ADP ATP NADP + NADPH Sugars Catabolism Biosynthesis Growth Maintenance Transport Microbial Metabolism VFA CO 2 CH 4 Heat

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

Microbial Interactions Secondary Fermentations CelluloseFibrobacter Cellulose fragments succinogenes Succinate + Acetate + Formate Selenomonas ruminantium Lactic acid + Propionate + Acetate + Formate + H 2 Megasphaera elsdenii Propionate + Acetate +H 2

Fermentation of Six Carbon Sugars (Glycolysis or Embden-Meyerhof) Glucose Fructose Starch Glu-1-PGlu-6-P Fru-6-P Fru-1,6-bisP Dihydroxyacetone-P PhospoenolpyruvateGlyceraldehyde-3-P PyruvateGlycerol Predominant pathway for six carbon sugars (2 ATP + 2 NADH 2 )/Glucose 6 carbon Fructose bisphosphate aldolase 3 carbon Accounts for 90% of fermentation in the rumen

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

Fermentation of Sugars Hexose Monophosphate Pathway Gucose Glu-6-P 6-P-Guconolactone Ribulose-5-P + CO 2 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 75% of xylan fermented by these pathways

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.

Acetic Acid 1. Pyruvate-formate lyase PyruvateAcetyl COAAcetate Formate 6HCH 4 + 2H 2 O 2. Pyruvate oxidoreductase (Most common pathway) FD FDH 2 (Flavin adenine dinucleotide) PyruvateAcetyl COAAcetate CO 2 3 carbon2 carbon

Acetic Acid AcetylCoAAcetyl-P ADP Phosphotransacetylase Acetate kinase ATP Acetate One pathway for AcetylCoA

Butyric Acid PyruvateAcetyl COA Acetaldyhyde CO2COA Acetoacetyl CoA Ethanol Malonyl COA NADH+H Acetyl CoA NAD COA +B-hydroxybutyryl COA Crotonyl COA NADH+H Butyryl COA NAD Acetate Butyrate Butyrate-PAcetyl COA FDFDH 2 CO 2 3 carbon 4 carbon ATP ADP A second pathway for AcetylCoA

Propionic Acid 1. Succinate or dicarboxylic acid pathway Accounts for about 60% of propionate production ATP PyruvateOxaloacetateMalate CO2ADP Fumarate NADH+H Propionly COASuccinate NAD Propionate Methylmalonly COASuccinyl COA CoVit B 12 Pyruvate carboxylase Uses H 3 carbon

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

Methane CO H 2 CH 4 + 2H 2 O The above is the overall reaction There are a number of enzymes and cofactors involved with combining CO 2 and H 2 to form CH 4 Formate + 3 H 2 CH 4 + 2H 2 O CO H 3H 2 Methane is the predominant hydrogen sink in the rumen Methanogens use H 2 as a source of energy LyasePreferred pathway

Fermentation of Glucose and Other Sugars Glucose PyruvateCO 2 FormateLactateOxaloacetate 2H Acetyl-CoAMalate AcrylateFumarate Acetoacetyl CoA Succinate Methane AcetateButyrate Propionate Succinyl CoA Propionyl CoA Methylmalonyl CoA CoVit B12

Fermentation Balance Low Acetate (High grain) Glucose2 Acetate + 2 CO H GlucoseButyrate + 2 CO H Glucose2 Propionate + 2 [O] CO H CH H 2 O

Fermentation Balance High Acetate (High forage) 3 Glucose6 Acetate + 6 CO H GlucoseButyrate + 2 CO H Glucose2 Propionate + 2 [O] 3 CO H 3 CH H 2 O

Fermentation Low Acetate Net: 3 Glucose2 Acetate + Butyrate + 2 Propionate + 3 CO 2 + CH H 2 O (Acetate:Propionate = 1 Methane:glucose =.33) High Acetate Net: 5 Glucose6 Acetate + Butyrate + 2 Propionate + 5 CO CH H 2 O (Acetate:Propionate = 3 Methane:Glucose =.60)

Energetic Efficiency VFA Production Heat of combustion kcal/mole kcal/mole of % of of acid glucose fermentedglucose Acetate Propionate Butyrate Glucose 673.0

Effect of Diet VFA Ratios Forage:Grain-----Molar ratios----- Acetate PropionateButyrate 100: : : : :

Branched-Chain Fatty Acids Propionyl CoA + Acetyl CoA Valerate ValineIsobutyrate + NH 3 + CO 2 LeucineIsovalerate + NH 3 + CO 2 Isoleucine2-methylbutyrate + NH 3 + CO 2 Fiber digesting bacteria have a requirement for branched-chain fatty acids.

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 (10 7 /ml) Grow very fast if sufficient glucose is present Double numbers within 20 min (up to 10 9 /ml) Produce lactic acid Lactobacillus ruminis & L. vitulinus also produce some lactic acid Methanobacter ruminantium in rumen (2 x 10 8 /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

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 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

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

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.

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

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

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

Adaptation to Grain Diets Two 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

Action of Ionophores Transmembrane Flux OutIN (High NA +, low K + ) (High K +, low Na + ) ATP H + H + ADP + P i H + H + K + K + Na + Na + H + H + M M

Gram Negative Ionophores Excluded M M Gram - positive Gram-negative

Effect of Ionophores Carbohydrates Sensitive toResistant toionophoreProduce more acetate & Hpropionate & less acetate CH 4

Ionophores - Continued Inhibit Rumminococcus albus Decreased acetate, Ruminococcus flavefaciens formate and CH 4 Butrivibrio fibrisolvens Increase Bacteroides succinogenes Increase propionate Bacteroides ruminicola Selanomonas ruminantium Also inhibit Streptococci Decrease lactate Lactobacilli production No effect Megasphaera Utilize lactate Selenomonas

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

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