ANS 520

Topics
•Amino acid metabolism
•Microbial protein contributions
•Ruminal N digestion

• Nitrogenous feed component, non-protein nitrogen components, endogenous
– Ammonia for bacterial growth
– Amino acids (AA) for animal needs (absorbed in small intestine)

Crude protein
DIP (RDP)
UIP (RUP)
SolP, % CP
NPN, % CP
NDFIP, % CP
ADFIP, % CP
B1, B2, B3, % hr
Feed Protein Acronyms
NRC Publications
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 NH
4
+
– Determine as NH
3
– Total N x 6.25 = crude protein
• Peptide bond:
NH
2
R
1
-C-C-NH
O C-C=O
R
2
N-C-COOH
H R
3

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
A - All degraded
B - Partly degraded
Slope = degradation rate
C - Not degraded
Digestion time, hr

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 A
Soluble
Insoluble
Sol Proteins - B1
Insoluble B2
Insoluble B3
Indigestible - C
Feed

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

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) 75%
Soybean meal ( Expeller processed) 50%
Alfalfa
Corn proteins
80%
62%
Corn gluten meal
Corn gluten feed
Dried distillers grains
Blood meal
Feather meal
Urea
42%
80%
55%
20%
30%
100%

Degradation of NPN Compounds
Activity associated with microorganisms
• Urea CO
2
+ 2 NH
3
High concentrations of urease activity in the rumen
Low concentrations of urea in the rumen
• Biuret 2 CO
2
+ 3 NH
Low activity in the rumen
3
• NO
3
NH
3

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
NH
3
CO
2
Amino acids Keto acids VFA
• Enzymes from microorganisms
Intracellular enzymes
• Peptides probably hydrolyzed to amino acids and then degraded
• NH
3
, VFA and CO
2 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 numbers
Low activity High activity
Butrivibrio fibrisolvens Clostridium aminophilum
Measphaera elsdenii Clostridium sticklandii
Selenomonas ruminantium Peptostreptococuss anaerobius
10 9 per ml
10 to 20 NMol NH
3 per min per mg protein
10 7 per ml
300 NMol NH
3 per mg protein per min
Monensin resistant Monensin sensitive
Involved in CHOH Ferment CHOH slowly or fermentation not at all

Microbial Protein Synthesis
•End product of protein degradation is mostly NH
3
•Protein synthesis
•Fixation of N in organic form
•Synthesis of amino acids
•Synthesis of protein(s)

• 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 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

• 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
NH
VFA
3
CHOH
Amino acids & Peptides
Amino acids
Fermentation
VFA
Microbial proteins
Microbial protein synthesis related to:
1. Available NH
3 and amino acids (DIP)
2. Fermentation of CHOH - Energy

Microbial Requirements
Bacteria
Nitrogen
• Mixed cultures
NH
3 satisfies the N requirement
Cross feeding can supply amino acids
• Pure cultures
Fiber digesters require NH
3
Starch digesters require NH
3 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 NH
3 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
TDN, % feed DM
Bacteria use energy to pump protons

Microbial Growth in The Rumen
Nutrients available to microbes
1. DIP - NH
3
, 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