The role of plant phenolics in ruminant nutrition. Bogor Agricultural University, Indonesia

Transcription

1 The role of plant phenolics in ruminant nutrition Anuraga Jayanegara Anuraga Jayanegara Bogor Agricultural University, Indonesia

2 Outline: 1. Phenolics in plants 2. Digestive systems of ruminants: an overview 3. The role of phenolics in ruminant nutrition 2

3 1. Phenolics in plants 3

4 Structure Definition of phenolics: a class of chemical compounds consisting of a hydroxyl group (-OH) bonded directly to an aromatic hydrocarbon group. Basic structure of phenolics 4

5 Classification 5

6 Source: McSweeney et al. (2001) Anim. Feed Sci. Technol. 91,

7 Diversity of tannin structures Source: Mueller-Harvey (2006) J. Sci. Food Agric. 86,

8 Synthesis Why plants produce phenolics? A strategy adopted by plants to deter attack by microorganisms, insects and higher animals. Factors affecting tannin levels: - Nutrient stress (N, P, K, S deficiencies) - High light intensity - High temperature - Severe drought - Tissue damage Increase tannin levels 8

9 Source: Jayanegara et al. (2011) Anim. Feed Sci. Technol. 163,

10 Interaction Multiple hydroxyl groups: enable phenolics to form complexes with proteins, polysachharides and minerals. Tannin-protein complex: 1. Hydrogen bonds: free phenolic hydroxyl groups 2. Hydrophobic bonds: aromatic ring structures t 3. Covalent bonds: polymerization reactions due to heating, exposure to UV radiation and the action of polyphenol oxidase 10

11 Illustration of protein precipitation by tannins Marangon et al. (2010) Analytica Chimica Acta 660,

12 2. Digestive systems of ruminants 12

13 Definition of ruminant: An even-toed ungulate mammal that chews the cud regurgitated from its rumen. The ruminants comprise the cattle, sheep, antelopes, deer, giraffes, and their relatives (Oxford Dictionary). Rumen microbes: Bacteria Protozoa Fungi Methanogens 13

14 Rumen fermentation Macromolecules: Fat/Lipids Carbohydrates: Starch Fibre Proteins rumen microbial fermentation: breakdown by microbial (saturation) enzymes to sugars glucose peptides amino acids major catabolic pathway: glycolysis (various) anaerobic! Fermentation products and resorption of short chain fatty acids (Gas) GI tract host metabolism: aerobic! Acetyl-CoA TCC Endoxidation CO2 H2 O ATP NAD(P)H 14

15 Pyruvate NADH 2 NAD Lactate CO 2 CO 2 CO CO 2 2 NAD NAD NAD!! Oxalacetat t NADH NADH 2! NADH 2 2 Acetyl-CoA Acetyl-CoA Acetyl-CoA NADH 2 ADP NAD ATP 2NADH 2 NADH 2 2NAD Acetate ADP ADP NAD Major SCFA: Acetate C2 Propionate C3 Butyrate C4 ATP ATP Butyrate CO 2 Propionate Formation of short-chain fatty acids in the rumen 15

16 Protein degradation PROTEIN PEPTIDE DIPEPTIDE (Endo-)proteases Oligopeptidases Dipeptidylpeptidases Dipeptidases AMINO ACIDS NH 3 Deaminases Synthesis of microbial protein R (C-skeleton) Fermentation for generating energy 16

17 Lipid metabolism Lipolysis Biohydrogenation of unsaturated fatty acids α-linolenic acid Linoleic acid Rumenic acid Vaccenic acid Stearic acid Source: Chilliard et al. (2007) Eur. J. Lipid Sci. Technol. 109,

18 Microbes involved in fatty acid biohydrogenation Source: Jenkins et al. (2008) J. Anim. Sci. 86,

19 Methane formation Global warming Loss of energy Source: Morgavi et al. (2010) Animal 4,

20 ADP ATP H 2 CO 2 Hydrogenase Formyl-MF 2(H) 2(H) Formyl-MPt Methyl-MPt Done by methanogens Uptake of CO 2 and H 2 detoxification of rumen ecosystem Stepwise reduction of CO 2 to a methyl-group, release of CH 4 No free intermediates Special cofactors (methanopterin, coenzyme M) Unknown energy balance Methyl-CoM- Reductase Methyl-CoM Methane ADP ATP 20

21 Source: 21

22 Method Methods to study rumen digestion and fermentation: 1. In vivo direct experiment to the animal 2. In sacco fistulated/cannulated rumen 3. In vitro mimicing rumen environment in a special apparatus a. Batch system (e.g., Hohenheim Gas Test, Tilley and Terry system, Reading Pressure Technique) b. Continuos culture (e.g., Rumen Simulation Technique/RUSITEC) 22

23 Hohenheim Gas Test IVOMD, energy (ME, NEL) ph, NH 3 Short-chain fatty acids (SCFA) Fatty acids (long-chain) Microbial population o 23

24 Buffer 39 o C Semi continuous in vitro fermentation system Feed Rumen Simulation Technique (RUSITEC) Substrate administration in nylon bags with defined pore sizes Substrate is completely removed and replaced at different time intervals Stirrer Overflow 24

25 3. The role of phenolics in ruminant nutrition 25

26 Digestibility and SCFA Source: Tiemann et al. (2008) J. Sci. Food Agric. 88, Call: 71 g CT/kg DM 26

27 Extractable CT (g/kg DM) n.d Source: Hess et al. (2008) Anim. Feed Sci. Technol. 147,

28 Kobe lespedeza: 151 g CT/kg DM Source: Animut et al. (2008) Anim. Feed Sci. Technol. 144,

29 Protein Rubisco protein (from white clover) Lotus pedunculatus Lotus corniculatus Source: Min et al. (2003) Anim. Feed Sci. Technol. 106,

30 Ruminal protein escape Source: Broderick and Albrecht (1997) Crop Sci. 37,

31 CT extract from L. pedunculatus Streptococcus bovis Eubacterium sp. Prevotella bryantii Butyrivibrio fibrisolvens Source: Molan et al. (2001) Can. J. Microbiol. 47,

32 Milk production Source: Benchaar et al. (2008) J. Dairy Sci. 91,

33 CT in diet (g/kg DM) Source: Hymes-Fecht et al. (2013) J. Dairy Sci. 96,

34 Source: Griffiths et al. (2013) Animal 7,

35 Dairy ygoat: - No difference in milk production - Decreased nematode infection Source: Hoste et al. (2005) Small Rum. Res. 59,

36 Methane 60 Relationships phenolic fractions and methane 27 tropical plant species CH4 4/IVOMD (ml/g) Y = X R 2 = 0.71*** Total phenols (g/kg DM) Source: Jayanegara et al. (2011) Anim. Feed Sci. Technol. 163,

37 CH4/IVOM MD (ml/g) Y = X R 2 = 0.21* CH4/IVOM MD (ml/g) Y = X R 2 = 0.55*** Non-tannin phenols (g/kg g DM) Total tannins (g/kg g DM) 37

38 D (ml/g) CH4/IVOM Y = X R 2 = 0.35** CH4/IVOM D (ml/g) Y = X R 2 = 0.36** Condensed d tannins (g/kg DM) Hydrolysable tannins (g/kg DM) 38

39 Principal component analysis: loading plot 1.0 Low CH4/ total gas Non-fiber ch PC2 (21.2% %) Low quality TP Bacteria TT HT CT NTP C2 C2/C3 Lignin (sa) Protozoa EE CP C3 C5isoC5 IVOMD Total SCFA Ammonia isoc4 High quality -0.5 ph C4 CH4/total gas ADFom andfom PC1 (37.0%) High CH4/ total gas

40 CP NH3 NTP EE C5 TP C2 isoc4 Bacteria Low CH4/total gas IVOMD 4 9.9%) PC2 ( isoc5 C3 CH4/total gas CT High CH 4 /total gas TT HT Protozoa NFC -0.5 NDF ADF C PC1 (33.8%) 18 alpine plant species Source: Jayanegara et al. (2011) J. Sci. Food Agric. 91,

41 Effects of plant mixtures on CH 4 emissions Chemical composition of the plants (g/kg DM) Treatment CP EE NDF ADF ADL TP NTP TT CT HT Carica papaya p Clidemia hirta Swietenia mahagoni Eugenia aquea CP, crude protein; EE, ether extract; NDF, neutral detergent fiber; ADF, acid detergent fiber; ADL, acid detergent lignin; TP, total phenols; NTP, non-tannin phenols; TT, total tannins; CT, condensed tannins; HT, hydrolysable tannins. Source: Jayanegara et al. (2013) Brit. J. Nutr. 109,

42 Carica papaya Swietenia mahagoni Eugenia aquea Clidemia hirta 42

43 Structure re of the treatments (as mg DM of plants incubated) No. Treatment Carica papaya Clidemia hirta Swietenia mahagoni Eugenia aquea 1 Cp Ch Sm Ea CpCh Single plants 6 CpSm CpEa ChSm plants 9 ChEa SmEa CpChSm CpChEa CpSmEa ChSmEa CpChSmEa plants 4 plants 43

44 60 j i hi gh ef d c bc ab a ab fg e e bc CH4 /digest tible OM (ml/g) Cp Ch Sm Ea CpC Ch CpSm CpE Ea ChSm ChE Ea SmE Ea CpChSm CpChE Ea CpSmE Ea ChSmE Ea 0 CpChSmE Ea Influence of the tropical plant mixtures on CH4/digestible OM 44

45 Associative effect (%) = 100 x (observed expected)/expectedexpected)/expected 50 CH 4 expecte ed value Synergistic effect The observed value is lower AB 1 Plant A AB 0 Plant B Additive effect AB 2 The observed value is higher Antagonistic effect CH 4 observed value Assessment of the associative effects 45

46 Source: Jayanegara et al. (2013) Brit. J. Nutr. 109,

47 CH4 de ecrease (%) 8 Purified tannins 7 )HT ,5 mg/ml 0,75 mg/ml 1,0 mg/ml Tannin concentration ti Chesnut Mimosa Quebracho Sumach CT Jayanegara et al. (2010) Sustainable Improvement of Animal Production and Health, FAO,

48 Phenolics and microbial population Source: Bhatta et al. (2009) J. Dairy Sci. 92,

49 Ruminal methanogens attached to protozoal species interspecies H transfer Protozoa-associated methanogens contribute up to 37% of total rumen methane emissions Removal of protozoa from the rumen (defaunation) may CH 4 emission Protozoa colonized by methanogens 49

50 Source: 50

51 OM (ml/g) CH 4 /digestible Database development from literatures 70 Y = X X 2 Total: 30 experiments, 171 treatments P<0.001; RMSE = 4.27; 60 In vitro batch: 15 experiments, 130 treatments R 2 = 0.659; n = 91 In vivo: 15 experiments, 41 treatments 50 Statistical analysis with mixed-model In vitro batch studies methodology Weighting data by number of replicates/animals in each experiment Dietary tannins (g/kg g DM) Meta-analysis study OM (ml/g) CH 4 /digestible Y = X P = 0.036; RMSE = 10.21; R 2 = 0.286; n = 30 In vivo studies Dietary tannins (g/kg DM) Source: Jayanegara et al. (2012) J. Anim. Physiol. Anim. Nutr. 96,

52 40 Change of CH 4 /dig gestible OM (%) In vitro batch studies In vivo studies Dietary tannins (g/kg DM) 52

53 Fatty acids Incubation of tropical plants in vitro Correlation coefficients between plant chemical composition and disappearance and appearance of C18 fatty acids (n = 27) Fatty acid Total phenols Non-tannin phenols Total tannins Condensed tannins Hydrolysable tannins Disappearance C18:3 n *** *** *** C18:2 n *** *** *** C18:1 n *** 0.42* 0.66*** 0.73*** 0.46* Appearance c9,t11-c18:2 0.55** ** 0.64*** 0.33 t11-c18: C18:0 0.47* * 0.44* 0.31 *, P<0.05; **, P<0.01; ***, P<0.001 Source: Jayanegara et al. (2011) Anim. Prod. Sci. 51,

54 Incubation of alpine plants in vitro Correlation coefficients between plant chemical composition and disappearance and appearance of C18 fatty acids (n = 18) Fatty acid Total phenols Non-tannin phenols Total tannins Condensed tannins Hydrolysable tannins Disappearance C18:3 n ** *** *** C18:2 n ** *** *** C18:1 n Appearance c9,t11-c18: * t11-c18:1 C *** *** *** C18:0 0.57* ** ** *, P<0.05; **, P<0.01; ***, P<0.001 Source: Jayanegara et al. (2012) Livest. Sci. 147,

55 CH: grass-clover hay (control) TF: dried sainfoin (7.9% CT) TH: CH + A. mearnsii extract (7.9% CT) SH: CH + Y. schidigera extract (1.1% saponins) Condensed tannins (CT) Source: Khiaosa-ard et al. (2009) J. Dairy Sci. 92,

56 Influence of tannins on biohydrogenating bacteria Containing 6.4% tannins from quebracho powder Source: Vasta et al. (2010) Appl. Environ. Microbiol. 76,

57 That s all. 57

58 Thank you very much for your attention! 58