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 AcronymsNRC Publications

Crude protein Total N x 6.25DIP (RDP) Degraded intake proteinUIP (RUP) Undegraded intake proteinSolP, % CP Soluble proteinNPN, % CP Nonprotein nitrogenNDFIP, % CP Neutral detergent fiber insoluble

proteinADFIP, % CP Acid detergent fiber insoluble

proteinB1, B2, B3, % hr 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-COOHH 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

A - All degraded

B - Partly degraded Slope = degradation rate

C - Not degraded

Digestion time, hr

Log,

% N

rem

aini

ng

Protein Degradation

DIP (RDP) = A + B[Kd/(Kd+Kp)] DIP = Degraded intake protein

Kd = degradation rate, %/hKp = passage rate, %/h

UIP (RUP) = B[Kp/(Kd+Kp)] + CUIP = Undegraded intake protein

Feed Protein Fractions (CNCPS & NRC)

Soluble

Insoluble

NPN - A

Sol Proteins - B1

Insoluble - B2

Insoluble - B3

Indigestible - CFeed

Protein Fractions In FeedsLaboratory Analysis

A - Soluble in buffer (borate-phosphate) and not precipitated by tungstic acidB1 - Soluble in buffer and precipitated by tungstic acidB2 - 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 Kd, %/hA InfinityB1 120 to 400B2 3 to 16B3 0.06 to 0.55C Not degraded

Kp Values

Wet foragesKp = 3.054 + 0.614X1

Dry foragesKp = 3.362 + 0.479X1 – 0.007X2 – 0.017X3

ConcentratesKp = 2.904 + 1.375X1 – 0.020X2

X1 = DMI, % Body WtX2 = Concentrate, % of ration DMX3 = 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 RuminalDegradation of Several Proteins

Used in Level 1

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

Degradation of NPN Compounds

Activity associated with microorganisms

• Urea CO2 + 2 NH3

High concentrations of urease activityin the rumenLow 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

NH3 CO2

Amino acids Keto acids VFA

• Enzymes from microorganismsIntracellular enzymes

• Peptides probably hydrolyzed to amino acids and then degraded

• NH3, VFA and CO2 absorbed from rumen

Amino Acid Fermentation

Valine IsobutyrateLeucine IsovalerateIsoleucine 2-methybutyrate

Alanine, glutamate, histidine, aspartate, glycine,serine, cystein and tryptophan pyruvate

Threonine, homoserine, homocyseine andmethionine Ketones

Control of Amino Acid Fermentation

When CHOH is ample for growth, incorporationof 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, aminoacids 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 activityButrivibrio fibrisolvens Clostridium aminophilumMeasphaera elsdenii Clostridium sticklandiiSelenomonas ruminantium Peptostreptococuss anaerobius

109 per ml 107 per ml10 to 20 NMol NH3 300 NMol NH3 per min per min per mg protein per mg proteinMonensin resistant Monensin sensitiveInvolved in CHOH Ferment CHOH slowly orfermentation 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 ProteinSynthesis in the Rumen

NH3 Amino acids & Peptides

VFA Amino acids MicrobialFermentation proteins

CHOH VFA

Microbial protein synthesis related to: 1. Available NH3 and amino acids (DIP)2. Fermentation of CHOH - Energy

Microbial RequirementsBacteria

Nitrogen• Mixed cultures

NH3 satisfies the N requirementCross feeding can supply amino acids

• Pure culturesFiber digesters require NH3

Starch digesters require NH3 and amino acidsPeptides 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 digestedRanges from 1.1 to 5.0

1. Kind of diet Forages > Grain2. Level of feeding High > Low3. Rate of passage Fast > Slow4. Turnover of microbial cells

Younger cells turnover less than aging cells

5. Maintenance requirement of cellsMicrobes use energy to maintain cellular integrity

6. Energy spillingDissipation of energy different from maintenanceMost apparent when energy is in excess

Efficiency of Microbial Growth

TDN, % feed DM

G B

CP

/100

g T

DN

813

Slow Low rumenpassage pH

Bacteria Low quality use energy to forages slow pump protons passage

Microbial Growth in The Rumen

Nutrients available to microbes1. 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 digestingnon-fiber and fiber CHOH both havea maximum yield of 50g cells/100gCHOH fermented

3. Other - branched-chain acids, minerals