1. Progress in forage-based strategies to improve the fatty acid composition of beef SA Morgan, S Huws and ND Scollan Aberystwyth, Wales, United Kingdom nigel.scollan@aber.ac.uk

2. Overview Plant-based strategies Ruminal lipolysis and biohydrogenation Chloroplast Beef omega-3’s relative to claims Conclusions

3. Nutritional aspects of health  consumers – diet, health and well-being  nutritional quality and product quality  high biological value  rich in micronutrients  but…..

4. Fatty acids in ruminant products PUFA linoleic acid (18:2n-6)  -linolenic acid (18:3n-3) eicosapentaenoic acid (20:5n-3; EPA) docosahexaenoic acid (22:6n-3; DHA) conjugated linoleic acids

5. Omega 3 & food chain

6. Families of polyunsaturated fatty acids n-3 Series 18:3 Alpha-linolenic acid 18:4 20:4 20:5 Eicosapentaenoic acid (EPA) 22:5 22:6 Docosahexaenoic acid (DHA) n-6 Series 18:2 Linoleic acid 18:3 20:3 20:4 Arachidonic acid 22:4 22:5 Docosapentaenoic acid (DPA)

7. Overview Plant-based strategies Ruminal lipolysis and biohydrogenation Chloroplast Beef omega-3’s relative to claims Conclusions

8. Grass a rich source of 18:3n-3

9. Factors that effect forage lipid forage species season plant maturity –leaf:stem fertiliser regime conservation method

10. 16:0 – Palmitic acid 18:2n-6 – Linoleic acid 18:3n-3 – Linoleic acid Other Dewhurst et al (2001) Forage Species

11. Season/Maturity 3 12 14 18 10 33 7 3 25 5 23 30 7 Stage of maturity Cell contents Cell wall Protein

12. Season/Maturity 3 12 14 18 10 33 7 3 25 5 23 30 7 Stage of maturity Lipid decreases as maturity increases Protein

13. Stems Fibre & Lignin Protein Leaves Lipid Minerals Low Medium High Vegetative Elongation Reproductive Composition (relative values) Season/Maturity

14. Cutting Date

15. Regrowth Interval

16. N Fertiliser

17. Conservation - Wilting

18. (Skøt et al. submitted) Identification of QTL for lipid constituents in Lolium perenne

19. Overview Plant-based strategies Ruminal lipolysis and biohydrogenation Chloroplast Beef omega-3’s relative to claims Conclusions

20. Mouth Abomasum Dietary Lipid FFA C18 PUFA Conjugated dienes and trienes Trans 18:1 18:0 Lipolysis Biohydrogenation Rumen Lipolysis and biohydrogenation 18:3 n-3 92% 18:2 n-6 86%

21.  Bacteria Lipolysis – Anaerovibrio lipolytica and Butyrivibrio fibrisolvens Biohydrogenation – Butyrivibrio fibrisolvens, B. hungatei, Clostridium proteoclasticum, Megasphaera elsdenii, Streptococcus bovis, Propionibacterium, Lactobacillus  Protozoa – little evidence but… - 75% of total microbial lipid - 50% biomass - 90% unsaturated fatty acids - Ingest PUFA-rich chloroplasts  Fungi Lipolysis – little evidence to date Biohydrogenation – yes, but slower than bacteria Micro-organisms involved in lipolysis and biohydrogenation

22. Group TVA1, TVA producers Group TVA2, TVA producers Group Atyp, TVA producers Group SA TVA and SA producers Butyrivibrio spp. phylogenetic tree ( Wallace et al. 2006) Butyrivibrio Pseudobutyrivibrio Clostridium proteoclasticum

23. Overview Plant-based strategies Ruminal lipolysis and biohydrogenation Chloroplast Beef omega-3’s relative to claims Conclusions

24. Ingested plant cells From mouth Abomasum Organelle phospho- & galacto lipid Free fatty acids Organelle phospho- & galacto lipid Intact cellRuptured cell PPO -ve Rumen microflora Bacterial lipolysis Free Pool C18 PUFA Conjugated dienes and trienes Trans monoenes 18:0 Protozoa Green odour compounds Plant lipolysis Lipolysis and biohydrogenation in fresh herbage Rumen

25. Ruminal protozoa are rich in polyunsaturated fatty acids due to ingestion of PUFA-rich chloroplast Scale bars – 20 µm Epidinium sp. isolated from the rumen of steers fed fresh grass Huws et al. (2009). FEMS Microbiology Ecology, 69: 461-471.

26. Protozoal contribution to fatty acid flow g/d e Challenge enhance chloroplast uptake whilst ensuring duodenal flow ( Huws et al., 2012 ) ZERO !!!

27. Overview Plant-based strategies Ruminal lipolysis and biohydrogenation Chloroplast Beef omega-3’s relative to claims Conclusions

28. European Food Safety Authority (EFSA, 2009) European Food Safety Authority –250 mg per day EPA and DHA or 2g per day 18:3n-3 –15% RDA – “source off” – 40 mg EPA/DHA –30% RDA – “high in” – 80 mg EPA/DHA

29. Studies assessing diet effects on beef fatty acids Grass silage v concentrate (Warren et al. 2008) Pasture v. 1 / 2 month concentrate (Aldai et al. 2011) Grass v red clover silage (Scollan et al. 2008) Lowland (< 400m) v ridge (400-700 m) (Costa et al. 2011) Control v protected fish oil (Dunne et al. 2011)

30. 18:3n-3 PUFA (mg/100 g muscle)

31. EPA (mg/100 g muscle)

32. DHA (mg/100 g muscle)

33. Studies assessing diet effects on beef fatty acids 18:3 n-3~ 50 mg/100g [ Compare 2g / 100g muscle] EPA ~ 20 mg/100 g (~ 36 veal) DHA~ 5 mg/100 g (~ 6 veal) [compare 40 mg/100 g muscle] 67mg EPA + DHA – protected lipids (Dunne et al. 2011)

34. 10kg DM d -1 Forage  200g lipid  100g 18:3n-3 10kg DM d -1 Forage  200g lipid  100g 18:3n-3 Lipolysis & BH Dietary lipid  Free C18:3n-3  C18 conjugated trienes/dienes  C18:1 isomers  C18:0 Lipolysis & BH Dietary lipid  Free C18:3n-3  C18 conjugated trienes/dienes  C18:1 isomers  C18:0 RUMEN Lipolysis Biohydrogenation Lipid reaching duodenum  16g lipid  8g/100g 18:3n-3 Lipid reaching duodenum  16g lipid  8g/100g 18:3n-3 Muscle FA content  3000 mg lipid  35 mg/100g 18:3n-3  25 mg/100g n-3LCPUFA Muscle FA content  3000 mg lipid  35 mg/100g 18:3n-3  25 mg/100g n-3LCPUFA To conclude…

35. Conclusions  Consumers – nutritional quality and product quality  Meat - source on n-3 PUFA – green forage  Reducing lipolysis and biohydrogenation  Advancing our understanding of bacterial lipolysis and biohydrogenation  Role of chloroplast

36. Dr Michael Lee Dr Sharon Huws Dr Alison Kingston-Smith Professor Jamie Newbold Leif Skot Matt Hegarety John Tweed Acknowledgements