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ADP ATP NADP + NADPH

Microbial Metabolism. Sugars. ADP ATP NADP + NADPH. Biosynthesis. Catabolism. VFA CO 2 CH 4 Heat. Growth Maintenance Transport. Fermentation in the Rumen. Mostly fermentation of sugars from polysaccharides Rumen is an anaerobic habitat

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ADP ATP NADP + NADPH

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  1. Microbial Metabolism Sugars ADP ATP NADP+ NADPH Biosynthesis Catabolism VFA CO2 CH4 Heat Growth Maintenance Transport

  2. Fermentation in the Rumen • Mostly fermentation of sugars from polysaccharides • Rumen is an anaerobic habitat • Disposal of reducing equivalents is a critical feature • of anaerobic fermentation • Production of lactic acid and ethanol • not extensively used in the rumen • Production of VFA major pathway • Hydrogenases produce hydrogen gas • from reduced cofactors • Methanogens use hydrogen to produce • methane

  3. Microbial Interactions Secondary Fermentations • Cellulose Fibrobacter Cellulose fragments • succinogenes • Succinate + Acetate + Formate • Selenomonas • ruminantium • Lactic acid + Propionate + Acetate + Formate + H2 • Megasphaera • elsdenii • Propionate + Acetate +H2

  4. Fermentation of Six Carbon Sugars (Glycolysis or Embden-Meyerhof) Accounts for 90% of fermentation in the rumen Glucose Fructose Starch Glu-1-P Glu-6-P Fru-6-P Fru-1,6-bisP Dihydroxyacetone-P Phospoenolpyruvate Glyceraldehyde-3-P Pyruvate Glycerol Predominant pathway for six carbon sugars (2 ATP + 2 NADH2)/Glucose 6 carbon Fructose bisphosphate aldolase 3 carbon

  5. An Alternate Pathway of Glucose Metabolism(Entner-Doudoroff & Pentose) Gucose Glu-6-P 6-P-Guconolactone Ribulose-5-P + CO2 6-P-gluconate Ribose-5-P 2-Keto-3-deoxy-6-P-gluconate Pyruvate Glyceraldehyde-3-P Pyruvate (1 ATP +1 NADPH)/Glucose Source of five carbon sugars NADP NADPH

  6. Fermentation of SugarsHexose Monophosphate Pathway 75% of xylan fermented by these pathways Gucose Glu-6-P 6-P-Guconolactone Ribulose-5-P + CO2 Xylulose-5-P Glyceraldehyde-3-P Ribose-5-P Acetyl-P Pyruvate Phosphoketolase Acetyl CoA Acetate Major pathway for five carbon sugars Source of five carbon sugars for biosynthesis 2 ATP, 2 NADPH, 1 NADH/Glucose NADP+ NADPH

  7. Pyruvate production is a central intermediate in ruminal bacteria and can be converted to variety of fermentation end products. The NADH produced during glycolysis must be re-oxidized so fermentation can continue.

  8. Acetic Acid 1. Pyruvate-formate lyase Pyruvate Acetyl COA Acetate Formate 6H CH4 + 2H2O 2. Pyruvate oxidoreductase (Most common pathway) FD FDH2 (Flavin adenine dinucleotide) Pyruvate Acetyl COA Acetate CO2 3 carbon 2 carbon

  9. Acetic Acid One pathway for AcetylCoA AcetylCoA Acetyl-P ADP Phosphotransacetylase Acetate kinase ATP Acetate

  10. Butyric Acid A second pathway for AcetylCoA FD FDH2 CO2 Pyruvate Acetyl COA Acetaldyhyde CO2 COA Acetoacetyl CoA Ethanol Malonyl COA NADH+H Acetyl CoA NAD COA +B-hydroxybutyryl COA Crotonyl COA NADH+H Butyryl COA NAD Acetate Butyrate Butyrate-P Acetyl COA 3 carbon ATP ADP 4 carbon

  11. Propionic Acid 1. Succinate or dicarboxylic acid pathway Accounts for about 60% of propionate production ATP Pyruvate Oxaloacetate Malate CO2 ADP Fumarate NADH+H Propionly COA Succinate NAD Propionate Methylmalonly COA Succinyl COA Co Vit B12 Pyruvate carboxylase Uses H 3 carbon

  12. Propionic Acid 2. Acrylate pathway (mostly by Megasphaera elsdinii) NADH NAD Pyruvate Lactic acid Acrylyl COA NADH+H Propionate NAD Propionyl COA This pathway becomes more important when ruminants adjusted to high starch diets Uses H

  13. Methane • CO2 + 4 H2 CH4 + 2H2O • The above is the overall reaction • There are a number of enzymes and cofactors involved • with combining CO2 and H2 to form CH4 • Formate + 3 H2 CH4 + 2H2O • CO2 + 2 H 3H2 • Methane is the predominant hydrogen sink in the rumen • Methanogens use H2 as a source of energy Lyase Preferred pathway

  14. Fermentation of Glucose and Other Sugars Glucose Pyruvate CO2 Formate Lactate Oxaloacetate 2H Acetyl-CoA Malate Acrylate Fumarate Acetoacetyl CoA Succinate MethaneAcetateButyratePropionate Succinyl CoA Propionyl CoA Methylmalonyl CoA Co Vit B12

  15. Fermentation Balance Low Acetate (High grain) Glucose 2 Acetate + 2 CO2 + 8 H Glucose Butyrate + 2 CO2 + 4 H Glucose 2 Propionate + 2 [O] CO2 + 8 H CH4 + 2 H2O

  16. Fermentation Balance High Acetate (High forage) 3 Glucose 6 Acetate + 6 CO2 + 24 H Glucose Butyrate + 2 CO2 + 4 H Glucose 2 Propionate + 2 [O] 3 CO2 + 24 H 3 CH4 + 6 H2O

  17. Fermentation Low Acetate Net: 3 Glucose 2 Acetate + Butyrate + 2 Propionate + 3 CO2 + CH4 + 2 H2O (Acetate:Propionate = 1 Methane:glucose = .33) High Acetate Net: 5 Glucose 6 Acetate + Butyrate + 2 Propionate + 5 CO2 + 3 CH4 + 6 H2O (Acetate:Propionate = 3 Methane:Glucose = .60)

  18. Energetic EfficiencyVFA Production Heat of combustion kcal/mole kcal/mole of % of of acid glucose fermented glucose Acetate 209.4 418.8 62.2 Propionate 367.2 734.4 109.1 Butyrate 524.3 524.3 77.9 Glucose 673.0

  19. Effect of DietVFA Ratios • Forage:Grain -----Molar ratios----- • Acetate Propionate Butyrate • 100:0 71.4 16.0 7.9 • 75:25 68.2 18.1 8.0 • 50:50 65.3 18.4 10.4 • 40:60 59.8 25.9 10.2 • 20:80 53.6 30.6 10.7

  20. Branched-Chain Fatty Acids Propionyl CoA + Acetyl CoA Valerate Valine Isobutyrate + NH3 + CO2 Leucine Isovalerate + NH3 + CO2 Isoleucine 2-methylbutyrate + NH3 + CO2 Fiber digesting bacteria have a requirement for branched-chain fatty acids.

  21. Rumen Acidosis • Animals gorge on grain • Decrease in rumen pH • Megasphaera elsdenii sensitive to acid pH • Decreased utilization of lactic acid • Streptococcus bovis usually not present in • high numbers (107/ml) • Grow very fast if sufficient glucose is present • Double numbers within 20 min (up to 109/ml) • Produce lactic acid • Lactobacillus ruminis & L. vitulinus also • produce some lactic acid • Methanobacter ruminantium in rumen (2 x 108/ml) • Sensitive to pH below 6.0 • Have no capacity to utilize more H+ • Excess H+accumulates • Some formation of ethanol • Most is used to produce lactic acid

  22. Rumen Acidosis • Increased production of lactic acid • Lactic acid poorly absorbed from rumen compared • with other VFAs • Lactic acid is a relatively strong acid • pK: Lactic acid 3.08 A, P, & B 4.75 - 4.81 • Very low rumen pH • Might be pH 5.5 or less • Both D and L isomers of lactic acid produced • – D is poorly metabolized in the body • Results in metabolic acidosis

  23. Acidosis • Subacute acidosis • Decreased fiber digestion • Depressed appetite • Diarrhea • Liver abscess • Feedlot bloat • Decreased milk fat • Acute acidosis • Laminitis • Death

  24. Acidosis • Liver abscess • Rumen epithelium not protected by mucous • Acid causes inflammation and ulceration (rumenitis) • Lactate promotes growth of Fusobacterium necrophorum • Fus. necrophorum infects ruminal ulcers • If Fus.necrophorum pass from rumen to blood, they • colonize in the liver causing abscesses • Incidence of liver abscess in feedlot cattle fed high • concentrate diets (>60% grain) ranges from 10 to over 50%. • Feeding antibiotic Tylosin (10 g/ton of feed) reduces incidence • of liver abscess in feedlot cattle.

  25. Acidosis • Laminitis (founder) • If rumen pH is chronically acidic • Epithelium releases metalloproteinases • Cause tissue degradation • If enter the blood stream causes inflammation • of laminae above the hoof • Feedlot bloat • Starch fermenting bacteria secrete polysaccharides • Produces a foam • Gas trapped in foam • Sudden death • If large amounts of starch escape the rumen • Overgrowth of Clostridium perfringens in the intestine • Produce enterotoxin that might cause death

  26. Acidosis • Diarrhea • Can be caused by some diseases • Often related to the diet in ruminats fed high-grain diets • Extensive fermentation in the hind gut • Acids produced • Absorbed but might cause damage to gut wall • Mucin secreted • Mucin casts can be observed in feces • Retention of water • Gas produced • Gas bubbles in feces

  27. Managing Acidosis • 1. Allow time for adjustment to diets with grain • Gradually increase grain in the diet • Program “step up” rations • Limit intake until adjusted • 2. Feed adequate roughage • Effective fiber (eNDF) • 3. Manage feed consumption • Prevent gorging of high starch feeds • “Read bunks” • System for knowing when to change • amount of feed offered • 4. Feed ionophores

  28. Adaptation to Grain DietsTwo to Four Weeks • Allow lactic acid utilizers to increase in numbers • Megasphaera elsdenii • Rarely present in rumen of hay fed animals • Selenomonas ruminantium • Propionibacter spp. • Not major populations in the rumen • Commercial preparations available • Maintain protozoa (lost at low pH, <5.5) • Ingest starch • Engulf bacteria producing lactic acid • Use glucose to make polysaccaride • Maintain methanogens • Use hydrogen • Growth of rumen papillae • Increased absorption of VFA

  29. Action of IonophoresTransmembrane Flux OutIN (High NA+, low K+) (High K+, low Na+) ATP H+ H+ ADP + Pi H+ H+ K+ K+ Na+ Na+ H+ H+ M M

  30. Gram NegativeIonophores Excluded M M Gram - positive Gram-negative

  31. Effect of Ionophores Carbohydrates Sensitive to Resistant to ionophore ionophore Produce more Produce more acetate & H propionate & less acetate CH4

  32. Ionophores - Continued Inhibit Rumminococcus albus Decreased acetate, Ruminococcus flavefaciens formate and CH4 Butrivibrio fibrisolvens Increase Bacteroides succinogenes Increase propionate Bacteroides ruminicola Selanomonas ruminantium Also inhibit Streptococci Decrease lactate Lactobacilli production No effect Megasphaera Utilize lactate Selenomonas

  33. Ionophores • Monensin sodium (Rumensin®) • 10 to 30 g per ton of 90% DM feed • Feedlot: 27 to 28 g per ton • Lasalosid (Bovatec®) • 10 to 30 g per ton of 90% DM feed • Feedlot: 30 g per ton • Laidlomycin propionate (Cattlyst®) • 5 to 10 g per ton of 90% DM feed • Feedlot: 10 g per ton

  34. Effects of Rumensin on Rumen Propionate Propionate production moles/day Roughage 5.96 Roughage + Rumensin 8.91 Concentrate 6.89 Concentrate + Rumensin 12.15

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