Metabolisme Protein pada

Ruminansia
(Rumen Nitrogen Metabolism)
Sri Suharti
Departemen Ilmu Nutrisi dan Teknologi Pakan
Fakultas Peternakan IPB
2016
Sistem Pencernaan Ternak Ruminansia
General Information
 No proteases in saliva
 No rumen secretions
 Microorganisms responsible for protein digestion in rumen (and reticulum)
 Bacteria
 Protozoa

NPN Utilization
 Urea (and most sources of NPN) rapidly degraded to NH3
 MO’s don’t care where NH3 comes from
 Sources of Rumen Nitrogen
 Feed
 Protein nitrogen
Protein supplements (SBM, CSM, grains, forages, silages...
 Nonprotein nitrogen (NPN)
Usually means urea
However, from 5% of N in grains to 50% of N in silage and
immature forages can be NPN
 Endogenous (recycled) N
 Saliva
 Rumen wall
 Ruminal Protein Degradation
 Fermentative digestion – enzymes of microbial origin
 MO proteases & peptidases cleave peptide bonds and release AA
 AA deaminated by microbes, releasing NH3 and C-skeleton
 MO’s use NH3, C-skeleton and energy to synthesize their own AA
Energy primarily from CHO’s (starch, cellulose)
 Formation of NH3 rapid...very few free AA in rumen
 Ruminant vs Nonruminant - Similarities

1. At tissue level – Metabolic pathways similar

2. Ruminant tissues can synthesize dispensable AA
3. Cannot synthesize indispensable AA
 Essential AA must be provided from digestive tract
4. Tissue proteins constantly undergoing turnover
5. AA not stored
6. Constant supply of AA required
 Ruminant vs Nonruminant - Dissimilarities
1. Microbial population has profound effect on AA reaching S.I.
a. AA profile at S.I. different from diet
 Up-grades low quality dietary protein
 Down-grades high quality dietary protein
b. Enables ruminants to use NPN efficiently
 Ruminants can be productive without a source of dietary true
protein
c. Animal can survive on low amounts of dietary protein by recycling
N (as urea) back to rumen
 Ruminant vs Nonruminant - Dissimilarities
1. Microbial population has profound effect on AA reaching S.I. (cont.)
d. Why we say nitrogen metabolism (vs protein metab.)
 Microbial intervention
 NH3 formation
e. Disadvantage: more protein can be destroyed in the rumen than
is synthesized
Result = Net loss of protein
Advantage: can have more protein leaving rumen than is in the
diet
Result = Net gain of protein
 Ruminant vs Nonruminant - Dissimilarities
2. In ruminant nutrition – generally not concerned with AA composition of
dietary protein
a. Type of feed does not affect AA comp. of bacteria and protozoa
leaving rumen
 AA comp. of MO’s reaching duodenum strikingly similar when
measured in labs around the world
b. Biological value (BV) of microbial protein ~80%
Protein Degradation in the Rumen

Feed proteins Peptides Amino acids

Undegraded feed proteins

Escaped feed proteins
“Bypass proteins”

Enzymes from protozoa and bacteria

Many species of bacteria involved
Bacterial enzymes are extracellular
Enzymes not in cell free rumen fluid
Both exopeptidase and endopeptidase activity
Assumption in CNCPS: Enzymes (microorganisms)
in excess – substrate limited
Factors Affecting Ruminal Protein Degradation
Chemical Nature of the proteins
• Solubility – More soluble proteins degraded faster
Some soluble proteins not extensively degraded
Egg ovalbumin, serum proteins
• 3-dimensional structure – Affects solubility & availability
• Chemical bonding
Disulfide bonds – Reduces degradation
Physical barriers
• Cell walls of plants
• Cross linking of peptide chains – Reduces degradation
Aldehydes, Tannins
Feed intake
Rate of passage – Time proteins remain in the rumen
Feed processing
• Rate of passage
• Heat damage – Complexes with carbohydrates
Feed Protein Fractions (CNCPS & NRC)

NPN - A

Soluble Sol Proteins - B1

Insoluble - B2

Insoluble - B3

Insoluble Indigestible - C
Feed
A - Soluble in buffer (borate-phosphate) and not precipitated by tungstic acid
B1 - Soluble in buffer and precipitated by tungstic acid
B2 - Insoluble in buffer = (Insol protein) - (protein insol in neutral detergent)
B3 - Insoluble in buffer = (Insol in neutral detergent) - (Insol in acid detergent)
C - Insoluble in buffer and acid detergent
“Bypass proteins”

Proteins that are not extensively degraded in the rumen

1. Natural
Corn proteins, blood proteins, feather meal

2. Modification of feed proteins to make them less

degradable
Heat - Browning or Maillard reaction
Expeller SBM, Dried DGS, Blood meal
Chemical
Formaldehyde
Polyphenols
Tannins
Alcohol + heat
Usually some loss in availability of amino acids - lysine
Degradation of nitrogenous compounds
by ruminal microorganisms
Bacteria
 30 to 50% of the bacteria are proteolytic.
 Most species have some activity with the exception of the main
cellulolytic bacteria (Fibrobacter succinogenes, Ruminococcus
flavefacians, R. albus).
 Major proteolytic bacteria: Ruminobacter amylophilus,
Butyrivibrio Fibrisolvens and Prevotella ruminicola.
 P. ruminicola is the most numerous proteolytic bacteria (> 60%
of ruminal bacteria) with strains that occur on both roughage
and mixed roughage-concentrate diets.
Bacteria cont’d
 R. amylophilus is the most active proteolytic bacteria. Important
on starch-based diets.
 Breakdown of both soluble and insoluble protein in the rumen.

Protozoa
 Minor involvement in soluble protein breakdown.
 Engulf and hydrolyze particulate proteins and bacteria.
 Predatory activity of protozoa against rumen bacteria
contributes to bacterial protein degradation and turnover in the
rumen.

Fungi
 Minor role in protein degradation.
 PROTEIN
D. ruminantium, B. fibrisolvens, E. caudatum
Clostridium spp, E. simplex, E. budayi
E. caudatum ecaudatum, E. ruminantium, E. maggii
Fusobacterium spp., E. medium
L. multipara O. caudatus, P. ruminicola
P. multivesiculatum, R. amylophilus, S. ruminantium
O. joyonii, N. frontalis, S. bovis, P. communis

OLIGOPEPTIDES
S. bovis, R. amylophilus, P. ruminicola
Dipeptidyl
peptidase
DIPEPTIDES
D. ruminantium, E. caudatum
F. succinogenes, M. elsdenii, P. ruminicola Dipeptidase
Isotricha spp., L. multipara, S. ruminantium

AMINO ACIDS
C. aminophilum, C. sticklandii
P. anerobius, B. fibrisolvens, P. ruminicola
M. elsdenii, S. ruminantium, E. caudatum
Isotricha spp.

AMMONIA
Properties of ammonia producing bacteria

High Numbers Low Numbers

Low Activity High Activity
Butyrivibrio fibrisolvens Clostridium aminophilum
Megasphaera elsdenii Clostridium sticklandii
Prevotella ruminicola Peptostreptococcus anaerobius
Selenomonas ruminantium
Streptococcus bovis

> 109 per ml 107 per ml

10 to 20 nmol NH3 min-1 300 nmol NH3 min-1

(mg protein) (mg protein)
Breakdown of NPN in the rumen
 Major sources of NPN include: dietary NPN, and recycled urea.
 Extremely rapid and releases ammonia.

Major end product of protein degradation in the rumen

 Ammonia

Influence of diet on proteolysis

Concentrate
 Increase in total microbial population, including several of the
more active protein degrading bacteria which are also amylolytic
(Prevotella rumincola, Ruminobacter amylophilus and
Streptococcus bovis).
Fresh forage
 Increase in the proportion of proteolytic bacteria relative to total
microbial population.
Degradation of NPN Compounds

Activity associated with microorganisms

• Urea CO2 + 2 NH3

High concentrations of urease activity in the rumen
Low concentrations of urea in the rumen

• Biuret 2 CO2 + 3 NH3

Low activity in the rumen

• NO3 NH3
Amino Acid Degradation in the Rumen

NH3 CO2

Amino acids Keto acids VFA

• Enzymes from microorganisms Intracellular enzymes

• Peptides probably hydrolyzed to amino acids and then degraded
• NH3, VFA and CO2 absorbed from rumen
Fate of Free Amino Acids in the Rumen

1. Amino acids not absorbed from the rumen

• Concentrations of free AA in the rumen very low

2. Amino acids and small peptides (up to 5 AA)

transported into bacterial cells
• Na pumped out of cells – Uses ATP
• Na gradient facilitates transport of AA by a carrier

3. Utilized for synthesis of microbial proteins

4. Amino acids metabolized to provide energy
Amino Acid Fermentation

Valine Isobutyrate
Leucine Isovalerate
Isoleucine 2-methybutyrate

Alanine, glutamate, histidine,

aspartate, glycine, serine, pyruvate
cystein and tryptophan

Threonine, homoserine,
homocyseine and methionine Ketones
Control of Amino Acid Fermentation
When CHOH is ample for growth, incorporation of amino acids into
protein is favored
• Majority of transported amino acids and peptides do not go
through ammonia pool

When CHOH supply is limiting growth, amino acids are fermented for
energy
• There is an increase in amino acids going through the ammonia
pool
Does Source of Carbohydrate Affect Amino
Acid Fermentation?
CHOH slowly fermented or with a significant lag time
• CHOH fermentation for growth might lag behind fermentation of AA

Rapidly fermented CHOH

• AA fermentation and CHOH might be more closely matched

Recycling of N into the rumen might offset disruptions in CHOH and AA

fermentations
Amino Acid Fermenters in the Rumen

High numbers Low numbers

Low activity High activity

Butrivibrio fibrisolvens Clostridium aminophilum

Measphaera elsdenii Clostridium sticklandii
Selenomonas ruminantium Peptostreptococuss anaerobius

109 per ml 107 per ml

10 to 20 NMol NH3 300 NMol NH3 per min
per min per mg protein per mg protein
Monensin resistant Monensin sensitive
Involved in CHOH Ferment CHOH slowly or not at all
fermentation
Rumen Microbial Protein

 Ruminally synthesized microbial protein supplies 50% OR MORE of

absorbable AA’s when rations are balanced properly.
 Digestibility of microbial protein is about 85% and has an EAA profile
similar to that of lean body tissue and milk.
 Microbial protein EAA is constant and not influenced by diet change.
 Microbial Protein Synthesis

End product of protein degradation is mostly NH3

Protein synthesis
Fixation of N in organic form
Synthesis of amino acids
Synthesis of protein(s)
Microbial Requirements
Bacteria
Nitrogen
• Mixed cultures
NH3 satisfies the N requirement
Cross feeding can supply amino acids
• Pure cultures
Fiber digesters require NH3
Starch digesters require NH3 and amino acids
Peptides can be taken up by cells
Branched-chain fatty acids
• Required by major rumen cellulolytic bacteria
Energy from fermentation
• Need energy for synthesis of macromolecules
Amino Acid Synthesis
Ammonia Fixation

1. Glutamine synthetase/glutamate synthase

• Glutamine synthetase
Glu + NH3 + ATP Gln

• Glutmate synthase
-ketoglutarate + glutamine + NADPH2 2 Glu

High affinity for NH3 - Concentrates NH3 in cells – Uses ATP

Because of N recycling this reaction may not be that important
Amino Acid Synthesis
Ammonia Fixation
2. Glutamic dehydrogenase
• -ketoglutarate + NH3 + NADH Glu

Low affinity for NH3 – High concentration of

enzyme in rumen bacteria – Does not use ATP

Probably predominant pathway

3. Other AA can be synthesized by transamination

reactions with glutamic acid

Estimates of NH3 requirements range from 5 (culture)

to 20 mg/100 ml (in situ digestion)
Sources of Amino Acids for Host Animal

1. Microbial proteins
Quantity determined by:
a) Fermentability of the feed
b) Quantity of feed consumed
c) Nitrogen available to microorganisms

2. Undegraded feed proteins (UIP)

Quantity will vary in relation to:
a) Degradability of feed proteins
b) Quantity of feed proteins consumed
Nutritional Value of Microbial Proteins

1996 NRC for Beef

Microbial protein 80% digestible in the intestine
UIP 80% digestible in the intestine

2001 NRC for Dairy and Level 1 CNCPS

Microbial protein 80% digestible in the intestine
Digestibility of RUP (UIP) is variable in Dairy NRC
UIP 80% digestible in Level 1 CNCPS
Matching Available Energy with
Rates of Protein Degradation

To maximize efficiency of microbial protein synthesis from

ammonia, available energy must be present.
Rumen NH3 Following Protein Ingestion
Rumen VFA from Carbohydrate Sources
Matching Protein
and
Energy Sources
Protein Pathways
History of Protein Systems for Ruminants

• ISU Metabolizable protein system

• Wisconsin system – When urea could be used
• Several European systems – Mostly MP systems
• 1985 NRC system – Summarized systems &
Proposed a MP system
Used in 1989 Dairy NRC
• Cornell CNCPS
• 1996 Beef NRC system – Mostly CNCPS system
Used in ISU Brands system
• 2001 Dairy NRC system
 Feeding Urea - Beef
 Feedlot cattle (fed grain or silage diets)
 Up to 650-750 lb, use natural protein (SBM, CSM)
Can’t consume enough for MO’s to meet protein needs
 >650-700 lb, urea = natural protein as N source
 Above 0.75% urea in diet DM, start observing palatability problems
( intake)
 General recommendation...
don’t exceed 1% urea in diet
 Feeding Urea - Dairy
 Dairy cows
 Upper limit ~1% of diet DM
 Palatability begins to limit intake

 Urea = 281% CP equivalent

 N = 45% of urea
 45%N x 6.25 = 281% CP
 Urea Toxicity (NH3 Toxicity)
 Mechanism
 Rumen [NH3]  Rumen pH 
 As pH , shift from NH4+ to NH3
 NH3 absorbed faster than NH4+
 Liver capacity to convert NH3 to urea is exceeded
 NH3 goes to blood
 2 mg NH3/100 ml plasma is toxic
 Urea Toxicity (NH3 Toxicity)
 Signs of toxicity
 Appear 20-30 min after urea ingestion
 Rapid and labored breathing
 Tremors
 Incoordination
 Inability to stand & tetany increasingly apparent
 Urea Toxicity (NH3 Toxicity)
 Treatment
 Orally dose with 5% acetic acid
(~1 gal. for 1,000 lb cow)
Shift equilibrium from NH3 to NH4+
 rate of absn

 Drench with cold water

 rumen temp. which  rate of urea hydrolysis
Dilutes NH3 concentration
Takes 6-12 gal.; not practical when several sick
 Urea Toxicity (NH3 Toxicity)
 Prevention
 Mix feeds well
 Don’t switch rapidly from natural protein to urea
 Always have feed available
 Don’t allow hungry animals access to highly palatable, high
urea diet, feed, or supplement (including lick tanks)
 Don’t use urea with low-energy feeds