Transcript 2010 Protein Metabolism I

Protein Metabolism I
ANS 520
Topics
•Amino acid metabolism
•Microbial protein contributions
•Ruminal N digestion
Protein Pathways
Ruminant Protein Metabolism
• Nitrogenous feed component, non-protein
nitrogen components, endogenous
– Ammonia for bacterial growth
– Amino acids (AA) for animal needs (absorbed in
small intestine)
Feed Protein Acronyms
NRC Publications
Crude protein
DIP (RDP)
UIP (RUP)
SolP, % CP
NPN, % CP
NDFIP, % CP
ADFIP, % CP
B1, B2, B3, % hr
Total N x 6.25
Degraded intake protein
Undegraded intake protein
Soluble protein
Nonprotein nitrogen
Neutral detergent fiber insoluble
protein
Acid detergent fiber insoluble
protein
Rate constants for degradable
fractions
Protein
• Analysis: Determine total N by Kjeldahl
– All N
NH4+
– Determine as NH3
– Total N x 6.25 = crude protein
• Peptide bond:
NH2
R1-C-C-NH
O C-C=O
R2 N-C-COOH
H R3
Nitrogenous Compounds in Feeds
• True proteins
 Polymers of AA (18 to 20 AA) linked by peptide
bonds
• Essential AA
– Have to be present in the diet (absorbed)
– Arg Lys Trp Leu Ile Val Met Thr Phy His
» PVT TIM HALL
• Nonessential amino acids (dispensable)
– Synthesized in body tissues
– Glu Gly Asp Pro Ala Ser Cys Tyr
 Proteins
Peptides
Amino acids
Nitrogenous Compounds in Feeds
• Nonprotein nitrogen
– Nitrogen not associated with protein
• Free amino acids, nucleic acids, amines,
ammonia, nitrates, nitrites, urea
• Crude protein
– Total nitrogen x 6.25
– Proteins on average contain 16% nitrogen
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
•Exceptions might include
•egg ovalbumin, serum proteins
• 3-dimensional structure – Affects solubility & availability
• Chemical bonding
•Disulfide bonds – Reduces degradation
Factors Affecting Ruminal Protein
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
Estimating Ruminal Protein Degradation
1. In situ digestion
Feed placed in Dacron bags suspended
in the rumen
Measure protein lost over time
2. Cannulated animals (rumen & duodenum)
Measure protein flowing through duodenum
Need to differentiate feed from microbes
3. In vitro incubation with rumen microbes
Relative differences among proteins
4. In vitro digestion with fungal enzymes
Protein Degradation In situ
Log, % N remaining
A - All degraded
B - Partly degraded
Slope = degradation rate
C - Not degraded
Digestion time, hr
Protein Degradation
DIP (RDP) = A + B[Kd/(Kd+Kp)]
DIP = Degraded intake protein
Kd = degradation rate, %/h
Kp = passage rate, %/h
UIP (RUP) = B[Kp/(Kd+Kp)] + C
UIP = Undegraded intake protein
Feed Protein Fractions (CNCPS & NRC)
NPN
Soluble
Feed
Insoluble
-
A
Sol Proteins - B1
Insoluble -
B2
Insoluble -
B3
Indigestible - C
Protein Fractions In Feeds
Laboratory Analysis
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
Kd Values for Feed Proteins
Fraction
A
B1
B2
B3
C
Kd, %/h
Infinity
120 to 400
3 to 16
0.06 to 0.55
Not degraded
Kp Values
Wet forages
Kp = 3.054 + 0.614X1
Dry forages
Kp = 3.362 + 0.479X1 – 0.007X2 – 0.017X3
Concentrates
Kp = 2.904 + 1.375X1 – 0.020X2
X1 = DMI, % Body Wt
X2 = Concentrate, % of ration DM
X3 = NDF of feedstuff, % DM
“Bypass proteins”
• Proteins that are not extensively degraded in the rumen
• Natural
•Corn proteins, blood proteins, feather meal
•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
Average Ruminal
Degradation of Several Proteins
Used in Level 1
Soybean meal (Solvent processed)
Soybean meal ( Expeller processed)
Alfalfa
Corn proteins
Corn gluten meal
Corn gluten feed
Dried distillers grains
Blood meal
Feather meal
Urea
75%
50%
80%
62%
42%
80%
55%
20%
30%
100%
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
Fate of Free Amino Acids in the Rumen
•
Amino acids not absorbed from the rumen
• Concentrations of free AA in the rumen very low
•
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
Utilized for synthesis of microbial proteins
Amino acids metabolized to provide energy
•
•
Amino Acid Degradation in the Rumen
Amino acids
NH3
CO2
Keto acids
VFA
• Enzymes from microorganisms
Intracellular enzymes
• Peptides probably hydrolyzed to amino acids
and then degraded
• NH3, VFA and CO2 absorbed from rumen
Amino Acid Fermentation
Valine
Leucine
Isoleucine
Isobutyrate
Isovalerate
2-methybutyrate
Alanine, glutamate, histidine, aspartate, glycine,
serine, cystein and tryptophan
pyruvate
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
Amino Acid Fermenters in the Rumen
High numbers
Low activity
Butrivibrio fibrisolvens
Measphaera elsdenii
Selenomonas ruminantium
109 per ml
10 to 20 NMol NH3
per min per mg protein
Monensin resistant
Involved in CHOH
fermentation
Low numbers
High activity
Clostridium aminophilum
Clostridium sticklandii
Peptostreptococuss anaerobius
107 per ml
300 NMol NH3 per min
per mg protein
Monensin sensitive
Ferment CHOH slowly or
not at all
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)
Rumen microbes
• Bacteria (50% CP)
• Protozoa (20-60%, avg 40% CP)
• Bacteria major player, % of microbial N
entering SI from protozoa < 10%
• N source for microbes
– Diet protein
– Non protein N
– Recycled N
Microbial N
• Microbial N entering SI (% of non-ammonia N)
– High protein diets -40%
– Low protein diets -60%
– Exclusive NPN diet -100%
• Limiting factors would include C and/or energy source
Nutritive Value of Microbial N
•
•
•
•
Increases value of low quality feed N
Decreases value of high quality feed N
Animal can survive on non-protein N
Can survive on low amounts of recycled N
Bacterial Protein
Synthesis in the Rumen
NH3
Amino acids & Peptides
VFA
Amino acids
Fermentation
CHOH
VFA
Microbial protein synthesis related to:
1. Available NH3 and amino acids (DIP)
2. Fermentation of CHOH - Energy
Microbial
proteins
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
Role of Protozoa
•Do not use NH3 directly
•Engulf feed particles and bacteria
• Digest proteins
• Release amino acids and peptides into rumen
• Use amino acids for protein synthesis
• Protozoa engulf bacteria
• Protozoa lyse easily – May contribute little
microbial protein to the animal
Efficiency of Microbial Growth
Grams microbial N/100 g organic matter digested
Ranges from 1.1 to 5.0
1. Kind of diet
Forages > Grain
2. Level of feeding
High > Low
3. Rate of passage
Fast > Slow
4. Turnover of microbial cells
Younger cells turnover less than aging cells
5. Maintenance requirement of cells
Microbes use energy to maintain cellular integrity
6. Energy spilling
Dissipation of energy different from maintenance
Most apparent when energy is in excess
Efficiency of Microbial Growth
Slow
passage
Low rumen
pH
Low quality
forages slow
passage
Bacteria
use energy to
pump protons
TDN, % feed DM
Microbial Growth in The Rumen
Nutrients available to microbes
1. DIP - NH3, peptides, amino acids
•
CNCPS adjusts for inadequate available N
2. Energy from the fermentation
•
•
Growth rate related to Kd of CHOH
Quantity of cells related to CHOH digested
CNCPS assumes microbes digesting
non-fiber and fiber CHOH both have
a maximum yield of 50g cells/100g
CHOH fermented
3. Other - branched-chain acids, minerals