USA) with a 0.25 µm film thickness and flame ionisation detector.The split/splitless injector was used with split 50.The samples were injected at 60 °C.The oven temperature was then raised by 6 °C/min to 120 °C, held for 9 min, subsequently increased by 3 °C/min to 242 °C, held for 90 min, then increased to the final temperature of 250 °C and held for 100 min.The injector was set at 270 °C and the detector at 280 °C.Hydrogen (Westfalen, Münster, Germany) was used as the carrier gas.Peaks were identified by comparison of retention times with known FAME standards.The fatty acids and the fatty acid distribution were analysed according to the DGF standard methods, C-VI 10a (DGF 2000) following the internal standard method.For this purpose the software Chromeleon (version 6.80) was used (Chromeleon, Dionex, Sunnyvale, USA).A FAME-Mix 37 Supelco reference standard (Sigma-Aldrich, Deisenhofen, Germany) was used.More fatty acid methyl ester was purchased from Sigma-Aldrich und conjugated linoleic acid (CLA) from Biotrend (Köln, Germany).
The fatty acid contents in meat were estimated using the IMF content of LD.

Statistical analysis
The statistical analysis was carried out with SPSS v. 20.0 (SPSS Inc., Chicago, IL, USA).All param eters were subjected to a four-factorial ANOVA according to the general linear model (GLM).Breed (A i ), grazing system (B j ), concentrate level (C k ) and type of concentrate (D l ) were used in the model as fixed elements.
where Y ijkl is the observed value of ijkl animal, µ is the population mean, e ijkl is the residual error.Differences were tested by means of the F-Test.After robust F-Test results the means were compared using the Tukey-Test.

Results and discussion
According to the data presented in the companion paper (Schmutz et al. 2013), the GS-breed proved to be significantly superior to the GH-breed in all essential parameters of the fattening and the carcass performance (final weight: 631 kg vs. 608 kg).Conversely, the GH-breed showed better meat quality parameters (IMF, tenderness, meat colour) (e. g.IMF: GH 3.89 % vs. GS 2.47 %).The grazing system had a significant impact on only a few parameters (e. g. carcass weight, dressing percentage, fat colour).The higher quality of the CGS pasture means a higher grazing yield and a better feedstuff quality.This led to a slightly improved carcass weight.The level of concentrate had no significant effect on the compiled parameters.
During the supplementation period in P4 (28 d before slaughter) some animals consumed little or no concentrate and so animals with a total concentrate intake of less than 5 kg (level low) and 7 kg (level medium) were not considered in the statistical analysis.As a effect, our results showed a slight increase in the IMF-content in comparison to the results for meat quality stated by Schmutz et al. (2013).
No statistical significance was found in the first, second and third order interactions for the parameters under consideration in this study.

Fatty acids profile of pasture samples and concentrates
According to Table 2, the most abundant fatty acids are LA, ALA and palmitic acid.This is in accordance with Clapham et al. (2005) and Dewhurst et al. (2001) who found the same tenden cy in the species under investigation.The same authors also stated that harvest date and interval have a significant impact on PUFA levels and that the concentrations of fatty acids decline as plants develop and mature.This was the case for the RGS-pasture and explains the presence of the higher amounts of PUFA and n-3 fatty acids (especially ALA) in the CGSpasture than in the RGS-pasture.These high contents implied high values of MUFA and n-6 fatty acids in RGS-pasture.Consequently, the n-6/n-3 ratio was twice as large in the RGS as in the CGS (0.47 vs. 0.20).
The botanical composition of the forage samples was not investigated further, but nearly all of the CGS was covered with perennial ryegrass and white clover.It should be noted that different botanical composition of both grazing systems could also explain the differences in their fatty acid composition.
In the concentrate mixtures in P4 (Table 2), higher levels of n-3 fatty acids (11.29 vs. 3.73) and lower levels of n-6 fatty acids (25.01 vs. 34.17)were detected in the linseed-oil con centrate than in the rapeseed-oil concentrate.Thus, the n-6/n-3 ratio was 2.2 in the linseed-oil concentrate and 9.2 in the rapeseed-oil concentrate.This is in accordance with the fatty acid composition of these oils.Thus, linseed oil presents a higher content of C 18:3 and lower content of C 18:2 than rapeseed oil (Noci et al. 2007, Pospišil et al. 2007).Additionally, the amount of oil present in the concentrate was higher for the linseed-oil concentrate than for the rapeseed-oil concentrate (4.9 % vs. 1.0 %).

Fatty acid profile in LD
The type of breed had a significant effect on most of the fatty acids of the LD.While the GH-breed showed high proportions of CLA, SFA and trans-FA, the GS-breed had significantly higher proportions of PUFA, n-3 (ALA, EPA, DPA, DHA) and n-6 (LA, C 18:3) fatty acids.Nevertheless, the GH-breed had a significantly lower n-6/n-3 ratio.This is in line with ear lier research, which has shown that, in addition to nutritional factors, genetic factors are con sidered to contribute to differences in fatty acid composition (Scollan et al. 2006).Such dif ferences between breeds occur due to a different gene expression or enzyme activity involved in fatty acid synthesis (De Smet et al. 2004).Differences between both breeds were also found in a study by Nuernberg et al. (2005) where German Simmental bulls showed higher proportions of n-6 and n-3 fatty acids and a higher n-6/n-3 ratio than German Holstein bulls.Choi et al. (2000) and Moreno et al. (2008) describe a significantly higher n-6/n-3 ratio in dairy breeds than in meat breeds.Conjugated linoleic acid deposition is also influenced by breed (Costa et al. 2012, Shen et al. 2007) and is related to vaccenic acid variations (Shen et al. 2007).Small proportions of CLA pass through the rumen and the main proportion of CLA in the fat tissue derives from local biosynthesis by vaccenic acid and by the effect of the ∆9-desaturase enzyme (Costa et al. 2012, Baumann et al. 1999).So this explains the significantly low values of linoleic acid in the GH-breed which exhibit significantly high values of trans-vaccenic acid and CLA.Grazing systems had a significant impact on few fatty acids.The CGS-group showed a significantly higher proportion of ALA and a significantly lower n-6/n-3 ratio in comparison with the RGS-group.As most of fatty acids stem from the microbiological synthesis in the rumen, the significant effects of grazing systems might possibly be explained by the differ ing rumen environments of the animals caused by different levels of raw fiber supply in pasture grass (raw fiber supply RGS>CGS, Schmutz et al. 2013).Razminowicz et al. (2006) found no evidence of the feeding practice having an influence on C 17:1, 18:1 12t and 22:1.The significant impact of grazing systems on the higher content of ALA and trans-vaccenic acid in the CGS-group is in line with the high content of ALA in the CGS-pasture.This heightened intake of ALA (Figure 1-Graphic A) might cause a higher proportion of ALA to be metabolized into trans-vaccenic acid and to then be accumulated in the intramuscular fat tissue; which leads to a significant lower n-6/n-3 ratio.In the case of C 22:1, its content in both grazing systems was probably different (which was not reported; Table 2) and caused the significant effect of grazing system on C 22:1 since this fatty acid is probably not subjected to the biological hydrogenation (Borgatti & Trigari 1979).

Fatty acid profile of the kidney fat
Similarly, the breed had a significant effect on the fatty acid profile of the kidney fat (Table 4) for C 18:1 9c, LA and C 20:3.The GS-breed had a significantly higher proportion of LA, which did not affect the n-6/n-3 ratio.Effects on LA and C 20:3 were also observed in the LD.The effect of breed on oleic acid might be attributed to the observed significant difference of the LA, as any consumed LA would be transformed into stearic acid in the rumen and finally metabolized into oleic acid by ∆9-desaturase in the fat tissue (Baumann et al. 1999).
The higher oleic acid level of LD compared to kidney fat could be due to a lower activity of the ∆9-desaturase in the kidney fat (Lee et al. 2011).The higher proportion of LA in the RGSpasture could explain the significant difference in trans-vaccenic acid (CGS-group<RGS-group).The observed higher proportion of oleic acid in the kidney fat of the CGS-group is ex plained by the high ruminal transformation of LA into trans-vaccenic acid, which is then metabolized into oleic acid in the kidney fat as would be the case in RGS-group.Scollan et al. (2001) found that a higher content of ALA in the feedstuff leads to higher contents of ALA in the muscles and the subcutaneous adipose tissue.This could explain the higher proportion of ALA in the CGS-group.
According to Ashes et al. (1992) heavier and fattier carcasses have higher proportions of neutral fat, which mainly contains SFAs.So this might explain the significantly higher proportion of ∑SFA in the kidney fat of the CGS-group which had heavier and fattier carcasses (Schmutz et al. 2013).
It is remarkable that, with the exception of C 20:1 11c, neither the level of the concentrate nor the type of concentrate had a significant effect on the analysed fatty acids of LD and kid ney fat.It is likely that the consumed concentrate was low and neither level M (275 kg/ animal) nor level L (191 kg/animal) could influence the fatty acid profile.The considered proportions of plant-oils in concentrates were probably low and as such they were unable to cause differences in the studied fatty acid profiles.

Fatty acid profile of the meat (LD)
The IMF of the GH-breed was significantly higher than that in the GS-breed (4.1 vs. 2.4 %).Consequently, the GH-breed presented higher nutritionally relevant fatty acid values (ALA, CLA, EPA, DHA) in meat (Table 5).High IMF leads to a high CLA level in muscle (Moreno et al. 2008), which is mainly located in the neutral fats and rises with increasing fatness (Scollan et al. 2003, Noci et al. 2007).
The GH-breed showed a significantly higher proportion of SFA and trans-fatty acids but nevertheless a lower PUFA/SFA ratio.Both genotypes show a very tight n-6/n-3 ratio.

Healthy fatty acids in the meat (LD)
A portion of 200 g of meat (LD) from GH-breed provides 47 mg CLA.Moreno et al. (2008) obtained 38 mg CLA with a similar calculation.The obtained n-6/n-3 ratios are under the recommended ratio by the German Association for Nutrition (DGE 2008) (<5:1).This value is difficult to reach due to the ruminal biohydrogenation (Warren et al. 2008).In order to be allowed to use the label »source of omega-3 fatty acids«, the food has to contain at least 0.3 g/100 g of ALA or 40 mg/100 g as the sum total of EPA and DHA at a maximum energy content of 100 kcal (Regulation [EC] 1924[EC] /2006[EC] and 432/2012).In the study only 17 % in the ALA and 40 % in the sum EPA+DHA of these recommendations were achieved (ALA: 56 mg [GH-breed], 41 mg [GS-breed]; sum of EPA+DHA: 17 mg [GH-breed], 14 mg [GS-breed]).So they do not qualify for nutrition and health related claims for beef produced under the conditions of the present study.A maximum of 60 mg ALA and 18 mg EPA+DHA for the GH-breed in the CGS-group was obtained.Warren et al. (2008) reported similar levels with 43 mg/100 g ALA and 24 mg EPA+DHA; and Nuernberg et al. (2002) reached 71 mg/100 g ALA.After considering the transformation rate of ALA into EPA (8-12 %) in the human body (Goyens et al. 2006), the value of EPA+DHA was 24 mg for GH-breed and 19 mg for GS-breed both in CGS-group.
The beef, besides milk and eggs, is the single natural source for long chain n-3 fatty acids for people who do not eat fish or fish products.In agreement to the results of our study, Nuernberg et al. (2002) and Razminowicz et al. (2006) determined that these products cover the human requirements only to a small extent.Hence it was possible to raise the proportions of nutritionally beneficial fatty acids in the meat of steers through a feeding practice based on small provisions of concentrate and high amounts of grass (grass silage and pasture).
In conclusion, in fattening steers fed with grass and grass products including limited supple mentation of concentrate, both genotype as well as grazing system had an effect on the fatty acid profile of the LD and kidney fat.While the genotype primarily affected the fatty acid profile of LD, the grazing system critically influenced the fatty acid profile of the kidney fat.Concentrate level and type of concentrate did not have a decisive influence.In general, the contents of healthy fatty acids in the meat, especially n-3 fatty acids could be raised.How ever, the legal requirements for a corresponding marketing could not be met.

Figure 1
Figure 1Calculated selected fatty acids intake (g/d/animal) according to the registered dry matter intake as forage and as concentrate (level medium) inSchmutz et al. (2013).A: from RGS or CGS, B: with or without oil supplemented concentrate under CGS, C: with or without oil supple mented concentrate under RGS (0.001) 0.339

Table 2
Fatty acid profiles (g/100 g FAME) during the last grazing period (P4) of pasture from the rotational and continuous grazing system also of concentrate with linseed and rapeseed oil

Table 3
Fatty acid profile (g/100 g FAME) of selected fatty acids of musculus longissimus dorsi of steers of the breeds GH and GS in relation to grazing system, concentrate level and type of concentrate (LS-Means [SEM])

Table 4
Fatty acid profile (g/100 g FAME) of selected fatty acids of kidney fat from steers of the breeds GH and GS in relation to grazing system, concentrate level and type of concentrate (LS-Means [SEM])

Table 5
Concentration of selected fatty acids (mg/100 g meat) in the musculus longissimus dorsi of steers of the breeds GH and GS in relation to grazing system, concentrate level and type of concentrate (LS-Means [SEM])