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SID 5 Research Project Final Report

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The SID 5 (Research Project Final Report) is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra website. A SID 5 must be completed for all projects.

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Project identification

1. Defra Project code LS3515

2. Project title

Optimising the eating quality of beef produced on sustainable forage systems

3. Contractororganisation(s)

Institute of Grassland and Environmental Research (IGER)Plas GogerddanAberystwythCeredigionSY23 3EB     

54. Total Defra project costs £ 1,297,565

5. Project: start date................ 01 July 2001

end date................. 30 June 2006

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6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing SID 5s contractors should bear in mind that Defra intends that they be made public. They

should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the SID 5 can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

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Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

In the UK, forage feeding systems based on grass and clover are important to the beef industry as they benefit from low inputs, are more sustainable and ensure that production costs are kept to a minimum and profitability is maximised. A distinct and under-exploited advantage of these systems is the potential for the production of beef with higher concentrations of “healthy” n-3 polyunsaturated fatty acids (PUFA) and other constituents derived from grass which may have health benefits and which also affect meat quality i.e. shelf-life and flavour. This research examined how these beneficial factors in forages may influence the nutritional value (n-3 PUFA and conjugated linoleic acids) and meat quality (shelf-life, colour and flavour) of beef. The research included a comparison between grass and concentrate feeding and examined how the fatty acid composition of beef may be further optimised on grass and/or clover feeding by the inclusion of beneficial ruminally protected oils. To compliment these studies, major rumen processes (lipolysis and biohydrogenation) which have a large effect on the fatty acid composition of beef have been investigated. Key management factors (wilting, use of inoculants) which influence important fatty acid composition of grass and clover during the ensiling process were investigated.

Results from the project illustrate that beef may be produced which is low fat (less than 4%), have a lower content of atherogenic saturated fatty acids (SFA), higher content of more beneficial monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) and lower n-6:n-3 PUFA ratio than was previously possible. Grass relative to concentrate feeding resulted in higher levels of n-3 PUFA (including the long chain C20 PUFA) resulting in very favourable n-6:n-3 ratios. Feeding concentrates richer in n-6 PUFA results in higher levels of linoleic acid (18:2n-6) and the longer chain n-6 PUFA. Grass grazing not only produces meat with an improved n-3 fatty acid content but also produces more yellow fat due to its carotene content and a greater lipid stability due to the natural intake of vitamin E with the diet. Supplementing animals with concentrates whilst grazing over a 100 day period tended to reduce the natural vitamin E content of the meat and reduce stability. Ruminally protected lipids (as noted in other studies at IGER and Bristol) resulted in large increases in 18:2n-6 and 18:3n-3 contributing to a very beneficial ratio of polyunsaturated : saturated fatty acids. This highlights the role of the rumen in regulating the ability to alter the fatty acid composition of beef. The protected lipids, however, caused a severe oxidative challenge in the meat (reduced stability) and which may be ameliorated by supplementing the diet with vitamin E. Relative to feeding grass silage, red clover beneficially enhanced the content of PUFA (both 18:2n-6 and 18:3n-3) but due to low content of vitamin E in the meat, red clover reduced colour shelf life. The reduction in shelf life could be ameliorated by feeding additional vitamin E with the red clover silage.

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A series of in vitro and in vivo studies on rumen lipid metabolism have extended substantially our knowledge base of the major mechanisms involved. In vitro studies have highlighted the importance of plant chemical constituents during lipid metabolism in the rumen most notability the role of (1) plant lipases in conducting the initial lipolysis of plant lipids and (2) the importance of red clover containing polyphenol oxidase in reducing lipolytic activity in the rumen and subsequently reducing biohydrogenation (and delivering higher levels of PUFA into meat as described above) and (3) the role of green odour compounds in manipulating the extent of biohydrogenation and the formation of metabolically important intermediates such as vacennic acid through their antimicrobial properties. In vivo studies have made excellent progress in characterising the process of ruminal lipolysis and biohydrogenation of dietary lipids, under a wide range of dietary conditions. The results confirm that the extent of biohydrogenation dietary PUFA from a range of different feed types, including forages, is very high, averaging approximately 86 and 92% for 18:2n-6 and 18:3n-3, respectively. However, we have noted lower levels of biohydrogenation, measured in vivo, when feeding red clover relative to grass silage. Red clover contains the enzyme polyphenol oxidase (PPO) which is activated when red clover tissue is damaged, reducing the extent of lipolysis and subsequently biohydrogenation. Feeding concentrates containing fish oil has also been found to reduce biohydrogenation resulting in increased production of vacennic acid, the major intermediate for the synthesis of CLA in tissue.

The proposed research is in support of DEFRA policy objectives of improving the eating quality (healthiness and flavour) of British beef by exploiting the many positive elements associated with grass and clover based production systems which are less intensive and which support more sustainable forms of beef production. The research contributes towards improving the competitiveness of the UK beef industry and also has major implications for the consumer by providing more healthy and wholesome food. The results will be used to help promote beef as a functional food, low in fat and containing beneficial fatty acids (such as n-3 PUFA and CLA) and to help create marketing opportunities through the identification of marketable points of difference, hence contributing to added value.

OUTCOMES AND FUTURE RESEARCH REQUIREMENTSThe effects of red clover on PUFA composition of meat most likely relates to the presence of polyphenol oxidase (PPO) in forage. This requires further investigation. Mechanisms which reduce the extent of biohydrogenation of dietary lipids by rumen micro-organisms are required and research should concentrate on these aspects. Understanding the major microbial species involved in biohydrogenation and how they are influenced by diet will permit an increased understanding of methods of modifying this process. Increasing our understanding and developing methods of altering lipolysis and biohydrogenation of dietary PUFA in the rumen is essential in terms of providing new opportunities for enhancing the fatty acid composition of beef and other ruminant products.

Grass feeding may have some beneficial effects on improving lipid and colour stability of raw meat but there is evidence that this merits further attention when these materials are further processed. The relationships between vitamin E (and other antioxidants) and n-3 PUFA in meat to ensure adequate stability during processing require attention. Evidence for beneficial relationships species-rich pastures and fatty acid composition and sensory attributes of meat is interesting and merits further attention. The transfer of linolenic acid from forage through to meat is dependent on two important processes: (1) increasing the level of linolenic acid in the forage (and hence into the animal) and (2) reducing the extent of ruminal biohydrogenation. Research should focus on increasing our understanding of these two major critical control points to increase delivery of linolenic from forage through to meat and milk. Greater integration of research across the various levels of the food chain and increased interaction with industry will help in the delivery of foods with higher nutritional and health benefits for consumers.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with

details of the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the scientific objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability;

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the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Transfer).

IntroductionIn the UK, forage feeding systems based on grass and clover are increasingly important to the beef industry as they benefit from low inputs, are more sustainable and ensure that production costs are kept to a minimum and profitability is maximised. These systems also have the potential to produce beef, which is healthier and of a more distinctive flavour, offering considerable advantages to the consumer. It is essential that research seeks to understand and exploit the positive aspects of these systems for the benefit of the whole industry. The fatty acid composition of ruminant products has become increasingly important in recent years as the interactions between nutrition and a number of lifestyle diseases such as heart disease and cancer has become more apparent. Although beef is a low-fat food (< 5% fat), the fatty acid composition is relatively saturated (approximately 45-50%). However, beef is a natural carrier of beneficial n-3 PUFA and conjugated linolenic acid (CLA). Opportunities exist to further promote the nutritional balance of beef, to increase the PUFA : saturated (P:S) fatty acid value and reduce the n-6:n-3 PUFA value. Nutritional manipulation is important, for example feeding diets which are rich in n-3 PUFA such as linseed (18:3n-3 alpha linolenic acid) or fish oil (20:5n-5, eicosapentaenoic acid and 22:6n-3 docosahexaenoic acid). Grass and legumes such as white and red clover are also very rich in n-3 PUFA (18:3n-3). Dietary PUFA are exposed to microbial lipolysis and biohydrogenation during passage through the gastro-intestinal tract. This process results in the production of saturated fatty acids which is one of the key reasons why ruminant fats tend to be highly saturated in nature. However, it also results in the formation of CLA and trans monoene intermediates. Understanding the events surrounding fatty acid metabolism in the rumen is central to enabling the development of effective strategies to manipulate the fatty acid composition of beef. Altering fatty acid composition may also change the shelf life and flavour characteristics of the meat.

Hence, the purpose of this project was to investigate key factors which influence the nutritional value and eating quality of beef, with a focus on forage-based feeding systems. Factors included forage management strategies and rumen metabolism of dietary fatty acids.

ObjectivesTo investigate dietary factors which influence the nutritional value in (n-3 PUFA and CLA) and eating quality (flavour) of beef. The aim is to offer added value to the consumer in terms of more healthy and wholesome food produced. The project has five main objectives:1. To examine the impact of forage feeding to produce beef with a fatty acid composition which is more

consistent with current human health recommendations and consumer requirements (increased P:S ratio, higher amounts of n-3 PUFA and CLA) and improved meat quality.

2. To understand events in the rumen (lipolysis and biohydrogenation) and post-absorption which determine the ability of the different forages/concentrates to manipulate fatty acid composition of beef.

3. To assess the impact of strategies imposed in objective 1 on the fatty acid composition of beef, focusing on long chain PUFA, CLA, trans and branched-chain fatty acids.

4. To assess the impact of strategies imposed in objective 1 on flavour attributes of beef and to establish relationships between tissue components which are influenced by diet and intensity of individual flavours.

5. To assess management factors which influence key chemical constituents (fatty acids and antioxidants) in grass and clover during the ensiling process.

One change was agreed with the project officer (Dr David Garwes) in April 2003. Experiment 2 under Workpackage 1 (To assess the temporal changes in fatty acid composition of beef post inclusion of grass and level of finish on the fatty acid composition of beef) was changed to an additional study in relation to ruminal lipid metabolism (experiments 5.4-5.7 below). This was because results in relation to the experiment 2 were acquired in the EU FPV project on "healthy beef". All other objectives were conducted as planned.

The project involves conducting studies to assess (1) the impact of forage-based “nutritional strategies” on the fatty acid composition, colour shelf life and sensory aspects of beef (linking objectives 1, 3 and 4) (2) impact of rumen processes on fatty acid metabolism (objective 2) and (3) the effect of ensiling on fatty acids (objective 5).

Nutritional Studies: large scale beef experiments have been conducted under objectives 1, 3 and 4.Experiment 1 The fatty acid composition of muscle fat and relationships to meat quality in Charolais steers: influence of level of red clover in the diet. Introduction Legumes are important constituents of ruminant diets and are a vital part of low input and organic systems. Previous studies have established the high intake characteristics and animal production potential of red clover silage. Studies with milk produced from cows fed on red clover silage was noted to have higher levels of polyunsaturated fatty acids (PUFA) but had a reduced shelf life which could be ameliorated by feeding additional vitamin E. This study considered the effects of incremental inclusion of red clover silage in the diet of beef cattle on the fatty acid composition of the m. longissimus dorsi and meat quality.

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Materials and methods Thirty two Charolais steers (initial live weight 490 kg (s.e.d. 6.7)) were randomly allocated to one of four dietary treatments (each consisting of eight animals) differing in forage type (grass or red clover silage or a mixture) and concentrate type (differing in vitamin E content, C1 and C2 containing 25 and 500 mg/kg vitamin E, respectively). The four diets were (1) grass + C1 (GS); (2) grass and red clover mix (50:50 DM basis) + C1 (GRS); (3) red clover + C1 (RC) and (4) red clover + C2 (RCVitE). Forage was offered ad libitum and feed levels were adjusted weekly to maintain a forage:concentrate ratio of 70:30 on a DM basis. Animals were slaughtered after 100 days on treatment and samples of m. longissimus thoracis et lumborum (LTL) were taken at 48h post-mortem for fatty acid analysis. Colour (L*a*b*) and lipid oxidative shelf life were determined on meat conditioned for 10 days at 1oC and then packed in a modified atmosphere for simulated retail display shelf-life at 4oC. Eating quality was determined on meat conditioned for 10 days by a 10-member trained sensory panel. An ANOVA with diet as the main factor was used to analyse the data.

Results Half carcass weights were similar across treatments and averaged 194 kg (s.e.d. 5.9). Total muscle fatty acids, saturated (SFA) and monounsaturated fatty acids (MUFA) were not different (Table 1). Increasing the amount of red clover relative to grass silage in the diet significantly increased total PUFA content and in particular the 18:2n-6 and 18:3n-3, resulting in beneficially higher P:S ratio and lower n-6:n-3 ratio. TBARS increased and colour saturation decreased incrementally with increasing amount of clover in the diet. The concentration of vitamin E in the muscle also decreased with increasing amount of clover. However, feeding additional vitamin E (diet RCVitE) alleviated this problem. Sensory attributes were largely not influenced by diet, although red clover (RC) produced the most tender meat.

Table 1. Fatty acid content (mg/100g muscle) of longissimus dorsi and meat quality attributes of LTL.

Forage typeGrass

(G)Grass/red

clover (GRS)

Red clover (RC)

Red clover + vitamin E (RCVitE)

sed P

Fatty acid composition mg/100 g muscleTotal fatty acids 3081 3639 4001 3074 0.36 NSSFA 1300 1535 1643 1270 261.7 NSMUFA 170.7 206.4 244.4 216.8 13.44 NSPUFA 170.7a 206.4b 244.4c 216.8bc 13.44 0.00118:2n-6 73.2a 92.8b 113.2c 99.3b 6.68 0.00118:3n-3 22.5a 34.1b 50.7c 37.5b 3.83 0.001P:S 0.07a 0.09ab 0.10bc 0.12c 0.01 0.01n-6:n-3 3.28c 2.73b 2.30a 2.66b 0.15 0.001TBARS (mg MDA/kg meat) day 7 0.64 a 1.31 b 4.88 c 1.13 b 0.45 0.001Colour saturation day 7 22.9 a 21.5 b 20.3 c 22.6 ab 0.57 0.001Vitamin E 3.47 a 2.92 b 1.80 c 3.32 a 0.18 0.001Toughness (0-100 line scale) 46.5ab 41.6a 42.0a 48.2b 2.80 0.05Beef flavour (0-100 line scale) 31.7 34.2 34.2 31.0 2.28 NSFishy (0-100 line scale) 0.6 0.2 1.1 0.4 0.5 NS

Conclusions Red clover increased the content of PUFA in meat and reduced colour shelf life. The latter was most likely associated with the low vitamin E content rather than the increased content of PUFA in the muscle, since a supra-nutritional supplement of vitamin E in the diet restored the vitamin E concentration and stability of the meat to that observed with other diets.

Experiment 2 temporal changes in fatty acid composition of beef post inclusion of grass and level of finish on the fatty acid composition of beef (as explained above this experiment was replaced with experiments 5.4 – 5.7).

Experiment 3 compared the effect of feeding grass silage vs. red clover plus/minus concentrate containing PUFA-rich linseed oil during a 120 day winter period followed by a finished period outdoors at grassIntroduction Experiment 1 demonstrated that feeding red clover relative to grass silage results in meat characterised by higher levels of polyunsaturated fatty acids (PUFA) but reduced shelf life which was associated with lower levels of vitamin E in the muscle. Colour shelf life could be ameliorated by feeding additional vitamin E. Feeding red clover silage followed by finishing off on pasture may help alleviate the problem of colour shelf life while maintaining the benefit of the legume in delivering higher PUFA into meat. Hence this study examined feeding red clover compared with grass silage during the winter, following by a summer finishing period at grass, on fatty acid composition, vitamin E content of meat, colour shelf life and sensory attributes of beef. (Please note this study also involved an additional 3 treatments (extra 27 animals in total) which were all 3 forage diets plus a

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concentrate containing 5% linseed oil. Since the results for the effects of concentrate feeding were similar to those noted in experiment 4 described below; only results for forage treatments are described below.

Materials and methods Twenty seven Charolais steers (initial live weight 496 kg (s.e.d. 14.9)) were randomly allocated to one of three ad libitum silage treatments (each consisting of nine animals) (1) grass silage, (2) red clover or (3) 50:50 mix (DM basis) of grass and red clover. Following ~ 120 days of silage treatments animals were turned outdoors and rotationally grazed on perennial ryegrass pastures. Animals were slaughtered after ~ 100 days at grazing and samples of m. longissimus thoracis et lumborum were taken at 48h post-mortem and analysed as described for Experiment 1 above.

Results Liveweight gain was similar across the different silages (mean 1.07 kg/d) and carcass weight and conformation and fat score were also similar (mean 191 kg, 90.2 (0-150 line scale) and 73.3 (0-150 line scale), respectively. Total muscle fatty acids and proportions of the major saturated fatty acids were not different (Table 1). The proportions of 18:2n-6 and 18:3n-3 were higher on red clover relative to grass silage resulting in higher P:S ratios on the red clover. There were no differences in vitamin E content of the muscle, all being higher than the recommended 3.5 required for optimum stability, resulting in similar colour and lipid stability for all treatments. There were no differences in eating quality between treatments as assessed by a trained sensory panel.

Table 2. Fatty acid composition of longissimus thoracis et lumborum and colour shelf life of loin steaks during simulated retail display and vitamin E content of muscle

Silage treatmentGrass Mix: grass and

red cloverRed clover SED P

Total fatty acids (mg/100g muscle)

2115 2607 1945 340.8 NS

16:0 23.7 24.7 23.5 0.81 NS18:0 14.8 15.8 15.7 0.70 NS18:1n-9 31.2 32.4 30.1 1.17 NS18:1 trans 2.58 2.22 2.17 0.274 NSCLA 0.49 0.41 0.39 0.064 NS18:2n-6 2.98 2.44 3.69 0.398 0.01618:3n-3 1.76 1.63 2.34 0.237 0.001EPA 0.89 0.67 0.95 0.148 NSDHA 0.17 0.12 0.22 0.064 NSDPA 1.02 0.75 1.10 0.127 NSP:S 0.12 0.10 0.15 0.017 0.024n-6:n-3 1.15 1.09 1.13 0.067 NSVitamin E (mg/100g muscle) 4.46 3.92 4.30 0.303 NSTBARS d10 (mg/100g muscle) 1.72 1.90 2.10 0.474 NSColour saturation day 9 19.06 18.46 18.96 1.22 NS

Conclusions Feeding red clover relative to grass silage followed by a period grazing fresh grass increased the content of PUFA in the meat. There were no differences in colour shelf life or sensory attributes between diets. This most likely relates to the fresh grass providing sufficient vitamin E to correct the lower amounts found with red clover feeding. The higher levels of PUFA in meat from red clover relates to the action of the plant enzyme polyphenol oxidase (PPO) protecting some of the beneficial plant PUFA from the biohydrogenation in the rumen (see objective 2 results).

Experiment 4 The fatty acid composition of muscle fat in Charolais steers: influence of grass versus concentrate feedingIntroduction Grass relative to concentrate feeding increases the content of n-3 polyunsaturated fatty acids (PUFA) resulting in a low n-6:n-3 PUFA ratio. Previous research by IBER and Bristol have demonstrated that ruminally protected plant lipids enhance PUFA content very significantly resulting in beneficial P:S and n-6:n-3 ratios. This study considered the effects of finishing steers (1) outdoors on grass ± concentrate versus (2) indoors on straw/concentrate ± a protected lipid supplemental with one of two levels of vitamin E on the fatty acid composition of the m. longissimus thoracis et lumborum.

Materials and methods Forty eight Charolais steers (initial live weight 506 kg (s.e.d. 4.7)) were randomly allocated to one of six dietary treatments (each consisting of eight animals) (1) ad libitum grazed perennial ryegrass (G) (2) grazed ryegrass + 2.5 kg concentrate (GC1), (3) grazed ryegrass + 5.0 kg concentrate (GC2), (4) straw plus concentrate (control); (5) straw + concentrate (standard vitamin E, 25 mg/kg)+ 600 g/d protected lipid supplement (PLS; n-6:n-3 ratio of 1:1) (PLSV1) and (6) straw + concentrate (high vitamin E, 500 mg/kg DM) + 600 g/d PLS (PLSV2). Straw was offered ad libitum. The grazed animals were maintained on rotational grazed paddocks while the straw/concentrate treatments (4, 5 & 6) were kept indoors. All animals were fed to achieve a

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similar rate of carcass gain by either restricting grass (treatments 2, 3) or restricting intake of the concentrate. The concentrate was based on barley, molasses, sugarbeet pulp, megalac and premix. Animals were slaughtered after 100 days on treatment and samples of m. longissimus thoracis et lumborum (LTL) were taken at 48h post-mortem for fatty acid analysis. An ANOVA with diet as the main factor was used to analyse the data.

Results Half carcass weights were higher for the outdoor relative to indoor animals (P < 0.001), but carcass fatness was similar. Total muscle fatty acids were similar across treatments (Table 1). Increasing concentrate outdoors (GC1 and 2) resulted in higher amounts of 18:2n-6 and lower 18:3n-3 relative to grass only. There was evidence that EPA was also reduced (GC2 v. G). Indoors, inclusion of the PLS resulted in large increases in 18:2n-6 and 18:3n-3, which did not impact on the longer chain C20 PUFA. PLS resulted in a large increase in P:S ratios (i.e PLSV1 v. control). Grass feeding resulted in the lowest n-6:n-3 ratios.

Table 3. Fatty acid content (mg/100g muscle) of longissimus thoracis et lumborum

Outdoor IndoorGrass

(G)GC1 GC2 Control PLSV1 PLSV2 SED P

Half carc. wt (kg)

161.5 164.6 166.6 157.0 156.8 156.0 2.42 0.001

Fatty acid composition (mg/100 g muscle)Total fatty acids

1725 1800 1637 1778 1880 1638 274.5 NS

16:0 405 432 383 435 456 383 73.7 NS18:0 254 256 225 266 260 234 43.1 NS18:1n-9 567 609 520 546 528 440 100.2 NS18:1 trans

33.0 31.0 32.5 26.5 29.6 28.0 7.01 NS

CLA 8.5 8.2 9.1 6.8 8.0 7.6 2.12 NS18:2n-6 58.4 64.6 77.5 91.2 178.3 171.8 10.01 0.001

18:3n-3 27.4 21.3 18.6 14.7 42.2 40.5 3.44 0.001EPA 14.5 13.2 12.4 11.3 11.9 10.8 1.18 0.039DHA 1.9 2.6 2.2 1.8 1.7 1.7 0.28 0.017DPA 19.1 19.7 18.8 16.9 13.5 13.2 1.21 0.001P:S 0.14 0.13 0.16 0.15 0.30 0.35 0.027 0.001n-6:n-3 1.44 1.83 2.29 2.96 3.10 3.12 0.223 0.001

As the amount of concentrates in the diet increased so the amount of vitamin E decreased, being almost replenished to that of grass-grazed animals by dietary addition in PLSV2. Reduced vitamin E led to an increased oxidation of lipids as shown by the TBARS values, particularly so by 10 days of retail display and when additional PUFA were incorporated into the meat when PLSV1 was fed, but the additional vitamin E in PLSV2 failed to produce the same meat stability as that for grass-grazed beef. Colour stability showed similar trends, though differences between diets were small and not statistically significantly different. The carotene content of the subcutaneous adipose tissue over the 10 th rib was highest in grass fed animals. That found in the indoor-fed animals may have been residual from the prior dietary regime and appeared to be reduced by the additional vitamin E in PLSV2. Carotene concentration was linearly related to the b* (yellowness) of the fat. The only difference found by the sensory panel was that the group fed grass and low concentrates produced meat which was significantly (P<0.05) tougher than the rest.

Table 4. TBARS (mg/100g muscle) and colour saturation of loin steaks during simulated retail display, vitamin E content (mg/100g) of muscle and carotene (mg/100g) content of adipose fat

Outdoor IndoorGrass (G) GC1 GC2 Control PLSV1 PLSV2 SED P

mg/100g lean tissueVitamin E 4.51c 3.86b 3.85b 2.72a 2.81a 4.04b 0.40 ***TBARS d5 § 0.36a 0.40a 0.62a 1.34b 2.07c 0.49a 0.297 ***TBARS d10§ 0.66a 0.95a 1.45ab 2.68b 5.70c 1.59ab 0.815 ***Carotenes in adipose tissue 0.65c 0.57bc 0.47bc 0.39ab 0.40ab 0.25a 0.086 ***Colour saturation day 8§ 17.8 18.0 17.5 17.8 16.3 17.9 0.91 NS

§ days of retail display

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Conclusions Total fatty acids were relatively low which contributed to higher P:S ratios across all treatments due to the acknowledged relationship between total lipid and P:S (Scollan et al., 2006). Grass feeding resulted in higher levels on n-3 PUFA resulting in very favourable n-6:n-3 ratios. However, these beneficial effects were diluted by feeding additional concentrate outdoors. Protected lipids resulted in large increases in 18:2n-6 and 18:3n-3 contributing to a very beneficial P:S ratio. Grass grazing not only produces meat with an improved n-3 fatty acid content but also produces more yellow fat due to its carotene content and a greater lipid stability due to the natural intake of vitamin E with the diet. Improving the P:S ratio by feeding protected lipids causes a severe oxidative challenge in the meat and this is overcome to a great extent by supplementing the diet with vitamin E. Supplementing animals with concentrates whilst grazing over a 100d period reduces the natural vitamin E content of the meat and reduces its stability, though non-significantly.

Objective 2 Workpackage 4 Rumen metabolism of fatty acidsRumen fatty acid metabolism studies: a series of in vitro and in vivo studies investigated how diet type (forage grass v. legumes; concentrate feeding; oil supplementation) influenced fatty acid metabolism in the rumen and absorption from the small intestine to help establish relationships between fatty acid composition of the diet and the meat. A number of in vitro studies (Experiment 5.1-5.3) examined the role of plant mediated lipolysis in the rumen and also the role of red clover polyphenol oxidase (PPO) in reducing lipolysis in the rumen. A further group of in vitro experiments (Experiment 5.4-5.7) were also carried out to determine the effect of plant attributes on the production of biohydrogenation intermediates and in particular cis-9 trans-11 conjugated linoleic acid (CLA). A series of in vivo studies investigated the effect of different dietary treatments on ruminal biohydrogenation of C18 PUFA and subsequent duodenal flow of fatty acids: i) grass versus clover silages and the potential of reduced biohydrogenation through the action of PPO (Experiment 6), ii) forage:concentrate ratio (Experiment 7), iii) fish oil concentrate (Experiment 8) and iv) the combined effect of red clover and fish oil (Experiment 9).

Experiment 5.1. In-vitro evidence for plant enzyme mediated lipolysis in the rumenIntroduction It is generally accepted that microbial enzymes are involved in lipolysis and are thus responsible for the destruction of plant membranes in the rumen. However, we questioned this assertion and tested the hypothesis that in ruminants grazing fresh pastures, the first stages of lipolysis could be mediated by plant lipases. These enzymes are ubiquitous in plants and their regulation might be altered as a consequence of the dual stress of elevated temperature and anoxia imposed on the plant metabolism of intact plant cells ingested by ruminants.

Materials and Methods Leaf blades of Lolium perenne (var. AberElan), grown under controlled conditions (25o

and 14h artificial light d-1), were harvested (3cm above ground level) after 5 weeks, cut into 5mm segments and incubated in 50 ml anaerobic phosphate buffer (50 mM Na2HPO4 and 50 mM KH2PO4) at 39oC for up to 360 min. At each time point, (0, 15, 30, 60, 120, 360 min) leaf segments were removed from each of three replicate incubations together with a 1ml sample of the buffer solution and taken for volatile fatty acid and lactate analysis. Internal standard was added to the buffer and leaf blades (1ml of 2.5mg C19:0/ml chloroform) and 20ml isopropanol to deactivate any plant enzymes. The sample was blended and filtered through a glass fibre filter. An isopropanol : chloroform (1:1 v/v) extraction was carried out on the filtered residue which was then recombined with the filtrate, rotary evaporated and resuspended in chloroform : methanol : saline (8:4:3) and extracted using the method of Folch. The lipid fractions were separated by thin layer chromatography, the individual layers methylated and analysed by gas chromatography. Both linoleic and linolenic acids were analysed to determine the level of biohydrogenation of the acids during the incubation period. The individual fractions are represented as percentage of total fatty acids and analysed by regression analysis and Student’s t-test between individual time points using Genstat.

Results and DiscussionTable 5. Changes in lipid fractions of grass incubated in a rumen like environment for 360 min.

Incubation time in minutes0 15 30 60 120 360 sed P

Lipid Fraction (%) Linear QuadraticTriacylglycerol 3.6 5.1 5.0 6.4 8.1 13.6 1.35 0.001Free Fatty Acids 3.5 4.1 4.5 3.9 3.5 9.8 0.78 0.001Polar Fraction 67.9 64.7 63.3 73.2 73.2 50.2 5.37 0.045 0.008Monoglycerides 1.7 2.7 1.7 2.2 3.2 5.1 1.25 NSDiglycerides 1.6 1.8 1.9 2.2 5.6 7.0 0.37 0.001Total Lipid (g/kg DM) 24.9 24.0 24.1 22.9 23.7 22.2 2.49 NS

The results show a change in the lipid fractions towards the end of the incubation period, with no change in total lipid recovery or percentage linoleic and linolenic acids (12.4 and 64.5, respectively). After 120 min there was a significant decline in the polar fraction and an increase in free fatty acids, triacylglycerol and diglycerides. It was concluded that a proportion of the polar fraction underwent lipolysis resulting in an increase in free fatty acids,

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which were then used for lipogenesis resulting in an increase in the triacylglycerol- and diglyceride fractions. Although trace amounts of acetate (propionate and butyrate were not detected) and lactate (0.71 and 1.21 mmol/l, respectively) were found in the buffer at the end of the incubation, their low levels made it unlikely that activity from contaminating bacteria (brought in with the leaf blades) could be responsible for the observed changes. Moreover as the linoleic or linolenic acids were not biohydrogenated, it appears that the lipolysis and lipogenesis was indeed plant-mediated and that the trace levels of acetate and lactate were of plant origin.

Experiment 5.2. Plant enzyme mediated lipolysis of Lolium perenne and Trifolium pratense in an in vitro simulated rumen environmentIntroduction Based on the positive conclusions in experiment 5.1, this study examined the difference in lipolytic activity between Lolium perenne and Trifolium pratense (red clover), as the latter has been shown to reduce biohydrogenation in the rumen (Lee et al. 2003).

Materials and Methods Lolium perenne (cv. AberElan) and Trifolium pratense (cv. Milvus) from experimental plots, were harvested (3cm above ground level), bruised by minimal crushing, cut into 5mm segments and incubated in 25ml antibiotic-containing (5.0mg chloramphenicol/ml in 50% v/v ethanol) anaerobic buffer at 39oC for up to 24 h. At different times (0, 1, 2, 3, 4, 5, 6 and 24 h) the incubation bottles were destructively harvested, the lipid extracted (isopropanol : chloroform; 1:1, v/v) and fractionated by thin layer chromatography. All fractionated lipid samples (membrane lipid, triacylglycerol, diglyceride and free fatty acid) were bimethylated using the procedure described by Kramer and Zhou, (2001). Lipolysis was calculated by expressing the percentage decrease in fatty acid content of membrane lipid (polar fraction, PF).

Results Lipid fluctuations in Lolium perenne and Trifolium pratense are shown in figures 1 and 2 respectively and degree of lipolysis is shown in Table 6. The data showed a curvilinear response for both Lolium perenne and Trifolium pratense suggesting a declining rate of lipolysis with time. Additionally, at 30%, Trifolium pratense had a significantly lower (P<0.05) overall lipolytic activity, in comparison to Lolium perenne at 35%. Increases in the amount of triacylglycerol (TG), diglycerides (DG) and free fatty acids (FFA) were observed with decreasing membrane lipid (PF) over time.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 10 20 30Time (hours)

Pro

porti

on o

f tot

al fa

tty a

cid

PF DGFFA TG

Figure 1. Fluctuation in Lipid fractions in Lolium perenne in an in vitro simulated rumen environment.

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0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 10 20 30Time (hours)

Pro

prot

ion

of to

tal f

atty

aci

d

PF DGFFA TG

Figure 2. Fluctuation in Lipid fractions in Trifolium pratense in an in vitro simulated rumen environment.

Table 6. Extent of lipolytic activity in the Lolium perenne and Trifolium pratense incubations at time Tx.

Incubation time (Tx) in hoursLipolysis 1 2 3 4 5 6 24Lolium p.(sed)

0.09(0.024)

0.27(0.033)

0.33(0.040)

0.37(0.017)

0.39(0.026)

0.40(0.056)

0.63(0.016)

Trifolium p.(sed)

0.11(0.013)

0.22(0.043)

0.26(0.025)

0.29(0.014)

0.35(0.034)

0.41(0.023)

0.55(0.011)

Sig. NS NS * * * NS *NS, Not significant; *, P<0.05.

Discussion These results support the view that plant lipases may play a role in lipolysis in the early stages of digestion of forages in the rumen and that the reduced level of lipolytic activity in Trifolium pratense may explain the reduction in ruminal biohydrogenation.

Experiment 5.3. Lipolysis of red clover with differing Polyphenol oxidase activities in batch culture.IntroductionPolyphenol oxidase (PPO) oxidises endogenous phenols to quinones, which react with nucleophilic sites of other compounds such as proteins. In red clover this complexing reaction has been shown to reduce both plant mediated proteolysis and lipolysis. This experiment investigated the role of red clover PPO on lipolysis in the presence and absence of rumen micro-organisms.

Materials and Methods Triplicate macerated shoot samples of two red clover lines, a wild type with a basal level of PPO activity (High PPO) and a mutant with reduced PPO activity (Low PPO) were incubated in anaerobic buffer, with and without strained rumen liquor inoculum (I+ and I-), at 39oC, over six time points (0, 1, 2, 4, 6 and 24 h). At each time point the samples were destructively harvested and lipolysis measured as percentage lose of membrane lipid. Lipolysis data was analysed using a general analysis of variance with repeated measurements (Genstat 8®).

Results and Discussion Table 7 shows the reducing effect of PPO on lipolysis (high vs Low) but also the elevated level of lipolysis when micro-organisms are present (I+ vs I-). If the PPO effect was solely due to the deactivation of plant lipases this difference should be neutralised through the addition of microbial lipases. The retention of the PPO effect in the I+ treatments suggests that PPO exerts some form of protection on the membrane lipids, in a similar manner to the complexing of protein. The lipid in forages is mainly in the form of polar membrane lipids, and polar lipid – phenol complexes could form due to the highly electrophilic nature of the PPO-produced quinones.

Table 7. Lipolysis of the two lines of red clover altering in their PPO activity incubated in the presence and absence of rumen fluid.

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High PPO Low PPO Sig.I+ I- I+ I- s.e.d PPO I PPO×I

Lipolysis (%) at 1h 10.6 6.6 37.4 8.5 2.97 * * NS 2h 16.7 8.9 37.5 17.2 5.10 * * NS 4h 36.0 14.6 44.6 21.9 3.68 * *** NS 6h 35.0 23.8 57.5 25.6 6.43 * ** NS 24h 71.5 28.3 82.4 41.9 5.94 * *** NS

*P<0.05, **P<0.01, ***P<0.001

Experiment 5.4. An in vitro investigation of forage factors which affect the production of conjugated linoleic acid and trans vaccenic acid in the rumen. I. Grass speciesIntroduction Extensive, pasture-based systems appear to offer the most cost-effective and natural means of cis 9 trans 11 conjugated linoleic acid (CLA) levels of ruminants lipids. However, since most regions in Europe rely increasingly on conserved forages for winter feeding of lactating animals, it is necessary to develop feeding systems for both fresh and conserved forage diets. An understanding of the mechanisms that cause the differences in CLA response to conservation is an essential pre-requisite to this task. This study investigated whether grass species affected CLA and trans vaccenic acid (TVA) production in an in vitro system.

Materials and methods Four week regrowths of eight different grass species were evaluated on two separate occasions: Diploid PRG (DPR); Tetraploid PRG (TPR); Italian ryegrass (IRG); Hybrid ryegrass (HRG); Timothy (TIM); Cocksfoot (COC); Meadow fescue (MF) and Tall fescue (TF). Fresh grass was cut from experimental plots (circa. 100grams FW) 3cm above soil level. The tissue was then crushed and cut into 5mm strips. Two and a half grams of fresh material was then loaded under CO2 into incubation bottles containing 10ml of strained rumen liquor and 10ml of Van Soest medium, the head space was gassed and the bottles sealed. Three bottles were used for each treatment in the first period and two in the second period. The bottles were incubated for 6 h in the dark at 39oC. At the end of the incubation the bottles were removed and 25ml of isopropanol : chloroform (1:1 v/v) was added along with 1ml of internal standard (2.5mg C19:0 / ml chloroform). This was then blended and the lipid extracted as described by Lee et al. (2004) and bimethylated as described by Kramer and Zhou (2001). Biohydrogenation of C18:2 and C18:3 were calculated as proportional loss of these fatty acids during the incubation. Genotypic differences in the concentrations of CLA, TVA and biohydrogenation were calculated using an unbalanced ANOVA with genotype × period as the treatment.

Results The production of CLA and TVA (as a proportion of C18 polyunsaturated fatty acid (PUFA) supply) and the extent of C18:2 and C18:3 PUFA biohydrogenation across the seven genotypes are shown in Table 8. Both Cocksfoot and Timothy produced significantly lower levels of CLA and TVA than the other genotypes, whilst Italian ryegrass resulted in the greatest formation of these biohydrogenation intermediates. Biohydrogenation of both C18:2 and C18:3 were lowest for Cocksfoot and highest for meadow fescue. The relatively large S.e.d’s, particularly in regards to CLA, were due to large variation between periods.

Table 8. CLA and TVA production (as a proportion of C18 PUFA supply; g/g) and the extent of C18 PUFA biohydrogenation (g/g)

COC DPR HRG IRG MF TF TIM TPR S.e.d Sig.CLAx 103 0.44a 0.75bc 0.89bc 1.39d 0.63b 0.95c 0.52ab 0.95c 0.135 ***TVA 0.14a 0.19b 0.21c 0.22c 0.19b 0.21c 0.12a 0.17b 0.011 ***BiohydrogenationC18:2 0.43a 0.53abc 0.57bc 0.52bc 0.59c 0.47ab 0.54abc 0.52abc 0.053 *C18:3 0.52a 0.60b 0.67cd 0.64cd 0.68d 0.59bc 0.55ab 0.63bc 0.039 *

Discussion The lower concentration of CLA and TVA with Cocksfoot may be due to the lower level of C18 PUFA biohydrogenation, although this does not explain the lower concentration of these fatty acids in Timothy. Species (genotype) appears to have an effect on the production of CLA and TVA, although numerically the differences were small and period/stage of growth had a dramatic effect on levels of production. The experiment also shows the low levels of cis 9 trans 11 CLA which are produced in the rumen and hence the importance of TVA in the formation of CLA in muscle and mammary gland lipid.

Experiment 5.5. An in vitro investigation of forage factors which affect the production of conjugated linoleic acid and trans vaccenic acid in the rumen. II. Wilting & cell damageIntroduction Studies with milk, have shown that cis 9 trans 11 conjugated linoleic acid (CLA) concentrations are higher for summer milk produced from cows grazing fresh pastures than for winter milk when conserved forages are fed (Jahreis et al., 1997). Furthermore, Offer (2003) showed that a similar depression in milk fat CLA occurred if grass was simply cut and fed after a short wilt. This experiment investigated the effect of wilting on the production of CLA and trans vaccenic acid (TVA) in an in vitro rumen simulation, and whether any differences

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could be related to changes in plant structure. It was hypothesised that fresh turgid grass may have a greater propensity for cell damage during mastication than the much more flaccid wilted grass, which in turn may increase microbial metabolism of the grass lipid.

Materials and method Fresh grass from eight different grass species, were cut from experimental plots on day 0 and day 1 of the trial (circa. 200grams FW) 3cm above soil level. The grass cut on day 0 was left to wilt for 24 hr on a laboratory bench. Half of the fresh and wilted tissue was then crushed and cut (C&C) into 5mm strips and 2 ½ g loaded into incubation bottles under CO2. The remaining grass was loaded into the incubation bottles and homogenised (H) also under CO2. Two bottles contained 10ml of strained rumen liquor and 10ml of Van Soest medium were used for each treatment. After gassing the head space and sealing, the bottles were incubated for 6 h in the dark at 39oC. At the end of the incubation the bottles were removed and the lipid extracted and analysed. Biohydrogenation of C18:2 and C18:3 were calculated as proportional loss of these fatty acids during the incubation. The effect of wilting and cell damage was assessed using an unbalanced ANOVA model with wilt × cell damage as the treatment and blocking according to grass species. The effect of grass species has previously been reported above (experiment 5.4).

Results The concentration of CLA and TVA in the incubation vessels and the extent of C18:2 and C18:3 biohydrogenation are shown in Table 9. Wilting had no significant effect on the concentration of CLA in the vessels but significantly reduced the concentration of TVA and the biohydrogenation of C18:2 and C18:3. The extent of cell damage appeared to have no effect on the concentration of either CLA or TVA but significantly increased the concentration of C18:0 (data not shown) and the biohydrogenation of C18:2 and C18:3.Table 9. CLA and TVA concentration (g/kg dry matter input) and C18:2 and C18:3 biohydrogenation

Unwilted Wilted SignificanceC&C H C&C H S.e.d Cell

damageWilt Interaction

CLA 0.011 0.011 0.010 0.011 0.0007 NS NS NSTVA 3.79 3.36 2.62 2.81 0.162 NS *** **

BiohydrogenationC18:2 0.37 0.52 0.33 0.43 0.037 *** * NSC18:3 0.48 0.65 0.46 0.58 0.027 *** * NS

Discussion The low level of CLA production in the batch cultures highlights the important role of TVA as a precursor of meat and milk CLA. Increasing the extent of cell damage significantly increased biohydrogenation, but surprisingly did not increase the concentration of the intermediates CLA and TVA. It, did, however increase the concentration of C18:0 which is the end point of biohydrogenation, which suggests that the reaction went further to completion. The fact that H wilted grass was not similar to H unwilted grass in terms of TVA concentration may suggest that structural differences alone between wilted and unwilted grass are unlikely to explain the large differences in CLA produced from fresh pasture and cut/wilted grass (Offer, 2003).

Experiment 5.6. The effect of fatty acid oxidation products on lipid metabolism during in vitro batch cultureIntroduction Studies in cows, have shown that milk fat conjugated linoleic acid (CLA) concentrations are higher for milk produced from cows grazing fresh pastures compared with conserved forages (Jahreis et al., 1997). This experiment investigated the hypothesis that this increased CLA production results from the action of volatile compounds (hydroperoxides, alcohols, aldehydes and ketones) released during the cutting of grass (De Gouw et al., 1999).These compounds have antimicrobial activities (Kubo et al., 1995) and may have an effect on rumen microbial populations.

Materials and Methods Van Soest anaerobic incubation medium was made up and 10ml dispensed into 21 incubation bottles containing 1 g of freeze-dried and ground grass silage. Each fatty acid oxidation product was evaluated in triplicate: i) tert-Butyl hydroperoxide, ii) cis-2-Hexen-1-ol, iii) Methyl vinyl ketone, iv) Hexanal, v) trans-2-Hexenal, vi) trans-2-Decenal along with a water control. These were added to a final concentration of 50µM. Incubation bottles were inoculated with 10ml of rumen fluid, from grazing cows with rumen fistulae, under CO2 then incubated at 39oC in the dark. The incubation period was set at 6 h, as an optimal time to maximise capture of biohydrogenation intermediates. At the end of the incubation, the bottles were removed from the incubator and 20ml of isopropanol : chloroform (1:1 v/v) added along with 1ml of internal standard (2.5mg C19:0 / ml chloroform). The lipid was extracted and bimethylated using 5N NaOH and 5% HCl in methanol. Fatty acid content of the vessels were analysed by ANOVA and compared against the water control. Biohydrogenation was determined as the proportional loss of C18 unsaturated fatty acids between time 0 h and 6 h. Results The results are reported as a mean percentage of total fatty acids in the vessels after 6 h incubation. Table 10 shows the major fatty acids and the biohydrogenation of C18:2 and C18:3. The addition of tert-Butyl hydroperoxide (TBH) and trans-2-Decenal (T2D) had the largest effect on the fatty acid proportions, with a significant reduction in C16:0, C18:1 cis, C18:2, C18:3 and BOC. They also produced a significant elevation in

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C18:0, C18:1 trans and in the case of TBH, total CLA proportions. The addition of cis-2-Hexen-1-ol reduced the proportions of C16:0 and C18:1 cis, and elevated both C18:0 and C18:1 trans. Methyl vinyl ketone addition resulted in a reduction in C16:0 and an increase in C18:1 trans. The addition of both TBH and T2D resulted in an elevation in the biohydrogenation of C18:2 and C18:3 compared to the control. Hexanal and trans-2-Hexenal appeared to have no effect on lipid metabolism in the incubations.

Table 10. Major fatty acids as a percentage of the total fatty acid content of the vessels after incubation.

Cis-2-Hexen-1-ol

Hexanal Methyl vinyl

ketone

Tert-butyl hydro

peroxide

Trans-2-Decenal

Trans-2-Hexenal

Water S.e.d Sig.

C16:0 14.7* 15.9 15.3* 13.3* 13.9* 16.2 17.1 0.62 ***C18:0 51.3* 46.7 49.5 54.5* 53.2* 47.0 45.6 2.06 **C18:1 trans† 9.00* 8.51 9.01* 9.83* 9.04* 8.64 8.30 0.292 **C18:1trans 11

6.16 6.03 6.16 6.75* 6.63* 6.12 5.83 0.181 **

C18:1 cis† 2.73* 3.23 3.02 2.45* 2.87* 3.29 3.41 0.162 ***C18:2n-6 3.30 4.21 3.53 2.74* 2.91* 4.05 4.25 0.446 *C18:3n-3 6.91 9.07 7.35 5.68* 6.23* 8.66 9.00 1.065 *CLA† 0.32 0.31 0.33 0.42* 0.35 0.32 0.35 0.021 **BOC‡ 4.14 4.49 4.34 3.50* 3.41* 4.30 4.51 0.302 **Biohydrogenation C18:2n-6 0.65 0.59 0.65 0.72* 0.78* 0.64 0.58 0.053 *C18:3n-3 0.70 0.65 0.71 0.77* 0.81* 0.69 0.64 0.045 *Figures with * superscript are significantly different from the control (Water), † Sum of all isomers, ‡ Branched and odd chain.

Discussion The increase in biohydrogenation of C18:2 and C18:3 and the associated increase in C18:1 trans and C18:0 may be attributable to the proliferation of biohydrogenating micro-organisms as a consequence of the toxic nature of TBH and T2D to competing micro-organisms reducing inter-specific competition. The results of this study show that fatty acid oxidation products affected rumen lipid metabolism. There is scope to investigate further the effect of long chain aldehydes and peroxides produced from freshly cut grass on the biohydrogenation of C18:2 and C18:3 in the rumen as a partial explanation for the increased flow of trans 11 C18:1 from the rumen of ruminants grazing fresh pasture and the consequential rise in meat and milk fat CLA.

Experiment 6. Duodenal flow and biohydrogenation of C18 polyunsaturated fatty acids in beef steers fed high sugar grass, red clover or grass/red clover mix silagesIntroduction Forage lipids contain a high proportion of the polyunsaturated fatty acids (PUFA) C18:2n-6 and C18:3n-3. However, microbial biohydrogenation in the rumen results in extensive loss of these beneficial fatty acids. This experiment investigated the duodenal flow of fatty acids in beef steers offered grass or red clover silage, the latter of which has been shown to reduce biohydrogenation of C18:3n-3 (Lee et al. 2003a). In addition the effect of silage prepared from a ryegrass with high water soluble carbohydrate (WSC) content was investigated, as previous research (Lee et al. 2003b) has shown that increasing the availability of WSC lowered rumen pH and increased the proportion of propionate, relative to other volatile fatty acids, conditions which have been linked to reduced biohydrogenation (Jenkins, 1993).

Materials and methods Six Hereford x Friesian steers 163 (se 5.9) kg, prepared with rumen and duodenal cannulae were allocated at random to receive one of five silage diets ad libitum; high sugar grass (HG); control grass (CG); HG and red clover (50:50 DM basis; HGR); CG and red clover (50:50 DM basis, CGR) and red clover (R). The experiment was conducted as a 5 x 5 incomplete Latin square with an additional randomly repeated sequence. There were four experimental periods each lasting 24 days, with a 14 day adaptation period to the diets, followed by a 10 day measurement period. Digesta flow at the duodenum was estimated using a dual phase marker system with Yb(CH3COO)3 and Cr EDTA as particulate and liquid phase markers, respectively. On days 20 and 21 of each period duodenal digesta was collected every three hours over a 24 h period. Lipid was extracted and analysed as described by Lee et al. (2004) (which also reports flows of C18:1 and the main CLA isomers). Statistical analysis was undertaken using an unbalanced analysis of variance, blocking according to period (Genstat 5; Lawes Agricultural Trust, 1997).

Results The principle chemical components of the silages were; DM: 277, 258, 289, 276, 309; NDF: 551, 587, 459, 482, 397; TN: 24.5, 25.4, 29.4, 29.2, 32.3 and WSC: 90.5, 55.3, 66.5, 43.8, 29.2 for HG, CG, HGR, CGR and R respectively. Silage pH ranged from 4.06 to 4.14 with a predominantly lactate fermentation. The control grass silage (CG) had a significantly lower dry matter intake than the other four silage treatments, with a consequently lower duodenal flow. There was a net increase in fatty acid flow between mouth and duodenum on the two grass diets and a net reduction when red clover was incorporated into the diet. Since the FA composition of the silages was similar the differences in intake of C18:0, C18:2n-6 and C18:3n-3 were related to differences in DM intake. Duodenal flows of C18:0 were higher (P < 0.01) and flows of C18:2n-6 and C18:3n-3 lower (P < 0.01)

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with the grass silage compared with the red clover mixtures or pure red clover. Biohydrogenation of C18:3 n-3 was reduced (P < 0.001) with increasing proportions of red clover in the diet.

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Table 11. Effect of forage type on intake and ruminal fatty acid metabolism.

HG CG HGR CGR R s.e.d PDM Intake (kg/d) 4.4b 3.6a 4.7b 4.6b 4.5b 0.42 *Fatty acid intake (g/day)C18:0 stearic 1.82b 1.41a 2.43c 2.37c 2.55c 0.207 ***C18:2n-6 linoleic 17.5ab 15.5a 21.6b 21.6b 21.2b 2.05 ***C18:3n-3 linolenic 57.9b 41.5a 58.1b 55.2b 50.1ab 6.01 ***Total fatty acids 105.7b 82.8a 113.8b 111.4b 101.7b 11.29 *Fatty acid flow to duodenum (g/day)C18:0 stearic 61.1c 41.1ab 49.0bc 45.2b 36.7a 6.28 **C18:2n-6 linoleic 1.94b 1.28a 2.34bc 2.15bc 2.62c 0.317 **C18:3n-3 linolenic 3.57b 2.34a 5.90c 5.29c 7.68d 0.661 ***Total fatty acids 126.4c 85.9a 106.6bc 98.5ab 83.7a 13.98 **Biohydrogenation (%)C18:2n-6 linoleic 89 92 89 90 88 1.7 NSC18:3n-3 linolenic 94c 95c 90b 90b 85a 1.5 ***

Discussion The decrease in duodenal flow of C18:0 and the increase in duodenal flow of C18:3n-3 is associated with a reduction in C18:3n-3 biohydrogenation when feeding red clover. The mechanism for this action may relate to intrinsic chemicals within red clover such as polyphenol oxidase and/or changes which occur in the microbial biohydrogenation pathways of C18 PUFA on red clover diets (Lee et al. 2004a).

Experiment 7. Duodenal flow and biohydrogenation of C18 polyunsaturated fatty acids in beef steers fed isonitrogenous and isoenergetic diets with contrasting forage : concentrate ratios Introduction Data on ruminal metabolism of lipids from forages have shown extensive biohydrogenation of C18 polyunsaturated fatty acids (Lee et al. 2003). Recent studies have noted a reduction in biohydrogenation an increase in duodenal flow of C18:1 trans-11 and a shift in the ruminal production of CLA from cis-9 trans-11 CLA towards trans-10 cis-12 CLA with increasing amounts of concentrate in the diet (Kucuk et al. 2001, Loor et al. 2004). The study assessed the effect of feeding steers on isonitrogenous and isolipid diets differing in forage concentration and supplemented with linseed oil on ruminal lipid metabolism.

Materials and methods Eight Hereford × Friesian steers (c. 533 kg), prepared with rumen and duodenal cannulae were offered one of four forage : concentrate (grass silage F: barley, sugarbeet and soya C) ratios: F80C20; F60C40; F40C60 and F20C80 on a dry matter basis. All diets were fed at 1.3% body weight and designed to be isonitrogenous and isoenergetic with total lipid made up to 6% dry matter intake with linseed oil. The experimental design was a four by four incomplete Latin square consisting of 3-periods with two animals per treatment. On days 20 and 21 of each 24 d period duodenal digesta was collected every 3 h over a 24 h period and on day 23 rumen fluid was collected hourly over a 12 h period. Lipid extraction and GC analysis was as described by Lee et al. (2003). Statistical analysis was undertaken using ANOVA, blocking according to period + animal (Genstat 7 2004).

Results Increasing the concentrate component in the diet from 0.20 to 0.60 reduced rumen pH from 6.58 to 6.37 and caused a small but significant shift in volatile fatty acid molar proportions increasing the glucogenic (propionate) : lipogenic (acetate + butyrate) ratio. Rumen ammonia-N concentration was also significantly reduced with increasing concentrate, from 156.8 to 101.0 mgN/l on F80C20 and F20C80, respectively. Dry matter intake was lower (P=0.016) on F80C20 than the other diets resulting in a reduced duodenal flow of nutrients. Intake and duodenal flow of oleic and linoleic acids were significantly higher with increasing concentrate level in the diet whereas linolenic acid intake and flow was not different, averaging 143.6 and 6.37 g/d, respectively. There were no differences in the flows of total C18:1 trans or CLA (47.8 and 1.79 g/d, respectively) across the diets. Biohydrogenation of linoleic dropped from 0.91 to 0.84 when increasing concentrate in the diet from 0.20 to 0.40 but thereafter concentrate had no effect, as with oleic and linolenic acids.

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Table 12. Effect of forage : concentrate ratio on intake and ruminal fatty acid metabolism.

F80C20 F60C40 F40C60 F20C80 s.e.d P valueDM Intake (kg/d) 6.1a 6.7b 6.7b 6.7b 0.18 *Fatty acid intake (g/d)C18:0 stearic 8.03a 9.11b 9.76c 10.4d 0.094 ***C18:2n-6 linoleic 54.7a 77.5b 98.0c 110.0d 0.52 ***C18:3n-3 linolenic 144.0 146.8 141.3 142.4 2.45 NSTotal fatty acids 283.0a 325.2b 352.4c 374.2d 4.73 ***Duodenal flow (g/d)C18:0 stearic 162.4 159.4 192.5 191.6 15.86 NSC18:2n-6 linoleic 4.12a 12.5b 13.7b 15.7b 1.75 ***C18:3n-3 linolenic 5.35 6.35 6.44 7.35 0.753 NSTotal fatty acids 298.9a 316.0ab 358.8b 369.5b 25.3 *Biohydrogenation (%)C18:2n-6 linoleic 91b 84a 86a 86a 2.3 *C18:3n-3 linolenic 96 96 95 95 0.5 NS

Values with different superscripts (abcd) differ significantly (p<0.05).

Discussion Increasing the proportion of concentrate in the diet when energy and nitrogen intakes were similar resulted in a drop in rumen ammonia and a small reduction pH with a consequent shift in VFA patterns towards propionate. The small drop in rumen pH on such contrasting F:C ratios was surprising. These results suggest that the F:C ratio had little effect on the flow of unsaturated fatty acids, C18:1 trans and CLA to the duodenum of beef steers, and that biohydrogenation of linoleic and linolenic acid is governed by other factors than forage : concentrate ratio.

Experiment 8. Effects of diets containing sunflower oil and fish oil on lipid metabolism and fatty acid flow to the duodenum of beef steers.Introduction Clinical research has shown that the intake of polyunsaturated fatty acids (PUFA) and in particular long chain PUFA such as C20:5n-3 and C22:6n-3 found in fish oil are beneficial to human health (Tapiero et al. 2002). Previous studies have shown that fish oil inclusion in the diet of ruminants has increased the concentration of long chain PUFA in milk (Shingfield et al. 2003) and muscle (Scollan et al. 2001a). Fish oil also significantly increased the post-ruminal flow of trans vaccenic acid (TVA), an intermediate in the biohydrogenation of linoleic and linolenic acid (Scollan et al. 2001b and Shingfield et al. 2003). This may have been responsible for the observed increase in the concentration of conjugated linoleic acid (CLA) in milk through the bioconversion of TVA to cis 9 trans 11 CLA in the mammary gland (Shingfield et al. 2003). This study was designed to report the effect of graded levels of fish oil on the flow of long chain PUFA and biohydrogenation intermediates such as TVA and CLA, when steers were offered a flat rate of linoleic acid, supplied from sunflower oil. This it was hoped would create a greater understanding of the effect of fish oil in the biohydrogenation of C18 PUFA.

Materials and methods Six Hereford x Friesian steers (c. 410 kg), prepared with rumen and duodenal cannulae were offered a first cut perennial ryegrass silage plus one of three concentrates: FISH0, FISH1 or FISH2 to contain 0, 10 and 40 g/kg fish oil respectively. The total daily feed allowance was 14 g DM/kg liveweight (c. 90% ad libitum) with a forage : concentrate ratio of 60:40 (DM basis). The experiment design was a Latin square consisting of 3-periods with two animals per treatment. Each 21d period consisted of 14d adaptation to the diet and 7d for digesta collection. Animals received their daily forage allocation at 09:00 and their daily concentrate allocation in 2 equal meals at 09:00 and 15:00. Digesta flow at the duodenum was estimated using a dual-phase marker. Separate samples of silage and concentrate were taken daily during the digestion periods. Sub-samples were freeze-dried, ground and retained for chemical analysis. Accumulated samples of daily duodenal digesta were thoroughly mixed and a whole and centrifuged fraction produced as described by Lee et al. (2003). Chemical and fatty acid compositions of the silages and digesta were determined as described by Lee et al. (2003). Digesta flows were calculated after mathematical reconstitution of true digesta as described by Faichney (1975). Biohydrogenation of C18 PUFA was assessed as the difference between daily intake and duodenal flow (g/day). Data were subjected to ANOVA (Genstat 7 ©, 2004) with diet as the treatment effect and blocking according to period + animal.

Results Dry matter and major fatty acid intake and duodenal flow are given in Table 13. There were no significant differences in nutrient and total fatty acid intake and duodenal flow. Increasing the concentration of fish oil in the diet significantly increased the intake and duodenal flow of long chain PUFA. It also increased the flow of TVA and CLA, but not the isomer cis 9 trans 11, and decreased the flow of stearic acid. Biohydrogenation of linoleic and linolenic acid were not significantly different across diets, averaging 90.8 and 91.8%, respectively.

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Table 13. Dry matter and major fatty acid intake and duodenal flow

FISH0 FISH1 FISH2 S.e.d. PIntake (g/d) Dry Matter (kg/d) 7.55 7.55 7.45 0.171 NS C16:0 Palmitic 67.2 66.1 58.7 0.81 0.001 C18:0 Stearic 25.1 22.7 13.8 0.48 0.001 C18:1n-9 Oleic 88.7 90.2 76.1 5.71 0.001 C18:2n-6 Linoleic 167.6 172.3 170.6 8.18 NS C18:3n-3 Linolenic 48.0 48.6 47.7 1.47 NS C20:5n-3 Eicosapentaenoic 0.17 1.34 5.99 0.155 0.001 C22:5n-3 Docosapentaenoic - 0.275 0.919 0.0179 0.001 C22:6n-3 Docosahexanoic 0.06 1.99 8.66 0.142 0.001 Total fatty acids 419.8 433.1 427.9 6.50 NSDuodenal Flow (g/d) Dry Matter (kg/d) 4.40 4.50 4.38 0.241 NS C16:0 Palmitic 87.1 89.5 80.3 3.95 NS C18:0 Stearic 275.4 259.1 169.8 10.49 0.001 C18:1 trans 11 50.6 74.2 83.2 9.46 0.022 Total trans C18:1 68.2 93.9 117.0 7.57 0.001 Total cis C18:1 40.6 41.3 42.1 2.54 NS CLA cis 9 trans 11 0.93 1.51 1.09 0.2441 NS Total CLA 2.65 3.89 6.59 0.398 0.001 C18:2n-6 Linoleic 15.8 17.5 13.5 1.01 0.012 C18:3n-3 Linolenic 4.07 4.06 3.65 0.287 NS C20:5n-3 Eicosapentaenoic 0.47 0.68 1.24 0.068 0.001 C22:5n-3 Docosapentaenoic 0.30 0.34 0.57 0.063 0.006 C22:6n-3 Docosahexanoic 0.41 0.53 1.21 0.082 0.001 Total fatty acids 543.7 568.3 507.6 26.94 NS

Discussion All three diets in the present study resulted in net synthesis of fatty acids across the rumen as previously reported by Scollan et al. (2001b) when feeding a fish oil supplement, and this maybe due to endogenous lipid or microbial synthesis. Fish oil had no effect on the extent of biohydrogenation of either linoleic or linolenic acid, but significantly increased the flow of the intermediate products TVA and total CLA and significantly reduced the flow of the end product stearic acid. However, there was no significant difference in the flow of cis 9 trans 11 CLA, the product of the initial isomerisation of linoleic acid in biohydrogenation, in these diets and so the effect of fish oil appeared to be an inhibition to the final reduction of TVA into stearic acid. Wallace et al. (2004) have identified two species of ruminal bacteria responsible for the biohydrogenation of both linoleic and linolenic acid due to their extreme sensitivity to PUFA, namely Butyrivibrio fibrisolvens and Fusocillus spp. These bacteria in conjunction but not in isolation can hydrogenate linoleic and linolenic acid to stearic acid. B. fibrisolvens hydrogenates the PUFA to cis 9 trans 11 CLA and TVA and Fusocillus completes the hydrogenation of TVA to stearic acid. It may be that the long chain PUFA in fish oil inhibits Fusocillus resulting in a significant elevation in TVA and consequently an increase in milk CLA through TVAs bio-conversion in the mammary gland (Shingfield et al. 2003).

Experiment 9. The effect of fish oil supplementation on ruminal C18 PUFA metabolism in beef steers offered either grass or red clover silage. Introduction Red clover and fish oil have been shown to alter ruminal lipid metabolism increasing PUFA and conjugated linoleic acid (CLA), respectively, in ruminant products. This study investigated the additive effect of these two feeds on C18 PUFA metabolism in beef steers.

Materials and methods Eight Hereford × Friesian steers prepared with rumen and duodenal cannulae were offered either grass or red clover silage at 90% ad libitum with one of three levels of fish oil 0, 1, 2, or 3 % DMI. The experimental design consisted of four 2 × 2 Latin squares within each oil level with an extra period. Flows of fatty acids at the duodenum were assessed using the dual phase-indigestible-marker technique.

Results and Discussion DMI was significantly (P < 0.001) higher for red clover silage (5.98) than grass silage (5.09 kg/d). Oil level had no effect on DMI with the exception of red clover at 3% oil which was significantly (P < 0.01) lower. C18:2 n-6 and C18:3 n-3 intakes averaged 13.2 and 25.1 for grass silage and 17.9 and 36.2 g/d for red clover silage, respectively. Biohydrogenation of C18:2 n-6 and C18:3 n-3 were significantly lower (P < 0.001) on red clover silage than grass silage with oil level increasing the extent of biohydrogenation in both diets (P < 0.05; 0.81 and 0.85 to 0.91 and 0.92 for grass silage and 0.76 and 0.73 to 0.87 and 0.83 for red clover silage at 0 and 3 % oil, respectively). C18:1 trans was significantly increased by oil level for both diets (4.6 to 15.0 and 9.4 to 22.5 for grass and red clover silage at 0 and 3 % oil, respectively). Oil level increased the proportion of C18:1 trans 11 in the duodenal digesta in both diets from 0.47 and 0.31 with no oil to 0.52 and 0.51 at 3 % oil for grass

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silage and red clover silage, respectively. CLA was also significantly increased on both diets by oil level (0.21 and 0.27 to 0.48 and 0.57 g/d for grass and red clover silage at 0 and 3 % oil, respectively). The results of this study show that red clover and fish oil have the potential to beneficially alter the fatty acid profile of ruminant products.

Objective 5 Effect of ensiling on fatty acids and antioxidants (experiments 10 and 11)Experiment 10 Forage type and ensiling Forages, such as grass and red clover, are a rich source of beneficial n-3 polyunsaturated fatty acids (PUFA), especially -linolenic acid (C18:3n-3), and may be used to improve the nutritional value of ruminant products. Silage is an important feed for cattle, however, few studies have investigated the effects of ensiling method, e.g. wilting and use of additives on the fatty acid composition of the resultant silage.

Materials and methods Grass and red clover were cut on the 24 th May with a hedge-trimmer and chopped immediately using a garden shredder (Bioline 1100, Atika). The pure grass and clover and a grass/clover mix (50:50 fresh basis) were wilted inside on a concrete floor for 0, 24 or 48 h then ensiled in 100ml boiling tubes with water (control, C), Powerstart (bacterial inoculant, P) or Addsafe (formic acid inoculant, A) applied at the recommended rate. All treatment combinations were prepared in triplicate. Tubes were left in a closed rack at room temperature and pressure for 120 days, after which the silages were analysed for freeze-dry matter (FDM). Fatty acids (FA) were analysed by gas chromatography (GC). Results were analysed by general analysis of variance (treatments = forage wilt additive).

Results Significant effects of forage type, wilting and additive were noted (P < 0.001). Wilting reduced total (FA) in the fresh material, 17.2, 18.4 and 14.1 gkg-1 FDM (s.e.d.=0.31; P<0.001) for 0, 24 and 48h, respectively. These results are reflected in the content of C18:2n-6, 3.4, 3.2 and 2.8 gkg-1 FDM (s.e.d.=0.06; P<0.001), and C18:3n-3, 9.6, 10.7 and 7.3 gkg-1 FDM (s.e.d.=0.17; P<0.001) for 0, 24 and 48h, respectively. Across all forages wilting also reduced the proportion of C18:3n-3, 55.8 v. 58.2 and 51.8 gkg-1 of total FA (s.e.d.=0.11; P<0.001) in the fresh material at 0, 24 and 48h, respectively. Averaged across all three forages, total FA were higher in red clover silage compared with grass silage averaging 15.6 v. 11.9 gkg-1 FDM (s.e.d.=0.38; P<0.001), respectively. Averaged across all 3 forages, wilting resulted in a 15% loss in total FA, 14.6 v. 12.4 gkg-1 FDM (s.e.d.=0.38; P < 0.001) for 0 and 48h, respectively, and losses in C18:3n-3, 8.1, 8.1 and 6.4 gkg-1 FDM (s.e.d.=0.28; P<0.001) for 0, 24 and 48h, respectively (Table 1). This is mirrored by results for that of C18:2n-6 (P<0.001). Averaged across all forage types, both additives reduced total FA on average 14.4, 13.7 and 13.3 gkg -1 FDM (s.e.d.=0.38; P < 0.01) for the C, P and A, respectively. The control silages had the highest content of C18:3n-3, 8.0 v. 7.5 and 7.2 gkg-1

FDM (s.e.d.=0.28; P<0.05) for P and A silages, respectively (). Across all three forages the additives did not affect the content of C18:2n-6, 2.6, 2.5 and 2.5 gkg-1 FDM (s.e.d.=0.04; P = 0.062) for C, P and A, respectively.

Discussion Marked differences were noted between 0, 24 and 48h wilted silages. Both additives and wilting induced a reduction in total FA, but the additives failed to have many significant effects on individual FA parameters. Wilting resulted in a loss in C18:3n-3 in both fresh and ensiled material possibly due to oxidation and the activity of plant lipases. Reduced in-silo dry matter losses from the A and P silages may have contributed to lower concentrations of total FA. Dewhurst and King, 1998, noted no effects of fermentation type on total FA concentrations.

Table 14. Effect of wilting time (hours) on selected fatty acids (gkg-1 FDM)

0h 24h 48hG GRc Rc G GRc Rc G GRc Rc s.e.d. sig.

FDM gkg-1 184 173 151 204 201 179 255 289 233 2.87 ***C16:0 2.5 2.5 2.9 2.3 2.8 3.1 2.2 2.6 2.8 0.08 **C18:2n-6 2.2 2.6 3.4 2.1 2.7 2.9 1.9 2.4 2.7 0.07 ***C18:3n-3 7.4 7.8 9.0 6.6 8.6 9.2 5.6 6.8 6.9 0.49 NS

Table 15. Effect of type of additive on selected fatty acids (gkg-1FDM)

Control Powerstart AddsafeG GRc Rc G GRc Rc G GRc Rc s.e.d. sig.

FDM gkg-1 204 204 171 215 232 198 224 226 194 2.87 ***C16:0 2.4 2.5 2.9 2.3 2.7 2.8 2.4 2.5 2.9 0.08 ***C18:2n-6 2.1 2.6 3.1 2.0 2.6 2.9 2.1 2.5 2.9 0.07 NSC18:3n-3 6.3 8.1 9.6 6.4 8.2 7.8 6.9 6.9 7.7 0.49 **

In Tables 14 and 15 NS, not significant; *P<0.05; **P<0.01; ***P<0.001; G = grass, Rc = red clover and GRc = grass/red clover mix.

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Experiment 11 Effect of herbage dry matter level and additive treatment on the chemical composition of silage prepared from ryegrass and red cloverIntroduction and methods Two further experiments (11a; grass only and 11b; red clover only) were conducted to investigate effect of herbage dry matter at ensiling and additive treatment on chemical composition of silage. Grass (11a) and red clover (1b) (second cut material July 2005) were cut on the 24 th May with a hedge-trimmer and chopped immediately using a garden shredder (see expt 10). Forages were wilted outside on a concrete floor to achieve a range of dry matter (DM) levels (1) direct as cut ~ 20% DM), (2) 25%, (3) 30%, (4) 0, 24 or 48 h then ensiled, in triplicate, in 100ml boiling tubes with water (control, C) or Powerstart (bacterial inoculant) applied at the recommended rate. Tubes were left at room temperature and pressure for 120 days, after which the chemical composition of the silages were analysed.

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Results and Discussion Only the results for red clover are presented (as trends were similar for grass treatments). In general inoculation had little effect on fatty acid and antioxidant composition of the silages and hence only the impact of dry matter is given in Table 16. As expected water soluble carbohydrates (WSC) increased with increasing DM content reflecting a restricted fermentation, as indicated by reduced lactic acid concentrations. Loss of C18:3 increased from 20 to 30% but thereafter further losses were not apparent. This supports the view that the PUFA losses are largely associated with the first period of wilting. Carotenoids were not significantly effected by DM at ensiling.

Table 16. Effect of dry matter on chemical composition of red clover silage

Dry matter20 % 25% 30% 35% 40% s.e.d P

DM (g/kg FM) 224.7 261.6 286.1 338.9 389.1 0.66 ***NH3-N 0.75 0.57 0.58 0.62 0.59 0.145 ***Nitrogen 26.0 26.0 24.7 26.1 26.4 1.26 NSWSC 30.4 34.3 44.9 52.8 62.1 3.85 ***NDF 350.7 383.1 401.2 380.8 385.2 14.35 **ADF 270.0 266.4 297.1 275.2 277.1 12.45 NSpH 3.65 3.69 3.67 3.80 3.79 0.108 ***Acetate (mMol/l) 5.44 6.17 7.96 10.6 6.81 1.021 *Lactate (mMol/l) 86.6 80.8 78.7 50.6 54.4 4.400 ***Butyrate (mMol/l) 1.15 1.15 1.15 3.36 1.15 0.835 ***C16:0 3.66 3.51 3.71 3.75 3.70 0.253 NSC18:0 0.52 0.51 0.51 0.52 0.50 0.042 NSC18:2n-6 4.59 4.39 4.61 4.60 4.60 0.265 NSC18:3n-3 8.84 7.72 7.96 8.13 8.00 0.845 *Total FA 19.7 17.9 18.7 18.9 18.7 1.51 NSPUFA loss (%)C18:2n-6 total 23.1 22.2 26.6 23.7 23.2 6.78 *C18:3n-3 total 26.2 31.5 36.5 33.1 33.4 7.45 *

Table 17. Effect of dry matter on the carotenoid composition (µg/g DM) of the red clover

20% 25% 30% 35% 40%Violaxanthine 0.05 0.06 0.08 0.05 0.07Antheraxanthine ND ND 0.14 ND NDLuteine 0.27 0.27 0.24 0.25 0.14Zeaxanthine 0.03 0.04 0.03 0.04 0.02ß-carotene 0.09 0.11 0.08 0.09 0.059-cis-ß-carotene 0.06 0.09 0.06 0.05 0.0313-cis-ß-carotene 0.02 0.03 0.02 0.03 0.01ND, not detected; tr, <0.005

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Overall discussionResults from the project have shown that beef may be produced which is low fat (less than 5%), have a lower content of atherogenic saturated fatty acids (SFA), higher content of more beneficial monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) and lower n-6:n-3 PUFA ratio than was previously possible. Grass relative to concentrate feeding resulted in higher levels on n-3 PUFA resulting in very favourable n-6:n-3 ratios. Grass grazing not only produces meat with an improved n-3 fatty acid content but also produces more yellow fat due to its carotene content and a greater lipid stability due to the natural intake of vitamin E with the diet. Supplementing animals with concentrates whilst grazing over a 100d period tended to reduce the natural vitamin E content of the meat and reduce stability. Ruminally protected lipids (as noted in other studies i.e Scollan et al., 2003) resulted in large increases in 18:2n-6 and 18:3n-3 contributing to a very beneficial P:S ratio. This highlights the role of the rumen in regulating the ability to alter the fatty acid composition of beef. The protected lipids, however, caused a severe oxidative challenge in the meat (reduced stability) and which may be ameliorated by supplementing the diet with vitamin E. Relative to feeding grass silage, red clover enhanced the content of PUFA (both 18:2n-6 and 18:3n-3) but due to low content of vitamin E in the meat, red clover reduces colour shelf life.

The in vitro and in vivo studies on rumen lipid metabolism have helped to extend substantially our knowledge of the major mechanism involved. In vitro studies have highlighted the importance of plant chemical constituents during lipid metabolism in the rumen most notability the role of: plant lipases in carrying out lipolysis of dietary lipids before and potentially during ingestion, polyphenol oxidase of red clover’s effect in reducing lipolytic activity in the rumen and subsequently reducing biohydrogenation and finally the role of green odour compounds in manipulating the extent of biohydrogenation and the formation of particular intermediates such as TVA through their antimicrobial properties. In vivo studies have made excellent progress in characterising the process of ruminal lipolysis and biohydrogenation of dietary lipids, under a wide range of dietary conditions. The results confirm that the extent of biohydrogenation dietary PUFA from a range of different feed types, including forages, is very high, averaging approximately 86 and 92% for 18:2n-6 and 18:3n-3, respectively. However, we have noted lower levels of biohydrogenation, measured in vivo, when feeding red clover relative to grass silage. Red clover contains the enzyme polyphenol oxidase (PPO) which is activated when red clover tissue is damaged, reducing the extent of lipolysis. Feeding concentrates containing fish oil has also been found to reduce biohydrogenation resulting in the elevation of TVA.

Future ResearchThe effects of red clover on PUFA composition of meat most likely relates to the presence of polyphenol oxidase (PPO) in forage. This requires further investigation. Mechanisms which reduce the extent of biohydrogenation of dietary lipids by rumen micro-organisms are required and research should concentrate on these aspects. Understanding the major microbial species involved in biohydrogenation and how they are influenced by diet will permit an increased understanding of methods of modifying this process. Increasing our understanding and developing methods of altering lipolysis and biohydrogenation of dietary PUFA in the rumen is essential in terms of providing new opportunities for enhancing the fatty acid composition of beef and other ruminant products. Grass feeding may have some beneficial effects on improving lipid and colour stability of raw meat but there is evidence that this merits further attention when these materials are further processed. The relationships between vitamin E (and other antioxidants) and n-3 PUFA in meat to ensure adequate stability during processing require attention. Evidence for beneficial relationships species-rich pastures and fatty acid composition and sensory attributes of meat is interesting and merit further attention. The transfer of linolenic acid from forage through to meat is dependent on two important processes: (1) increasing the level of αLNA in the forage (and hence into the animal) and (2) reducing the extent of ruminal biohydrogenation. Research should focus on increasing our understanding of these two major critical control points to increase delivery of αLNA from forage through to meat and milk. Greater integration of research across the various levels of the food chain and increased interaction with industry will help in the delivery of foods with higher nutritional and health benefits for consumers.

Dissemination and exploitation of results The results from the project have been communicated through a number of means, from the publication of scientific results in high quality peer-reviewed learned journals through to articles in the popular farming press and technical fact-sheets. Results have been disseminated at national and international scientific meetings, and local and national agricultural shows and events. For full list of associated publications please see below. The research and associated outputs will directly underpin the production of safe and healthy beef for consumers. The research will encourage a more competitive and sustainable beef food industry delivering products which consumers require and helping to stimulate market differentiation. It is evident, that the beef industry in the UK cannot compete with other key beef producing countries on price and will have to focus more on "quality" to achieve a competitive and sustainable industry.

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ReferencesDe Gouw, J.A., Howard, C.J., Custer, T.G. and Fall, R. 1999. Emissions of volatile organic compounds from cut grass and clover are enhanced during the drying process. Geophys. Res. Letters 26:811-814.Dewhurst, R. J. and King, P. J. (1998). Effects of extended wilting, shading and chemical additives on the fatty acids in laboratory grass silages. Grass and Forage Science, 53(3): 219-224.Faichney, G.J, 1975. The use of markers to partition digestion within the gastro-intestinal tract of ruminants. In: I.W. McDonald, A.C.I. Warner (Editors). Digestion and metabolism in the ruminant. University of New England, Armidale, pp. 277-291.Folch, J., Lees, M. and Stanley, G.H.S. 1957. A simple method for the isolation and purification of total lipids from animal tissue. J. Biol. Chem. 226:497-509.Jahreis, G., Fritsche, J. and Steinhart, H. 1997. Conjugated linoleic acid in milk: high variation depending on production system. Nutr. Res. 17:1479-1484.Jenkin, T.C. 1993. Lipid metabolism in the rumen. J. Dairy Sci. 76: 3851-3863Kramer, J.K.G. and Zhou, J. 2001. Conjugated linoleic acid and octadecenoic acids: Extraction and isolation of lipids. Euro. J. Lipid Sci. Technol. 103: 594-632.Kubo, A., Lunde, C.S. and Kubo, I. 1995. Antimicrobial activity of the olive oil flavour compounds. J. Agric. Food Chem. 43:1629-1633.Kucuk, O., Hess, B.W., Ludden, P.A. and Rule, D.C. 2001. Effect of forage : concentrate ratio on ruminal digestion and duodenal flow of fatty acids in ewes. Journal of Animal Science 79: 2233-2240.Lee, M.R.F., Harris, L.J., Dewhurst, R.J., Merry, R.J. and Scollan, N.D. 2003a. The effect of clover silages on long chain fatty acid rumen transformations and digestion in beef steers. Ani. Sci. 76: 491-501.Lee, M.R.F., Merry, R.J., Davies, D.R., Moorby, J.M., Humphreys, M.O., Theodorou, M.K., MacRae, J.C., Scollan, N.D. 2003b. Effects of increasing availability of water-soluble carbohydrates on in vitro fermentation. Ani. Feed Sci. Technol. 104: 59-70.Lee, M.R.F., Tweed, J.K.S., Connelly, P.L., Merry, R.J., Dewhurst, R.J., Scollan, N.D. 2004a. Duodenal flow of C18:1 and conjugated linoleic acid isomers in beef steers fed high sugar grass, red clover or grass/red clover mix silages. Pro. Bri. Soc. Ani. Sci. 67.Lee, M.R.F., Winters, A. L., Scollan, N.D., Dewhurst, R.J., Theodorou, M.K. and Minchin, F.R. 2004b. Plant-mediated lipolysis and proteolysis in red clover with different polyphenol oxidase activities. J. Sci Food Agric. 84:1639-1645Lee, M.R.F., Hodgkins, C., Tweed, J.K.S., Scollan N.D., and Dewhurst R.J. 2005. An in vitro investigation into forage factors which may influence the production of conjugated linoleic acid and trans vaccenic acid in the rumen. I. Genotype. XX International Grassland Congress, June 26th – 1st July, Dublin, Eire, pp179.Loor, J.J., Ueda, K., Ferlay, A., Chilliard, Y. and Doreau, M. 2004. Biohydrogenation, duodenal flow, and intestinal digestibility of trans fatty acids and conjugated linoleic acids in response to dietary forage : concentrate ratio and linseed oil in dairy cows. J. Dairy Sci. 87: 2472-2485.Offer, N.W. 2002. Effect of cutting and ensiling grass on levels of CLA in bovine milk. XIII International silage conference, September 11th-13th, Auchincruive, Scotland, pp. 16-17.Scollan, N.D., Choi, N.J., Kurt, E., Fisher, A.V., Enser M., and Wood, J.D. 2001a. Manipulating the fatty acid composition of muscle and adipose tissue in beef cattle. Brit. J. Nut. 85, 115-124.Scollan, N.D., Dhanoa, M.S., Choi, N.J., Maeng, W.J., Enser, M. and Wood J.D. 2001b. Biohydrogenation and digestion of long chain fatty acids in steers fed on different sources of lipid. J. Agric. Sci. 136, 345-355Scollan, N.D., Enser, M. Gulati, S., Richardson, I. and Wood, J.D. (2003). Effects of including a ruminally protected lipid supplement in the diet on the fatty acid composition of beef muscle in Charolais steers. Brit. J. Nutr. 90, 709-716.Shingfield, K.J., Ahvenjarvi, S., Toivonen, V., Arola, A., Nurmela, K.V.V., Huhtanan, P. and Griinairi, J.M. 2003. Effect of dietary fish oil on biohydrogenation of fatty acid and milk fatty acid content in cows. Ani. Sci. 77, 165-179.Tapiero, H., Nguyen Ba, G., Couvreur, P. and Tew K.D., 2002. Polyunsaturated fatty acid (PUFA) and eicosanoids in human health and pathologies. Biomed. Pharmecother. 56, 215-222.Wallace R.J., Walker, N.D., Richardson, A.J., Chaudhary, L.C., Koppova,, I., McEwan N.R., McKain, N., King, T.P. and Newbold C.J., 2004. Re-isolation, identification, and propertied of ‘Fusocillus’, the only species of ruminal bacteria known to convert linoleic acid to stearic acid. Appl. Environ Microbiol.

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References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.Reviews and Refereed Papers1. Wright, T.C., Odongo, N.E., Scollan, N.D. and McBride, B.W. (2007). Nutritional Manipulation of

Functional Foods Derived from Herbivores for Human Nutritional Benefit. China Scientific Press 2007, Proceedings of the 7th International Symposium on the Nutrition of Herbivores, Beijing. Eds. Q.X. Meng, J.X. Liu, and W.Y. Zhu.

2. Scollan, N.D. and Wood, J.D. (2006). Enhancing the nutritional value of beef and its relationships with meat quality. Page 83-84 in Proceedings of British Society of Animal Science, Linking up the meat chain: ensuring quality and safety for the consumer, 19-20 October 2006, Krakow, Poland.

3. Scollan, N. D. and Huws, S.A. Enrichment of ruminant products with beneficial fatty acids, Recent Advances in Nutrition, Australia, 15, 21-31.

4. Scollan, N.D., Richardson, R.I. and Moloney, A.P. (2005). Effect of beef systems on meat composition and quality. Proceedings of British Society of Animal Science, pages 1-5 (The science of beef quality 8th Annual Langford Food Industry Conference).

5. Scollan, N. D., Dewhurst, R. J., Moloney, A. P., Murphy, J. J. (2005). Improving the quality of products from grassland. In: Grassland a global resource, DA McGilloway (editor), Wageningen Academic Publishers, The Netherlands, pages 41-56.

6. Scollan, N. D., Richardson, I., De Smet, S., Moloney, A. P., Doreau, M., Bauchart, D. and Nuernberg, K. (2005). Enhancing the content of beneficial fatty acids in beef and consequences for meat quality. In: Indicators of milk and beef quality, JF Hocquette and S Gigli (editors), EAAP Publ. 112, Wageningen Academic Publishers, The Netherlands, pages 151-162.

7. Scollan, N. D. Opportunities for improving the nutritional characteristics of beef: a role for grassland agriculture. 27th Congress of Animal Production Argentina, Tandil, Argentina, 20-22 October 2004

8. Lee, M.R.F., Huws, S.A., Scollan, N.D. and Dewhurst, R.J. (2007). Effects of fatty acid oxidation products (green odour) on rumen bacterial populations and lipid metabolism in vitro. Journal of Dairy Science, 90, 3874-3882.

9. Scollan, N.D., Hocquette, J.F., Nuernberg, K., Dannenberger, D., Richardson, I. and Moloney, A.P. (2006). Innovations in beef production systems that enhance the nutritional value of beef and its relationship with meat quality. Meat Science 74, 17-33.

10.Yanez-Ruiz, D.R., Scollan, N.D., Merry, R.J. and Newbold, C.J. (2006). Contribution of rumen protozoa to duodenal flow of nitrogen, conjugated linoleic acid and vaccenic in steers fed silages differing in their water-soluble carbohydrate content. British Journal of Nutrition 96, 861-869.

11.Lee, M.R.F., Connelly, P.L., Tweed, J.K.S., Dewhurst, R.J., Merry, R.J., Scollan, N. D. (2006). Effects of high sugar ryegrass and mixtures with red clover silage on rumen function. 2. Lipid metabolism. Journal of Animal Science 84, 3061-3070.

12.Lee, M.R.F., Parfitt, L.F., Scollan, N.D. and Minchin, F.R. (2006). Lipolysis in red clover with different polyphenol oxidase activities in the presence and absence of rumen micro-organisms. Journal of the Science of Food and Agriculture 87 (7), 1308-1314.

13.Lee, M.R.F., Olmos Colmenero, J. de J., Winters, A.L., Scollan, N.D., Minchin, F.R. (2006). Polyphenol oxidase activity in grass and its effect on plant mediated lipolysis and proteolysis of Dactylis glomerata (Cocksfoot) in a rumen simulated environment. Journal of the Science of Food and Agriculture 86, 1503-1511.

14.Hocquette, J.F., Richardson, R.I., Prache, S., Medale, F., Duffy, G. and Scollan, N.D. (2005). Future trends for research on quality and safety of products from animal origin. Italian Journal of Animal Science, 4 (3)   49-72.

15.Lee, M.R.F., Tweed, J.K.S., Dewhurst, R.J., Scollan, N.D. (2006). Effect of forage: concentrate ratio on ruminal metabolism and duodenal flow of fatty acids in beef steers. Animal Science, 82, 31-40.

16.Lee, M.R.F., Tweed, J.K.S, Moloney, A.P. and Scollan, N.D. (2005). The effects of fish oil supplementation on rumen metabolism and the biohydrogenation of unsaturated fatty acids in beef steers given diets containing sunflower oil. Animal Science 80, 361-367.

17.Lee, M.R.F., Winters, A.L., Scollan, N.D., Dewhurst, R.J., Theodorou, M.K. and Minchen, F.R. (2004). Plant-mediated lipolysis and proteolysis in red clover with different polyphenol oxidase activities. Journal of Science of Food and Agriculture, 84 (13), 1639-1645.

18.Scollan, N.D., Lee, M.R.F. and Enser, M. (2003). Biohydrogenation and digestion of long chain fatty acids in steers fed on Lolium perenne bred for elevated levels of water-soluble carbohydrate. Animal Research, 52, 501-511.

19.Lee, M.R.F., Harris, L., Merry, R.J., Dewhurst, R.D. and Scollan, N.D. (2003). Effect of clover silages on long chain fatty acid rumen transformations and digestion in steers. Animal Science 76, 491-501.

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Edited contributions to conference or learned societies1. Scollan, N.D., Gibson, K., Ball, R. and Richardson, R.I. (2008). Meat quality of Charolais steers: influence of

feeding grass versus red clover during winter followed by finish off grass. Proceedings of the British Society of Animal Science, submitted.

2. Richardson, R.I., Wood, J.D., Ball, R., Hallett, K.G. and Scollan, N.D. (2007). Effect of grass and concentrate feeding systems on fatty acid composition, lipid and colour shelf life of beef loin muscle. 53 rd International Congress of Meat Science and Technology August 2007, China.

3. Scollan, N.D., Hallett, K.G., Wood, J.D. and Richardson, I. (2007). The fatty acid composition of muscle fat in Charolais steers: influence of grass versus concentrate feeding. Proceedings of the British Society of Animal Science, p 14.

4. Richardson, R.I., Wood, J.D., Ball, R., Nute, G.R. and Scollan, N.D. (2007). Influence of grass and concentrate feeding systems on lipid and colour shelf life of loin steaks from Charolais steers. Proceedings of the British Society of Animal Science, p109.

5. Kim, E.J., Wood, J.D., Richardson, I., Huws, S.A. and Scollan, N.D. Effect of level of fish oil in the diet on flow of fatty acids to the small intestine in steers.  Proceedings of the British Society of Animal Science, p151.

6. Lee M. R. F., Shingfield K. J. and Scollan N. D. (2006). The effect of fish oil supplementation on ruminal C18 PUFA metabolism in beef steers offered either grass or red clover. Proceedings of the American Society of Animal Science.

7. Scollan, N.D., Costa, P., Hallett, K.G., Nute, G.R., Word, J.D. and Richardson, R.I. (2006). The fatty acid composition of muscle fat and relationships to meat quality in Charolais steers: influence of level of red clover in the diet. Proceedings of the British Society of Animal Science, p23.

8. Doreau, M., Lee, M.R.F., Ueda, K. and Scollan, N.D. (2005). Ruminal metabolism and absorption of fatty acids from forages. Rencontres Recherches Ruminants 12, 101-104.

9. Richardson, R.I., Costa, P., Nute, G.R. and Scollan, N.D. (2005). The effect of feeding clover silage on polyunsaturated fatty acid and vitamin E content, sensory, colour and lipid oxidative shelf life, of beef loin steaks. 51st International Congress of Meat Science and Technology August 7-12, 2005 – Baltimore, Maryland USA

10. Lee, M. R. F., Tweed, J. K. S., Scollan, N. D. , Dewhurst, R. J. (2005). An in vitro investigation of forage factors which affect the production of conjugated linoleic acid and trans vaccenic acid in the rumen. II. Wilting & cell damage. Page 180 in XX International Grassland Congress,, F.P. O’Mara, R.J. Wilkins, L. ‘t Mannetje, D.K. Lovett, P.A.M. Rogers, T.M. Boland (editors), Wageningen Academic Publishers, The Netherlands.

11. Lee, M.R.F., Hodgkins, C., Tweed, J.K.S., Scollan, N.D., Dewhurst, R.J. (2005). An in vitro investigation of forage factors which affect the production of conjugated linoleic acid and trans vaccenic acid in the rumen. I. Grass species. Page 179 in XX International Grassland Congress,, F.P. O’Mara, R.J. Wilkins, L.‘t Mannetje, D.K. Lovett, P.A.M. Rogers, T.M. Boland (editors), Wageningen Academic Publishers, The Netherlands.

12. Scollan, N.D., Enser, M., Hallett, K.G. , Ball, R., Nute, G.R. , Wood, J.D.   et al. (2005). The fatty acid composition of muscle fat and relationships to meat quality in Charolais steers: influence of level of fish oil in the diet. Proceedings of the British Society of Animal Science (BSAS) Winter Meeting, p21.

13. Lee, M.R.F., Tweed, K.J.S., Neville, M.A., Scollan, N.D., Dewhurst, R.J. (2005). The effect of fatty acid oxidation on lipid metabolism during in vitro batch culture. Proceedings of the British Society of Animal Science (BSAS) Annual Meeting, York, p 73.

14. Lee, M.R.F., Tweed, J.K.S., Scollan, N.D. (2005). Duodenal flow and biohydrogenation of C18 polyunsaturated fatty acids in beef steers fed isonitrogenous and isoenergetic diets with contrasting forage: concentrate diets. Proceedings of the British Society of Animal Science Annual Meeting, p71.

15. Scollan, N.D., Enser, M., Richardson, R.I., Gulati, S., Hallett, K.G., Nute, G.R. and Wood, J.D. (2004). The effects of ruminally-protected dietary lipid on the fatty acid composition and quality of beef muscle. Page 116 in the Proceedings of the International Congress of Meat Science and Technology, Helsinki, Finland (8-13 th

August 2004).16. Lee, M.R.F., Tweed, J.K.S., Connelly, P.I., Merry, R.J., Dewhurst, R.J. and Scollan, N.D. (2004). Duodenal

flow and biohydrogenation of C18 polyunsaturated fatty acids in beef steers fed high sugar grass, red clover or grass/red clover mix silages. British Society of Animal Science, York. April 2004.

17. Lee, M.R.F., Tweed, J.K.S., Connell, P.I., Merry, R.J., Dewhurst, R.J. and Scollan, N.D. (2004). Duodenal flow of C18:1 and conjugated linoleic acid isomers in beef steers fed high sugar grass, red clover or grass/red clover mix silages. British Society of Animal Science, York. April 2004.

18. Warren, H.E., Enser, M., Hallett, K., Wood, J.D., Dhanoa, M.S. and Scollan, N.D. (2004). Effect of age on the fatty acid classes of beef muscle. British Society of Animal Science, York. April 2004.

19. Whittington, F.M., Nute, G.R., Scollan, N.D., Richardson, R.I. and Wood, J.D. (2004). Effect of diet and breed on skatole deposition in cattle slaughtered at 19 and 24 months. British Society of Animal Science, York. April 2004.

20. Lee, M.R.F., Martinez, E.M., Scollan, N.D. and Theodorou, M.K. (2003). In vitro rumen plant enzyme mediated lipolysis: grass v. red clover. Conference on Gastrointestinal Function, Chicago, Illinois, 10-12 March 2003   Abstract 3.

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21. Lee, M.R.F., Martinez, E.M., Scollan, N.D. and Theodorou, M.K. (2003). Plant enzyme mediated lipolysis of Lolium perenne and Trifolium pratense in an in vitro simulated rumen environment. Annual Meeting of the European Association for Animal Production, Rome, Italy. 31 Aug-3 Sept 2003.

22. Nurernberg, K., Dannenberger, D., Nuernberg, G., Ender, K. and Scollan, N.D. (2003). N-3 fatty acids and conjugated linoleic acids of longissimus muscle in different cattle breeds. Annual Meeting of the European Association for Animal Production, Rome, Italy. 31 August-3 September, 2003.

23. Richardson, R.I., Enser, M., Whittington, F.W., Hallet, K.G., Wood, J.D. and Scollan, N.D. (2003). Effects of product type and fatty acid composition on shelf life of nutritionally modified beef. British Society of Animal Science Winter meeting, March 2003.

24. Scollan, N.D. (2003). The role of grassland agriculture in producing products of enhanced nutritive value. First International Congress on the Atlantic Diet, 17-19th July 2003, Viana do Castelo, Portugal.

25. Scollan, N.D., Enser, M., Richardson, R.I., Wood, J.D. (2003). Optimising the fatty acid composition of beef muscle. British Society of Animal Science Winter meeting, March 2003.

26. Scollan, N.D., Enser, M., DeSmet, S., Moloney, A., and Nuerberg, K. (2002) Factors influencing the fatty acid composition of beef. Proceedings of Meat fatty acids conference, July 2002, Axbridge, Nr. Bristol.

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