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?