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Transcript
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 Soluble sugars Cellulose Hemicellulose Pectin Starch Lignin Alfalfa Grass Corn 5 4 2 25 30 22 26 6 6 4 2 1 72 12 9 - DDGS 1-5 16-18 26-34 15-19 - • Fibrous carbohydrates – Cellulose • A chain of glucose units bound by beta-1,4-linkages Starch-Groups are axial Cellulose-Groups are equatorial • Intramolecular hydrogen bonding – Poor flexibility – Good tensile strengh – Low solubility in water or dilute acid – Intermolecular hydrogen bonding From Van Soest (1994) • 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 • Hemicellulose – Heterogeneous mixture of pentose, hexose and uronic acids bound to a beta-1,4-linked core composed primarily of xylose Monomer, % Hemicellulose Arabinose Xylose Glucose Galactose Rhamnose Glucuronic acid Alfalfa Bromegrass 10.4 12.0 58.5 59.2 6.9 20.9 6.9 7.8 3.9 13.5 - Location Branch point Chain Chain Chain Chain Branch point – Monomers of xylose chain are twisted at 60o Van Soest (1994) – 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-1,4glycosal or ester linkages • Significance of branch points – Increased branch points > Greater digestibility > Greater solubility – Hemicellulose is more closely bound to lignin than cellulose – Pectin • Polymers of galacturonic acid bound by alpha-1,4linkages – Chains are coiled – Very digestible by microorganisms • Rhamnose units are substituted in the chains – Chains twist • Arabinose and galactose side chains bind by alpha-1,4linkages Van Soest (1994) • Adjacent chains of rhamnogalactans may be crosslinked through Ca+ ions bridged across galactouronyl residues • Locations of fiber carbohydrates • Lignin – A poorly defined polymer of phenylpropane units – 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) – 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 – 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 80 Grasses CF dig, % Legumes 40 10 20 Lignin/ADF, % – Mechanisms of lignin’s effects on digestion » Physically encrusting the fiber » Altering the stereochemistry of the polysaccharides » Toxicity to cellulolytic bacteria – 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 – 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 • Cellulose digestion – In reticulorumen • Approximately 90% of cellulose digestion – Requires two steps • Microbial attachment • Hydrolysis Miron et al. JDS 84:1294 – 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 – Structure of the cellulosome – 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 • 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 – Starch • Chief storage polysaccharide in plants • Two components – Amylose (Glucose units bound by alpha-1,4-linkages) O CH2OH CH2OH O O OH CH2OH O OH OH O O OH OH O OH – Amylopectin (Glucose units by alpha-1,4-linkages with alpha-1,6-branch points) CH2OH OH O O H CH2OH O CH2OH CH2 O O O OH OH OH O O O O OH OH OH • Composition varies between: – Variety Amylose 30 100 Normal Waxy – Maturity » Maturity increases amylose Amylopectin 70 0 • 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% • 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 – 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 – 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 – 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 – Factors affecting starch digestion • DM intake – Increased dry matter intake decreases starch digestion F:C 75:25 25:75 Multiple of Maintenance 1 3 1 3 Starch intake, gm/d 1056 3291 2460 9463 Starch dig, % 96.8 84.7 96.8 84.3 – Percentage of grain in diet Results of rates of passage and digestion. Physical form of forage plays a role 75 Ruminal Starch Dig, % 60 52 % corn in diet • 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 • VFA production – Importance of VFA Endproduct % of digested energy VFA 49-58 Heat 6-12 Gas 4-8 Microbial mass 26-32 • 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 – Pyruvate is immediately converted to VFAs • Acetate production – Pyruvate oxidoreductase (Most common) Fd FDH2 Pyruvate Coenzyme A Acetyl CoA CO2 Acetate ADP ATP – Pyruvate-formate lyase Coenzyme A Pyruvate ADP Acetyl CoA Acetate Formate CH4 + H2O 6H+ ATP • Butyrate (60% Butyrate from acetate) – Condensation ATP ADP Pyruvate CoA Acetyl CoA Acetyl CoA ATP CO2 ADP Malonyl CoA CoA Acetoacetyl CoA NADH2 CoA NAD B-Hydroxybutyryl CoA Crotonyl CoA NADH2 NAD Butyryl CoA Acetyl CoA Acetate Butyryl P ADP ATP Butyrate • Propionate – Succinate or dicarboxylic acid pathway • 60-90% of propionic acid production CO2 ATP ADP Pyruvate NADH2 NAD Oxaloacetate CO2 Malate H 2O Fumarate Propionyl CoA ADP NADH2 ATP NAD Succinate Propionate Methylmalonyl CoA Succinyl CoA – 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 • Fermentation of intermediates – Lactate • In forage-fed animals; • In grain-fed animals; Lactate > Butyrate 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 • 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 > H 2 O » Anerobic organisms CO2 > CH4 Pyruvate > Propionate Acetate > Butyrate NO3 > NO2 > NH3 SO4 > S – 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 – 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 • 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 • Examples of diet effects on VFA production – Forage:Concentrate VFA, Molar% Acetate Propionate Butyrate Methane, Mcal/d Forage:Concentrate 60:40 40:60 20:80 66.9 62.9 56.7 21.1 24.9 30.9 12.0 12.2 12.4 3.1 2.6 1.8 – Physical form of forage VFA, Molar% Acetate Propionate Butyrate Long 62.5 23.8 10.8 Alfalfa hay Grind Coarse Fine 56.8 47.5 27.1 28.5 13.6 23.9 Pelleted 18.2 45.7 32.8 – 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?