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Ruminant Carbohydrate Digestion

Ruminant Carbohydrate Digestion. References Church 145-171; 260-297 Van Soest 95-117; 118-128; 160-165, 171-177 Sejrsen 139-143 Journal of Dairy Science 84:1294-1309 Journal of Animal Science 80:1112- Carbohydrates in common feedstuffs Carbohydrate, %DM Alfalfa Grass Corn DDGS

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Ruminant Carbohydrate Digestion

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  1. Ruminant Carbohydrate Digestion • References • Church 145-171; 260-297 • Van Soest 95-117; 118-128; 160-165, 171-177 • Sejrsen 139-143 • Journal of Dairy Science 84:1294-1309 • Journal of Animal Science 80:1112- • Carbohydrates in common feedstuffs Carbohydrate, %DMAlfalfaGrassCornDDGS Soluble sugars 5 4 2 1-5 Cellulose 25 30 - 16-18 Hemicellulose 22 26 6 26-34 Pectin 6 4 - - Starch 2 1 72 15-19 Lignin 12 9 - -

  2. Fibrous carbohydrates • Cellulose • A chain of glucose units bound by beta-1,4-linkages • Intramolecular hydrogen bonding • Poor flexibility • Good tensile strengh • Low solubility in water or dilute acid Starch-Groups are axial Cellulose-Groups are equatorial

  3. Intermolecular hydrogen bonding • Allows the development of a crystalline lattice • In cellulose digestion, intermolecular bonds must first be broken converting crystalline to amorphous cellulose • More intermolecular bonds in pure cellulose than native cellulose From Van Soest (1994)

  4. Hemicellulose • Heterogeneous mixture of pentose, hexose and uronic acids bound to a beta-1,4-linked core composed primarily of xylose Monomer, % HemicelluloseAlfalfaBromegrassLocation Arabinose 10.4 12.0 Branch point Xylose 58.5 59.2 Chain Glucose 6.9 20.9 Chain Galactose 6.9 7.8 Chain Rhamnose 3.9 - Chain Glucuronic acid 13.5 - Branch point • Monomers of xylose chain are twisted at 60o Van Soest (1994)

  5. Arabinose and uronic acid branch points • Arabinose binds by Beta-1,3-linkages • Uronic acids bind by Beta-1,2-, Beta-1,3-, or Beta-1,4- glycosal or ester linkages • Significance of branch points • Increased branch points > Greater digestibility > Greater solubility • Hemicellulose is more closely bound to lignin than cellulose

  6. Pectin • Polymers of galacturonic acid bound by alpha-1,4-linkages • Chains are coiled • Very digestible by microorganisms • Rhamnose units are substituted in the chains • Chains twist • Arabinose and galactose side chains bind by alpha-1,4-linkages • Adjacent chains of rhamnogalactans may be cross-linked through Ca+ ions bridged across galactouronyl residues Van Soest (1994)

  7. Locations of fiber carbohydrates

  8. Lignin • A poorly defined polymer of phenylpropane units

  9. Lignin in plants is composed of a highly condensed ‘core’ lignin and a ‘non-core’ lignin composed of low molecular weight phenolics, primarily ferulic and p-coumaric acids. • Ratios very with plant species • Binding is random • Relation to cell wall carbohydrates • Only binds to hemicellulose • Forms a matrix around cellulose Van Soest (1994)

  10. Linkages between carbohydrates and lignin vary with plant species • Ester linkages • Between carbohydrates and ferullic and hydroxycinnamic acid • Found in grasses • Saponifiable with alkali • Ether linkages • Directly between carbohydrates and core lignin • Found in dicotyledenous plants • Difficult to hydrolyze • Biological function • Strength against compression forces • Disease resistance • Factors affecting lignin content • Maturity • Ambient temperature • Increasing temperature increases lignin synthesis and reduces photosynthesis

  11. 80 Grasses CF dig, % Legumes 40 10 20 Lignin/ADF, % • Effects of lignification • Lignin is the major factor limiting digestion of forage cell walls • Protects up to 1.4 – 2.0 x its weight in CHO and up to 8 CHO units from the lignin bond • Mechanisms of lignin’s effects on digestion • Physically encrusting the fiber • Altering the stereochemistry of the polysaccharides • Toxicity to cellulolytic bacteria

  12. Delignification treatments • Alkali treatments • Treatments • 4% NaOH • 3% NH3 • Saponifies ester linkages • Only effective on grasses • Increase digestibility and intake 10-20% • Alkaline hydrogen peroxide lignin • Increases digestibility by 60% • Effective on all forages • Biological delignification • White rot fungi

  13. Other factors affecting cell wall digestion • Arabinose:Xylose ratio • Decreases with maturity, decreasing digestibility • Tannins • May reduce digestibility by: • Cellulase inhibition • Protein binding • Cutin • Waxy coating, decreasing digestibility • Silica • High in forages from arid environments, decreasing cellulose digestibility • Oils • Toxic to cellulolytic bacteria • Bacterial nutrition • N, S, and isoacids increase fiber digestion • Grain in diet • Increasing grain>Decreased pH and starch>Reduce cellulose digestibility • Increased rate of passage

  14. Cellulose digestion • In reticulorumen • Approximately 90% of cellulose digestion • Requires two steps • Microbial attachment • Hydrolysis Miron et al. JDS 84:1294

  15. Attachment of cellulolytic bacteria on fiber • Results in a lag period in digestion • Phases • Transport of bacteria to fiber • Slow • Dependent on number of bacteria • Nonspecific adhesion of bacteria to sites on substrate • Binds with Glycocalyx Mixture of polysaccharide, glycoprotein and protein on outside of cell membrane of gram- bacteria Peptidoglycan of gram+ bacteria • Occurs mainly at cut or macerated sites of the plant • Specific adhesions of bacteria with digestible cellulose • Structures Cellulosome: Large, multienzyme complexes specialized for adhesion and hydrolysis of cellulose Fimbriae or Pili: Small (5-7 nm in width and 100-200 nm in length) structures in both gram + and – bacteria • Proliferation and colonization of bacteria

  16. Structure of the cellulosome

  17. Cellulose hydrolysis • Cellulases are extracellular • Enzymes • Endo-B-1,4-glucanase > Cleaves cellulose chains • Exo-B-1,4-glucanase > Cleaves cellobiose units • Cellobiase > Cleaves cellobiose • Hemicellulose digestion • Hemicellulose > Lignin-hemicellulose > Monosaccharides complexes • Enzymes found in cell-free rumen fluid and within cells • Endoxylanase > Hydrolyzes xylose linkages • Xylosidase > Hydrolyzes xylose linkages • Arabinofuranosidase > Hydrolyzes arabinoxylans • Glucuronidase > Hydrolyze Glucuronxylan • Pectin digestion • Rapid Pectic lyase & Pectin methylesterase Polygalacturonase Pectin > Polygalacturonic acid > Galacturonic acid

  18. Lower GIT tract digestion of fiber carbohydrates • Abomasum and small intestine • Little digestion • Large intestine • Fermentation of both cellulose and hemicellulose • Greater % of hemicellulose digestion than cellulose digestion occurs in LI • % of fiber carbohydrate digested in the LI increases with factors that reduce ruminal digestion

  19. CH2OH OH O O OH CH2OH CH2OH CH2OH CH2OH CH2 CH2OH O O O O O O OH OH OH OH OH OH O O O O O O O O OH OH OH OH OH OH • Starch • Chief storage polysaccharide in plants • Two components • Amylose (Glucose units bound by alpha-1,4-linkages) • Amylopectin (Glucose units by alpha-1,4-linkages with alpha-1,6-branch points)

  20. Composition varies between: • Variety AmyloseAmylopectin Normal 30 70 Waxy 100 0 • Maturity • Maturity increases amylose • Components are arranged in concentric spheres in granules • Held together by hydrogen bonds • Bonds limit ability to swell in water and allow access of enzymes to material in center of granules • Digestion proceeds from outside to center of granule • Bolds broken by heating, particularly in water, destroying granule structure • Gelatinization • Basis for processes like Steam-flaking Popping • Processes also affect seedcoat and protein matrix • Increases digestibility 10-20%

  21. Starch digestion • Rumen • 47-95% digested in rumen • Digested by alpha-amylase to oligosaccharides • Found in cell-free rumen fluid, but 70% associated with particulate-bound microorganisms • Activity increases in high grain diets • Microorganisms • Prevotella amylophilus • Streptococcus bovis • Oligosaccharides degraded to glucose by maltases near cells • Protozoa uptake • Primarily holotrichs • Stabilizes fermentation • Do not readily pass from rumen • Bacterial uptake • Storage polysaccharide • May accounts for as much as 50% of carbohydrate leaving rumen

  22. Small intestine • Mechanisms similar to nonruminants Pancreatic IntestinaTranl amylase maltase Starch > Oligosaccharides > Glucose • Glucose absorption • Active transport by a secondary active glucose and galactose tranporter (SGLT1) at the apical membrane • Activity greater in pre-ruminants than ruminants • Activity greater in concentrate selecting species than roughage selectors • Increases with glucose infusions • Transport at the basolateral membrane of epithelium is by facilitated diffusion using a GLUT2 transporter • Limitations of small intestinal starch digestion • 45-90% digested in the small intestine • Limitations • Inadequate amylase activity • Inadequate maltase • Intestinal pH • Rate of passage

  23. Large intestine • Only significant when high levels of starch escape ruminal digestion • Fermentation similar to rumen • VFAs are absorbed • Microbial protein is produced and excreted

  24. Importance of location of starch digestion • Since small intestinal digestion is limited, digestion in the rumen is most valuable • Ruminal escape starch may be associated with hemorrhagic bowel syndrome • Hemorrhaging in the jejunum occurs in the first 100 days of lactation • Symptoms • Abdominal distention • Bloody feces • Dehydration • Shock • Death • Possible causes • Ruminal escape starch causes growth of Clostridium perfringens type A • Moldy feed

  25. Results of rates of passage and digestion. Physical form of forage plays a role 75 Ruminal Starch Dig, % 60 52 % corn in diet • Factors affecting starch digestion • DM intake • Increased dry matter intake decreases starch digestion • Percentage of grain in diet

  26. Type of starch • Barley > Corn > Sorghum • Waxy > Normal • Processing • Cracking or grinding increases digestibility 2 – 5% • Steam-flaking, popping etc improves starch digestion by: • 6-10% in corn • 15-20% in sorghum

  27. VFA production • Importance of VFA Endproduct% of digested energy VFA 49-58 Heat 6-12 Gas 4-8 Microbial mass 26-32

  28. VFA production • VFA produced from pyruvate • Net production • Glycolysis (/ glucose) 2 ATP 2 NADH2 • Pentose PO4 pathway (/pentose) 1.67 ATP 2 NADPH2 1 NADH2 1 pentose ATP ATP ATP ATP ATP

  29. Pyruvate is immediately converted to VFAs

  30. Acetate production • Pyruvate oxidoreductase (Most common) Fd FDH2 Pyruvate Acetyl CoA Acetate Coenzyme A CO2 ADP ATP • Pyruvate-formate lyase Coenzyme A ADP ATP Pyruvate Acetyl CoA Acetate Formate CH4 + H2O 6H+

  31. Butyrate (60% Butyrate from acetate) • Condensation ATP ADP Acetyl CoA CoA Pyruvate Acetyl CoA Acetoacetyl CoA ATP CO2 NADH2 CoA ADP CoA NAD Malonyl CoA B-Hydroxybutyryl CoA Crotonyl CoA NADH2 NAD Butyryl CoA Acetyl CoA Acetate Butyryl P ADP ATP Butyrate

  32. Propionate • Succinate or dicarboxylic acid pathway • 60-90% of propionic acid production CO2 ATP ADP NADH2 NAD Pyruvate Oxaloacetate Malate H2O CO2 Fumarate Propionyl CoA ADP NADH2 ATP NAD Succinate Propionate Methylmalonyl CoA Succinyl CoA

  33. Acrylate pathways • Important on high grain diets • Accounts of 40% of propionate production • Associated with Megasphaera elsdenii NADH2 NAD Pyruvate Lactate Acrylyl CoA NADH2 Propionate NAD Propionyl CoA

  34. Fermentation of intermediates • Lactate • In forage-fed animals; Lactate > Butyrate • In grain-fed animals; Lactate > Propionate • Succinate • Supplies at least 1/3 of the propionate • Formate • Rapidly converted to H2 + CO2 • H2 • 4H2 + CO2 > CH4 + 2H2O • Ethanol • Rapidly converted to acetate

  35. Factors controlling fermentation endproducts • Maximum ATP yields for the microorganisms • Maintenance of Reduction-Oxidation balance • In glycolysis, 2 NADH2 are produced per glucose. • Must be oxidized to maintain Redox balance • Electron acceptors • Aerobic organisms O2 > H2O • Anerobic organisms CO2 > CH4 Pyruvate > Propionate Acetate > Butyrate NO3 > NO2 > NH3 SO4 > S

  36. Thermodynamic order of preference for electron acceptors • NO3 > NO2 • NO2 > NH4 • Crotonyl CoA>Butyryl CoA • Fumarate > Succinate • Acrylyl CoA > Propionyl CoA • SO4 > HS • Acetoacetyl CoA > B-OH-Butyryl CoA • CO2 > CH4 • Pyruvate > Lactate • CO2 > Acetate • Why does CH4 supercede Propionate or Butyrate production • Greater ATP produciton • Greater affinity for H at low concentrations • Low amounts of other acceptors

  37. Redox balance in the rumen • 2H (Reducing equivalents) produced: • Glucose > 2 Pyruvate + 4H (as 2 NADH2) • Pyruvate + H2O > Acetate + CO2 + 2H (as 1 FADH2) • 2H accepted: • CO2 + 4H2 > CH4 + 2H2O • Pyruvate + 4H (as 2 NADH2) > Propionate + H2O • 2 Acetate + 4H (as 2 NADH2) > Butyrate + 2H2O • Fermentation balance • High forage diets • 5 Glucose > 6Acetate + Butyrate + 2Propionate + 5CO2 + 3CH4 + 6H2O • Acetate:Propionate = 3 • CH4:Glucose = .60 • High grain diets • 3 Glucose > 2Acetate + Butyrate + 2Propionate + 3CO2 + CH4 + 2H2O • Acetate:Propionate = 1 • CH4:Glucose = .33

  38. VFA production • Usually peaks 4 hours after feeding • Concentration does not equal production • Factors that increase propionate, decrease acetate and methane • Factors affecting VFA produced • Diet forage:concentrate ratio • Decreased forage and increased concentrate • Decreased acetate and methane, increased propionate • Dietary buffers • Increased acetate and methane, decreased propionate • Decreased physical form of diet (Grinding, pelleting etc) • Decreased acetate and methane, increased propionate • Ionophores • Decreased acetate and methane, increased propionate • Unsaturated fatty acids • Decreased methane, increased propionate

  39. Examples of diet effects on VFA production • Forage:Concentrate Forage:Concentrate VFA, Molar%60:4040:6020:80 Acetate 66.9 62.9 56.7 Propionate 21.1 24.9 30.9 Butyrate 12.0 12.2 12.4 Methane, Mcal/d 3.1 2.6 1.8 • Physical form of forage Alfalfa hay Grind VFA, Molar%LongCoarseFinePelleted Acetate 62.5 56.8 47.5 18.2 Propionate 23.8 27.1 28.5 45.7 Butyrate 10.8 13.6 23.9 32.8

  40. Methane inhibitors • Nitrates, sulfates, and alkaloids will inhibit CH4, but decreases propionate and butyrate as well • Chloral hydrate (CCl4) • Reduces CH4 and increases propionate • H2 accumulates and microbial growth is reduced • Myristic acid (Brit. J. Nutr. 90:529-540) • A 14-carbon saturated fatty acid • Reduced CH4 production by 58% while increasing propionate concentration (mmol/l) by 86% • Did not affect DM intake • Tended to decrease NDF digestion • Acetogenesis • 2CO2 + 2H2 > CH3COOH • Thermodynamically unfavorable to methane production • Doesn’t usually occur in the rumen • Does occur in the large intestine of various species and in termites • Why doesn’t it occur in the rumen?

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