Ruminant Carbohydrate Digestion

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 841294-1309
• Journal of Animal Science 801112-
• Carbohydrates in common feedstuffs
• Carbohydrate, DM Alfalfa Grass Corn DDGS
• 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, Hemicellulose Alfalfa Bromegrass Locati
  on
• 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 gt Greater digestibility
• gt 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
• Saponfiable 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

• 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 lignins 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
• ArabinoseXylose ratio
• Decreases with maturity, decreasing digestibility
• 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 graingtDecreased pH and starchgtReduce
  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 841294
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-
  bacteris
• 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

16

• Structure of the cellulosome

17

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

• 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
• Amylose Amylopectin
• 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 my 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 gt Oligosaccharides
  gt 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 selectores
• 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

• Factors affecting starch digestion
• DM intake
• Increased dry matter intake decreases starch
  digestion
• Percentage of grain in diet

26

• Type of starch
• Barley gt Corn gt Sorghum
• Waxy gt 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

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 gt Butyrate
• In grain-fed animals Lactate gt Propionate
• Succinate
• Supplies at least 1/3 of the propionate
• Formate
• Rapidly converted to H2 CO2
• H2
• 4H2 CO2 gt 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 gt H2O
• Anerobic organisms
• CO2 gt CH4
• Pyruvate gt Propionate
• Acetate gt Butyrate
• NO3 gt NO2 gt NH3
• SO4 gt S

36

• Thermodynamic order of preference for electron
  acceptors
• NO3 gt NO2
• NO2 gt NH4
• Crotonyl CoAgtButyryl CoA
• Fumarate gt Succinate
• Acrylyl CoA gt Propionyl CoA
• SO4 gt HS
• Acetoacetyl CoA gt B-OH-Butyryl CoA
• CO2 gt CH4
• Pyruvate gt Lactate
• CO2 gt 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 gt 2 Pyruvate 4H (as 2 NADH2)
• Pyruvate H2O gt Acetate CO2 2H (as 1 FADH2)
• 2H accepted
• CO2 4H2 gt CH4 2H2O
• Pyruvate 4H (as 2 NADH2) gt Propionate H2O
• 2 Acetate 4H (as 2 NADH2) gt Butyrate 2H2O
• Fermentation balance
• High forage diets
• 5 Glucose gt 6Acetate Butyrate 2Propionate
  5CO2
• 3CH4 6H2O
• AcetatePropionate 3
• CH4Glucose .60
• High grain diets
• 3 Glucose gt 2Acetate Butyrate 2Propionate
  3CO2
• CH4 2H2O
• AcetatePropionate 1
• CH4Glucose .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 forageconcentrate 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
• ForageConcentrate
• 
  ForageConcentrate
• VFA, Molar 6040 4060 2080
• 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 Long Coarse Fine Pelleted
• 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. 90529-540)
• 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 gt CH3COOH
• Thermodynamically unfavorable to methane
  production
• Doesnt usually occur in the rumen
• Does occur in the large intestine of various
  species and in termites
• Why doesnt it occur in the rumen?