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Rumen Metabolism of 22:6n-3 In Vitro is Dependent on its Concentration and Inoculum Size, but Less Dependent on Substrate Carbohydrate Composition

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Lipids

Abstract

Ruminal disappearance of linoleic and linolenic acid has been studied extensively. Less is known of the metabolism of docosahexaenoic acid (22:6n-3). The aim of this study was to identify factors which affect the disappearance of 22:6n-3 during in vitro batch incubations using rumen fluid from sheep. In experiment 1, the effect of the rumen fluid/buffer ratio (0.2 or 0.4), substrate (cellulose or cellulose/glucose), time of 22:6n-3 addition (0.08 mg/mL after 0 or 6 h of incubation) and incubation time (24 or 48 h) was evaluated. A mixture design was used in experiment 2 to evaluate the effect of carbohydrate type (cellulose, glucose, cellobiose and starch) on 22:6n-3 disappearance (0.08 mg/mL). In experiment 3, several concentrations of 22:6n-3 (0.05–0.30 mg/mL) were evaluated with different substrate mixtures (combinations of cellobiose, starch and cellulose). In a final experiment, the effect of the rumen fluid/buffer ratio (0.20, 0.35 and 0.50) and substrate (glucose, cellobiose and starch) was evaluated. In this experiment, 22:6n-3 was added as a proportion of rumen fluid ranging from 0.1 to 0.4 mg/mL rumen fluid, contrary to former experiments where concentrations were relative to culture medium. Low levels of 22:6n-3 (0.05 mg/mL) allowed extensive metabolism whereas increasing amounts of 22:6n-3 hampered its disappearance. A greater proportion of rumen fluid resulted in increased disappearance of 22:6n-3. The effect of carbohydrate type was small compared with the former two factors. These results suggest that in vitro metabolism of 22:6n-3 is mostly dictated by the conditions at the start of the incubation, i.e., inoculum, probably reflecting the density of bacteria able to metabolize 22:6n-3.

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Abbreviations

AUC:

Area under the curve

h:

Hour

RF:

Rumen fluid/buffer ratio

SEM:

Standard error of the mean

References

  1. Jenkins TC, Wallace RJ, Moate PJ, Mosley EE (2008) Board-invited review: recent advances in biohydrogenation of unsaturated fatty acids within the rumen microbial ecosystem. J Anim Sci 86:397–412

    Article  CAS  PubMed  Google Scholar 

  2. Shingfield KJ, Griinari JM (2007) Role of biohydrogenation intermediates in milk fat depression. Eur J Lipid Sci Tech 109:799–816

    Article  CAS  Google Scholar 

  3. Chilliard Y, Glasser F, Ferlay A, Bernard L, Rouel J, Doreau M (2007) Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat. Eur J Lipid Sci Tech 109:828–855

    Article  CAS  Google Scholar 

  4. Kairenius P, Toivonen V, Shingfield KJ (2011) Identification and ruminal outflow of long-chain fatty acid biohydrogenation intermediates in cows fed diets containing fish oil. Lipids 46:587–606

    Article  CAS  PubMed  Google Scholar 

  5. Fievez V, Vlaeminck B, Jenkins T, Doreau M (2007) Assessing rumen biohydrogenation and its manipulation in vivo, in vitro and in situ. Eur J Lipid Sci Technol 109:740–756

    Article  CAS  Google Scholar 

  6. AbuGhazaleh AA, Jenkins TC (2004) Disappearance of docosahexaenoic and eicosapentaenoic acids from cultures of mixed ruminal microorganisms. J Dairy Sci 87:645–651

    Article  CAS  PubMed  Google Scholar 

  7. Klein CM, Jenkins TC (2011) Docosahexaenoic acid elevates trans-18:1 isomers but is not directly converted into trans-18:1 isomers in ruminal batch cultures. J Dairy Sci 94:4676–4683

    Article  CAS  PubMed  Google Scholar 

  8. Aldai N, Hervas G, Belenguer A, Frutos P, Mantecon AR, Kramer JK (2012) Evaluating the in vitro metabolism of docosahexaenoic acid in sheep rumen fluid. Lipids 47:821–825

    Article  CAS  PubMed  Google Scholar 

  9. Mould FL, Morgan R, Kliem KE, Krystallidou E (2005) A review and simplification of the in vitro incubation medium. Anim Feed Sci Tech 123–124:155–172

    Article  Google Scholar 

  10. Vlaeminck B, Mengistu G, Fievez V, de Jonge L, Dijkstra J (2008) Effect of in vitro docosahexaenoic acid supplementation to marine algae-adapted and unadapted rumen inoculum on the biohydrogenation of unsaturated fatty acids in freeze-dried grass. J Dairy Sci 91:1122–1132

    Article  CAS  PubMed  Google Scholar 

  11. Hassim HA, Lourenco M, Goel G, Vlaeminck B, Goh YM, Fievez V (2010) Effect of different inclusion levels of oil palm fronds on in vitro rumen fermentation pattern, fatty acid metabolism and apparent biohydrogenation of linoleic and linolenic acid. Anim Feed Sci Tech 162:155–158

    Article  CAS  Google Scholar 

  12. Van Ranst G, Fievez V, Vandewalle M, Van Waes C, De Riek J, Van Bockstaele E (2010) Influence of damaging and wilting red clover on lipid metabolism during ensiling and in vitro rumen incubation. Animal 4:1528–1540

    Article  PubMed  Google Scholar 

  13. De Weirdt R, Coenen E, Vlaeminck B, Fievez V, Van den Abbeele P, Van de Wiele T (2013) A simulated mucus layer protects Lactobacillus reuteri from the inhibitory effects of linoleic acid. Benef Microbes 4:299–312

    Article  PubMed  Google Scholar 

  14. Udén P, Robinson PH, Mateos GG, Blank R (2012) Use of replicates in statistical analyses in papers submitted for publication in animal feed science and technology. Anim Feed Sci Tech 171:1–5

    Article  Google Scholar 

  15. Scheffe H (1963) Simplex-centroid design for experiments with mixtures. J R Stat Soc Ser B Stat Methodol 25:235–263

    Google Scholar 

  16. AbuGhazaleh AA, Jenkins TC (2004) Short communication: docosahexaenoic acid promotes vaccenic acid accumulation in mixed ruminal cultures when incubated with linoleic acid. J Dairy Sci 87:1047–1050

    Article  CAS  PubMed  Google Scholar 

  17. Wasowska I, Maia MRG, Niedzwiedzka KM, Czauderna M, Ribeiro JMCR, Devillard E, Shingfield KJ, Wallace RJ (2006) Influence of fish oil on ruminal biohydrogenation of C18 unsaturated fatty acids. Br J Nutr 95:1199–1211

    Article  CAS  PubMed  Google Scholar 

  18. Desbois AP, Smith VJ (2009) Antibacterial free fatty acids: activities, mechanisms of action and biotechnological potential. Appl Microbiol Biotechnol 85:1629–1642

    Article  PubMed  Google Scholar 

  19. Maia, Chaudhary LC, Figueres L, Wallace RJ (2007) Metabolism of polyunsaturated fatty acids and their toxicity to the microflora of the rumen. Antonie Van Leeuwenhoek 91:303–314

    Article  CAS  PubMed  Google Scholar 

  20. Maia MR, Chaudhary LC, Bestwick CS, Richardson AJ, McKain N, Larson TR, Graham IA, Wallace RJ (2010) Toxicity of unsaturated fatty acids to the biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens. BMC Microbiol 10:52

    Article  PubMed Central  PubMed  Google Scholar 

  21. Tan C, Phillip Smith R, Srimani JK, Riccione KA, Prasada S, Kuehn M, You L (2012) The inoculum effect and band-pass bacterial response to periodic antibiotic treatment. Mol Syst Biol 8:617

    Article  PubMed Central  PubMed  Google Scholar 

  22. Dubos RJ (1947) The effect of lipids and serum albumin on bacterial growth. J Exp Med 85:9–22

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  23. Zaldivar J, Ingram LO (1999) Effect of organic acids on the growth and fermentation of ethanologenic Escherichia coli LY01. Biotechnol Bioeng 66:203–210

    Article  CAS  PubMed  Google Scholar 

  24. Kim YJ, Liu RH, Bond DR, Russell JB (2000) Effect of linoleic acid concentration on conjugated linoleic acid production by Butyrivibrio fibrisolvens A38. Appl Environ Microbiol 66:5226–5230

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  25. Shingfield KJ, Kairenius P, Arola A, Paillard D, Muetzel S, Ahvenjarvi S, Vanhatalo A, Huhtanen P, Toivonen V, Griinari JM, Wallace RJ (2012) Dietary fish oil supplements modify ruminal biohydrogenation, alter the flow of fatty acids at the omasum, and induce changes in the ruminal Butyrivibrio population in lactating cows. J Nutr 142:1437–1448

    Article  CAS  PubMed  Google Scholar 

  26. Loor JJ, Ueda K, Ferlay A, Chilliard Y, Doreau M (2005) Intestinal flow and digestibility of trans fatty acids and conjugated linoleic acids (CLA) in dairy cows fed a high-concentrate diet supplemented with fish oil, linseed oil, or sunflower oil. Anim Feed Sci Tech 119:203–225

    Article  CAS  Google Scholar 

  27. Doreau M, Fievez V, Troegeler-Maynadier A, Glasser F (2012) Ruminal metabolism and digestion of long chain fatty acids in ruminants: recent advances in knowledge. INRA Prod Anim 25:361–373

    CAS  Google Scholar 

  28. Klein CM (2011) Biohydrogenation of docosahexaenoic acid. Ph.D. Thesis, Clemson University

  29. Kramer JKG, Hernandez M, Cruz-Hernandez C, Kraft J, Dugan MER (2008) Combining results of two GC separations partly achieves determination of all cis and trans 16:1, 18:1, 18:2 and 18:3 except CLA isomers of milk fat as demonstrated using Ag-ion SPE fractionation. Lipids 43:259–273

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The study was funded by the Research Foundation-Flanders (FWO) and the Special Research Fund (BOF, Bijzonder Onderzoeksfonds). B.V. benefits from a post-doctoral grant from the Research Foundation-Flanders (FWO-Vlaanderen).

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Authors and Affiliations

  1. Laboratory for Animal Nutrition and Animal Product Quality, Ghent University, Proefhoevestraat 10, 9090, Melle, Belgium

    B. Vlaeminck, T. Braeckman & V. Fievez

Authors
  1. B. Vlaeminck
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  2. T. Braeckman
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  3. V. Fievez
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Correspondence to V. Fievez.

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Vlaeminck, B., Braeckman, T. & Fievez, V. Rumen Metabolism of 22:6n-3 In Vitro is Dependent on its Concentration and Inoculum Size, but Less Dependent on Substrate Carbohydrate Composition. Lipids 49, 517–525 (2014). https://doi.org/10.1007/s11745-014-3905-8

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  • DOI: https://doi.org/10.1007/s11745-014-3905-8

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