Dynamics of protein metabolism in the ruminant
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Dynamics of Protein Metabolism in the Ruminant. 2.2. 2.3. Analysis of Dietary Protein. Crude protein (CP %) = total N (%)  6.25 Factor is based on 16% N in protein. True protein varies between 13 to 19% N. Source %N in protein Conversion factor oilseed proteins 18.5 5.40

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Analysis of dietary protein l.jpg
Analysis of Dietary Protein

  • Crude protein (CP %) = total N (%)  6.25

  • Factor is based on 16% N in protein.

  • True protein varies between 13 to 19% N.

    Source %N in protein Conversion factor

    oilseed proteins 18.5 5.40

    cereal proteins 17.0 5.90

    meat or fish 16.0 6.25

    alfalfa 15.8 6.33

    true microbial protein 15.0 6.67

  • Not all N in protein is present as true protein.


Classification of protein and nitrogen fractions in feedstuffs l.jpg
Classification of protein and nitrogen fractions in feedstuffs

Total

Borate

Buffer

Neutral

Detergent

Acid

Detergent

Sol

A

B1

Insol

B2

B3

C

Sol

A1

B1

B2

Insol

B3

C

Sol

A1

B1

B2

B3

Insol

C


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Crude Protein feedstuffs

True protein

(60 to 80%)

Non-protein

nitrogen

Lignified

nitrogen

Essential amino acids

Arginine (Arg)

Histidine (His)

Isoleucine (Ile)

Leucine (Leu)

Lysine (Lys)

Methionine (Met)

Phenylalanine (Phe)

Threonine (Thr)

Tryptophan (Trp)

Valine (Val)

Non-essential

amino acids

Alanine (Ala)

Asparagine (Asn)

Aspartic acid (Asp)

Cysteine (Cys)

Glutamic acid (Glu)

Glutamine (Gln)

Glycine (Gly)

Proline (Pro)

Serine (Ser)

Tyrosine (Tyr)

Amides

Amines

Amino acids

Peptides

Nucleic acids

Nitrates

Ammonia

Urea


Calculations l.jpg
Calculations feedstuffs

Protein Fraction

A, B1

B2

B3 C

Log of % nutrient

remaining

Hours

Calculate slope (change per hour) of each line.

Slope = kd, has units of % of pool remaining that is lost per hour.


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Terms for describing nitrogen components of feedstuffs feedstuffs

  • Degradable Intake Protein (DIP): dietary crude protein degraded in the rumen.

  • Undegraded intake protein (UIP): dietary crude protein that is not degraded in the rumen and escapes or bypasses the rumen to the intestine. It is largely true protein but also contains ADFIP.

  • Soluble protein (SolP): Contains non-protein nitrogen, amino acids and peptides. Soluble protein is degraded instantaneously in the rumen.


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Terms for describing nitrogen components of feedstuffs feedstuffs

  • Non-protein nitrogen (NPN): Includes amides, amines, amino acids, some peptides, nucleic acids, nitrates, urea, ammonia. Degraded instantaneously in the rumen.

  • Acid detergent fiber insoluble protein (ADFIP): Consists of heat damaged protein and nitrogen associated with lignin. Resists ruminal fermentation and is indigestible in the small intestine.


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Protein content of common feedstuffs feedstuffs

CP DIP UIP SolP NPN ADFIP

Feedstuff %DM %CP %CP %CP %SolP %CP

Alfalfa silage 19.5 92 8 50 100 15

Barley silage 11.9 86 14 70 100 6.1

Corn silage 8.6 77 23 50 100 9

Alfalfa hay 22 84 16 28 93 14

Timothy hay 10.8 73 27 25 96 5.7

Barley straw 4.4 30 70 20 95 65

Barley grain 13.2 67 33 17 29 5


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Protein content of protein supplements feedstuffs

CP DIP UIP SolP NPN ADFIP

Plant sources %DM %CP %CP %CP %SolP %CP

Canola meal 40.9 67.9 32.2 32.4 65 6.4

Soybean meal 52.9 80 20 33 27 1

Soypass* 52.6 34 66 6.8 50 1

Brewer’s grains 29.2 34.1 65.9 4 75 12

Corn distiller’s gr. 30.4 26.6 73.7 6 67 18

Corn gluten meal 66.3 41 59 4 75 2

*Commercial product: LignoTech USA, Inc.


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Protein content of protein supplements feedstuffs

CP DIP UIP SolP NPN ADFIP

%DM %CP %CP %CP %SolP %CP

Animal sources

Blood meal 93.8 25 75 5 0 1

Feather meal 85.8 30 70 9 89 32

Fishmeal 67.9 40 60 21 0 1

Meat and bone 50 47 53 16.1 93.8 4.9

Non-protein nitrogen sources

Urea 291 100 0 100 100 0


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Ruminally Protected Protein feedstuffs

  • A nutrient(s) fed in such a form that provides an increase in the flow of that nutrient(s), unchanged, to the abomasum, yet is available to the animal in the intestine

  • Methods to decrease the rate and extent of ruminal degradation involved the use of heat, chemical agents, or combination of both


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Heat Processing feedstuffs

  • Heat processing decrease rumen protein degradation by denaturation of proteins and by the formation of protein-CHO (Millard reactions) and protein cross-links. Commercial methods that rely solely on heat include: cooker-expeller, roasting, extrusion, pressure toasting, and micronization.

  • Heat processing reduced fraction A, increases fraction B, and C, and decreases in the fractional rates of degradation of the fraction B


Heat processing cont l.jpg
Heat Processing cont. feedstuffs

  • Over heating also causes significant losses of lysine, cysine, and arginine.

  • Among those AA, lysine is the most sensitive to heat damage and undergoes both destruction and decreased availability


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Chemistry of the Maillard reaction between reducing sugars and lysine residues during heat treatment of proteins


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Heat Processing and lysine residues during heat treatment of proteins

  • Careful control of heating conditions is required to optimize the content of digestible RUP.

  • Under heating results in only small increase

    in digestible RUP.

    . Over heating reduces the intestinal digestibility of RUP through the formation of indigestible Millard products and protein complexes.


Chemical treatment l.jpg
Chemical Treatment and lysine residues during heat treatment of proteins

  • Chemical treatment of feed proteins can be divided into three categories: 1) chemicals that combine with and introduce cross-links in proteins, (2) chemicals that alter protein structure by denaturation (e.g., acids, alkalis, and ethanol), and (3) chemicals that bind to proteins but with little or no alteration of protein structure (e.g., tannins).


Chemical treatment cont l.jpg
Chemical Treatment and lysine residues during heat treatment of proteinscont.

  • For a variety of reasons, often including less than desired levels of effectiveness, use of chemical agents as the sole treatment for increasing the RUP content of feed proteins has not received commercial acceptance.

  • A more effective approach involving “chemical” agents has been to combine chemical and heat treatments.

  • An example of this approach is the addition of lignosulfonate, a byproduct of the wool pulp industry that contains a variety of sugars (mainly xylose), to oilseed meals before heat treatment.


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Chemical Treatment and lysine residues during heat treatment of proteinscont.

  • The combined treatments enhance non-enzymatic browning (Millard reactions) because of the enhanced availability of sugar aldehydes that can react with protein.


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Characterization of Protein Sources and lysine residues during heat treatment of proteins

  • Common protein supplements that are high in RUP are:

  • Fish meal

  • Meat and bone meal (MBM)

  • Feather meal (FtM)

  • Blood meal (BM)

  • Corn gluten meal (CGM)

  • Distillers dried grains (DDG)

  • DDG with solubles (DDGS)

  • Brewers dried grains (BDG)

  • Brewers wet grains (BWG)


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Nitrogen transactions in the rumen and lysine residues during heat treatment of proteins

Sources of nitrogen in the rumen

  • Dietary crude protein (true protein and NPN).

  • Recycled microbial protein (bacteria and protozoa).

  • Endogenous N (urea, abraded epithelial cells, salivary proteins).


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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.


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Bacteria cont’d microorganisms

  • 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.


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PROTEIN microorganisms

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

Dipeptidyl

peptidase

S. bovis, R. amylophilus, P. ruminicola

DIPEPTIDES

D. ruminantium, E. caudatum

F. succinogenes, M. elsdenii, P. ruminicola

Isotricha spp., L. multipara, S. ruminantium

Dipeptidase

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 l.jpg

High Numbers microorganisms

Low Activity

Butyrivibrio fibrisolvens

Megasphaera elsdenii

Prevotella ruminicola

Selenomonas ruminantium

Streptococcus bovis

> 109 per ml

10 to 20 nmol NH3 min-1

(mg protein)

Low Numbers

High Activity

Clostridium aminophilum

Clostridium sticklandii

Peptostreptococcus anaerobius

107 per ml

300 nmol NH3 min-1

(mg protein)

Properties of ammonia producing bacteria


Slide52 l.jpg

Breakdown of NPN in the rumen microorganisms

  • 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 l.jpg
Influence of diet on proteolysis microorganisms

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.



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Factors Influencing Microbial Protein Synthesis microorganisms

Ammonia

  • Most important source of N for bacterial protein synthesis.

  • 50 to 80% of bacterial N is derived from ammonia.

  • Bacteria hydrolyzing structural carbohydrates utilize ammonia as N source.

  • Several mechanisms for the uptake of ammonia:

    • high affinity, low Km (ammonia concentration) enzyme system

    • glutamate synthetase - glutamate synthase (GS-GOGAT)

    • lower affinity, higher Km system

    • NADP-glutamate dehydrogenase (NADP-GDH), NAD-GDH and alanine dehydrogenase.

  • Minimum level of ammonia is necessary for maximum growth and efficiency (5 mg/100 ml of rumen fluid).


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Peptides and amino acids microorganisms

  • 20 to 50% of ruminal microbial N is derived from this pool.

  • Supplying preformed peptides and amino acids spares the cost associated with synthesizing amino acids.

  • Rapidly fermenting organisms, bacteria hydrolyzing non-structural carbohydrates (starch, pectin, sugars), utilize peptides, amino acids and ammonia.

  • Availability of peptides improves microbial growth.


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Synchronization of protein and carbohydrate degradation microorganisms

  • Microbial protein synthesis is maximized when the release of N from protein occurs with the release of energy from the degradation of carbohydrates.

    Fractional Outflow Rates

  • Increasing the rate of passage removes the more mature organisms, reducing the median age of the microbes.

  • Reduces the amount of energy expended on maintenance so more energy can be used for growth.


Slide58 l.jpg

Efficiency of Microbial Growth microorganisms

14

12

10

8

BCP/100 gm TDN

pH

6

Rate of passage

4

2

0

55

70

Diet % of TDN (DOM)




Intraruminal nitrogen recycling l.jpg
Intraruminal nitrogen recycling protein turnover

Turnover of bacteria and protozoa.

  • 30 to 55% of bacterial N

  • 75 to 90% of protozoal N

    Causes of microbial N recycling

  • Engulfment and subsequent digestion of bacterial cells by protozoa

  • Lysis due to autolytic enzymes, bacteriocins, or other soluble compounds in response to nutrient deprivation or interspecies competition

  • Activity of bacteriophages and mycoplasmas.


Ammonia accumulation in the rumen l.jpg
Ammonia accumulation in the rumen protein turnover

  • Ammonia concentration exceeds the capacity of the ruminal bacteria to utilize it.

  • Absorbed across the ruminal wall into the blood where it is transported to the liver and metabolized to urea.

  • Urea is filtered by the kidney and excreted in urine as waste N.

  • In addition to poor N retention, the synthesis of urea from ammonia also has an energetic cost (12 kcal/g N) to the animal.


Urea recycling l.jpg
Urea recycling protein turnover

  • Blood urea originates from the endogenous metabolism of tissue protein, the deamination of excess absorbed amino acids and the absorption of ruminal ammonia.

  • Recycled to the rumen primarily through the rumen wall and to a lesser extent via saliva (approx 15% of urea recycled to the rumen is via saliva)

  • Facultative microorganisms located on the rumen epithelium wall have urease activity



Composition of microbial protein reaching the intestine l.jpg
Composition of microbial protein reaching the intestine wall to urea

Hay Hay and Conc

sheep 1 sheep 2 sheep 1 sheep2

N in rumen digesta, g

Fungi .21 .60 .42 .69

Protozoa 8.0 5.86 18.3 11.5

Bacteria 11.5 10.4 9.03 8.07

N in rumen digesta, % of total microbial N

Fungi 1.1 3.6 1.5 3.4

Protozoa 40.7 34.7 65.9 56.7

Bacteria 58.3 61.7 32.6 39.8

N in duodenal digesta, g

Fungi .10 .22 .21 .49

Protozoa .55 1.08 1.83 1.63

Bacteria 13.1 14.6 10.1 15.9

N in duodenal digesta, % of total microbial N flow

Fungi .73 1.4 1.7 2.7

Protozoa 4.0 6.8 15.1 9.0

Bacteria 95.3 91.8 83.2 88.2


Undegraded dietary protein l.jpg
Undegraded dietary protein wall to urea

  • Protein that escapes microbial degradation passes to the lower digestive tract where it will be largely degraded. Only the very refractive N component such as N bound to lignin or products of the Maillard reaction will not be degraded.

  • Benefit to the animal of supplying UIP will depend on the provision of essential amino acids that are required in excess of what is supplied by microbial protein.


Protein digestion in the abomasum l.jpg
Protein digestion in the abomasum wall to urea

HCl

Denatured protein

disruption of non-covalent bonds

uncoiling of protein

Protein

HCl

Pepsinogen

(inactive)

Pepsin

(hydrolysis bonds at

carboxylic end of

aromatic AA and Leu)

Pepsin

Small polypeptides

few amino acid

Denatured

protein

pH 1.6 to 3.2


Secretion and activation of pancreatic and intestinal proteolytic enzymes l.jpg
Secretion and activation of pancreatic and intestinal proteolytic enzymes

Polypeptides

Short peptides

AA

Intestinal

endocrine cell

CCK and Secretin

Cholecystokinin (CCK)

Pancreatic

acinar cell

Intestinal

mucosal cell

Enterokinase

Trypsinogen

Trypsin

Chymotrypsin

Elastase

Carboxypeptidase

Chymotrypsinogen

Proelastase

Procarboxypeptidase


Sites of hydrolysis of proteolytic enzymes l.jpg
Sites of hydrolysis of proteolytic enzymes proteolytic enzymes

  • Pancreas

  • Trypsin Dibasic AA (Arg, Lys),

  • C-terminal end

  • Chymotrypsin Aromatic C terminal peptides

  • Elastase Neutral C terminal peptides

  • Carboxypeptidase C-terminal end

  • Intestine

  • Enteropeptidase N-terminal end


Digestion in the small intestine l.jpg
Digestion in the small intestine proteolytic enzymes

Pancreatic and intestinal

proteases

Amino acids

Dipeptides

Tripeptides

Polypeptides

Oligopeptides

Intestinal di- and tripeptidases

(cell membrane and cytosol)

Amino

acids

Dipeptides

Tripeptides


Protein absorption l.jpg
Protein absorption proteolytic enzymes

Small intestine

  • Major site of absorption

  • Amino acids absorbed in the ileum

  • Dipeptides and tripeptides absorbed in the jejunum

  • Active transport (energy dependent)

  • 9 carrier systems for amino acids

  • specific for certain amino acids


Protein metabolism l.jpg
Protein metabolism proteolytic enzymes

  • Intestinal cell

    • Glu, Asp, Gln metabolized by intestinal cell

    • provides 40% of energy requirements

  • Liver

    • protein synthesis

    • synthesis of non-essential amino acids

    • C-skeletons catabolized for energy and the amine group metabolized to urea


Nitrogen metabolism in the large intestine l.jpg
Nitrogen metabolism in the large intestine proteolytic enzymes

  • N supplied to the lower tract comes from the recycling of urea and other endogenous protein (sloughed epithelial cells, enzymes and glycoproteins of mucus).

  • Energy substrates come from the residual fermentable fibre, the glycocalyx of rumen microorganisms, starch and other polysaccharides that have resisted rumen and enteric digestion.

  • As the amount of fermentable energy from the diet reaching the lower tract increases, microbial synthesis increases and fecal N excretion increases.


Routes of nitrogen excretion l.jpg
Routes of nitrogen excretion proteolytic enzymes

Urine (urea)

  • Endogenous urinary N from the catabolism of tissue proteins

  • Absorption and metabolism of excess ruminal ammonia.

  • Catabolism of excess absorbed amino acids

    Feces

  • Microbial N synthesized in and passed from the large intestine.

  • Sloughed cells and secretions of the GI tract.

  • Undigested unabsorbed dietary protein.


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Undegradable in rumen proteolytic enzymes

Degradable

in rumen

Feed

NPN

Indigestible

Digestible

Peptides

amino

acids

Plasma urea

NH3

Rumen

Energy

Microbial N

Tissues

Maintenance

Growth

Conceptus

Lactation

Wool

NH3

Peptides

amino

acids

Small intestine

Endog N

Energy

NH3

Large intestine

Microbial N

Endog N

Urine

Feces


Meeting the protein requirements of ruminant animals l.jpg
Meeting the protein requirements of ruminant animals proteolytic enzymes

  • Degradable intake protein in the rumen for ruminal microorganisms to maximize digestibility of the diet and feed intake.

  • Absorbable essential amino acids at the intestine from the digestion of microbial protein produced in the rumen and dietary intake protein that escapes rumen fermentation.


Defaunation protozoal removal l.jpg
Defaunation (protozoal removal) proteolytic enzymes

  • Removal of protozoal predation of bacteria.

  • Increases substrates (starch) available for fermentation and growth by bacteria.

  • Increases amount of bacterial protein synthesized in the rumen.

  • Increases the flow of microbial protein from the rumen.

  • Reduction in ammonia concentration.


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Chemistry of the Maillard reaction between reducing sugars and lysine residues during heat treatment of proteins


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