Transcript Dynamics of Protein Metabolism in the Ruminant

Dynamics of Protein
Metabolism in the
Ruminant
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.
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2.2
2.3
Analysis of Dietary Protein

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
Crude protein (CP %) = total N (%)  6.25
Factor is based on 16% N in protein.
True protein varies between 13 to 19% N.
Source
oilseed proteins
cereal proteins
meat or fish
alfalfa
true microbial protein

%N in protein
18.5
17.0
16.0
15.8
15.0
Conversion factor
5.40
5.90
6.25
6.33
6.67
Not all N in protein is present as true protein.
Research shows...
 Researchers
at Cornell University reported
that in 67 lactation trials evaluating the UIP
approach, milk production significantly
increased in 19%, decreased in 9% and did
not change in 73%.
 Emphasized the need to consider the quality
of UIP sources and AA balance when
utilizing the protein partitioning system.
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Cornell Protein Partitioning
System
 This
system, introduced in the late 1980’s
differentiated between the various N forms
by breaking the protein content of feed
down into fractions based on:
– availability
– site of digestion
– degree of rumen solubility
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Protein Categories (Based on the
Cornell system)
CRUDE PROTEIN
AVAILABLE
PROTEIN
DEGRADEABLE
PROTEIN
UNAVAILABLE
PROTEIN (ADF-N)
UNDEGRADEABLE
PROTEIN
METABOLIZABLE
PROTEIN
RAPIDLY
SOLUBLE PROTEIN
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UNUSED
PROTEIN
SLOWLY
SOLUBLE PROTEIN
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Classification of protein and
nitrogen fractions in feedstuffs
Total
Borate
Buffer
Sol
A
B1
Insol
B2
B3
C
Neutral
Detergent
Sol
A1
B1
B2
Insol
B3
C
Acid
Detergent
Sol
A1
B1
B2
B3
Insol
C
Crude Protein
True protein
(60 to 80%)
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)
Non-protein
nitrogen
Amides
Amines
Amino acids
Peptides
Nucleic acids
Nitrates
Ammonia
Urea
Lignified
nitrogen
Classification of Protein and Nitrogen in Feedstuffs
Fraction
A1
Composition
NH3, NO3, AA, peptides
B1
True soluble protein
Globulins and some
albumins
Most albumins and
glutelins
Prolamines, extensins
B2
B3
C
Denatured proteins
Heat damaged protein
and N bound to lignin
Ruminal
Degradation (%/h)
Instantaneous
200 to 300
Intestinal
Digestion(%)
None reaches
intestine
100
5 to 15
100
0.1 to 1.5
80
0
0
Log of % nutrient
remaining
Calculations
Protein Fraction
A, B1
B2
B3 C
Hours
Calculate slope (change per hour) of each line.
Slope = kd, has units of % of pool remaining that is
lost per hour.
Terms for describing nitrogen
components of 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.
Terms for describing nitrogen
components of 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.
Protein content of common feedstuffs
Feedstuff
CP
%DM
DIP
%CP
UIP
%CP
Alfalfa silage
Barley silage
Corn silage
Alfalfa hay
Timothy hay
Barley straw
Barley grain
19.5
11.9
8.6
22
10.8
4.4
13.2
92
86
77
84
73
30
67
8
14
23
16
27
70
33
SolP
%CP
50
70
50
28
25
20
17
NPN ADFIP
%SolP %CP
100
100
100
93
96
95
29
15
6.1
9
14
5.7
65
5
Protein content of protein supplements
Plant sources
CP
DIP
%DM %CP
UIP
%CP
SolP
NPN ADFIP
%CP %SolP %CP
Canola meal
Soybean meal
Soypass*
Brewer’s grains
Corn distiller’s gr.
Corn gluten meal
40.9
52.9
52.6
29.2
30.4
66.3
32.2
20
66
65.9
73.7
59
32.4
33
6.8
4
6
4
67.9
80
34
34.1
26.6
41
*Commercial product: LignoTech USA, Inc.
65
27
50
75
67
75
6.4
1
1
12
18
2
Protein content of protein supplements
CP
DIP
%DM %CP
UIP
%CP
SolP
NPN ADFIP
%CP %SolP %CP
93.8
85.8
67.9
50
25
30
40
47
75
70
60
53
5
9
21
16.1
0
89
0
93.8
1
32
1
4.9
Non-protein nitrogen sources
Urea
291
100
0
100
100
0
Animal sources
Blood meal
Feather meal
Fishmeal
Meat and bone
Ruminally Protected
Protein


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
Heat Processing


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.
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

Chemistry of the Maillard reaction between reducing
sugars and lysine residues during heat treatment of
proteins
Heat Processing


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

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.


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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.
Chemical Treatment cont.

The combined treatments enhance nonenzymatic browning (Millard reactions)
because of the enhanced availability of
sugar aldehydes that can react with
protein.
Characterization of Protein
Sources
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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)
Nitrogen transactions in the rumen
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).
Degradation of nitrogenous compounds
by ruminal microorganisms
Bacteria


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
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 roughageconcentrate 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
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
High Numbers
Low Activity
Low Numbers
High Activity
Butyrivibrio fibrisolvens
Megasphaera elsdenii
Prevotella ruminicola
Selenomonas
ruminantium
Streptococcus bovis
Clostridium aminophilum
Clostridium sticklandii
Peptostreptococcus
anaerobius
> 109 per ml
107 per ml
10 to 20 nmol NH3 min-1
(mg protein)
300 nmol NH3 min-1
(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.
Microbial protein synthesis in the rumen
Factors Influencing Microbial
Protein Synthesis
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).
Peptides and amino acids
 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.
Synchronization of protein and carbohydrate
degradation
 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.
Efficiency of Microbial Growth
14
BCP/100 gm TDN
12
10
8
6
Rate of passage
pH
4
2
0
55
Diet % of TDN (DOM)
70
Effect of dilution rate on YATP.

Reduces the amount of intraruminal N
recycling (microbial protein turnover).
Intraruminal nitrogen recycling
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




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



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
Factors involved in increasing the permeability of the
rumen wall to urea
Composition of microbial protein reaching the intestine
Hay
sheep 1
sheep 2
Hay and Conc
sheep 1
sheep2
N in rumen digesta, g
Fungi
.21
.60
.42
Protozoa
8.0
5.86
18.3
Bacteria
11.5
10.4
9.03
N in rumen digesta, % of total microbial N
Fungi
1.1
3.6
1.5
Protozoa
40.7
34.7
65.9
Bacteria
58.3
61.7
32.6
N in duodenal digesta, g
Fungi
.10
.22
.21
Protozoa
.55
1.08
1.83
Bacteria
13.1
14.6
10.1
N in duodenal digesta, % of total microbial N flow
Fungi
.73
1.4
1.7
Protozoa
4.0
6.8
15.1
Bacteria
95.3
91.8
83.2
.69
11.5
8.07
3.4
56.7
39.8
.49
1.63
15.9
2.7
9.0
88.2
Undegraded dietary protein


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
HCl
Denatured protein
disruption of non-covalent bonds
uncoiling of protein
Protein
HCl
Pepsinogen
(inactive)
Denatured
protein
Pepsin
(hydrolysis bonds at
carboxylic end of
aromatic AA and Leu)
Pepsin
pH 1.6 to 3.2
Small polypeptides
few amino acid
Secretion and activation of pancreatic and
intestinal proteolytic enzymes
Polypeptides
Short peptides
AA
Intestinal
endocrine cell
CCK and Secretin
Pancreatic
acinar cell
Cholecystokinin (CCK)
Intestinal
mucosal cell
Enterokinase
Trypsinogen
Chymotrypsinogen
Proelastase
Procarboxypeptidase
Trypsin
Chymotrypsin
Elastase
Carboxypeptidase
Sites of hydrolysis of proteolytic enzymes


Pancreas
Trypsin
Chymotrypsin
Elastase
Carboxypeptidase
Dibasic AA (Arg, Lys),
C-terminal end
Aromatic C terminal peptides
Neutral C terminal peptides
C-terminal end
Intestine
Enteropeptidase
N-terminal end
Digestion in the small intestine
Pancreatic and intestinal
proteases
Polypeptides
Oligopeptides
Dipeptides
Tripeptides
Amino acids
Dipeptides
Tripeptides
Intestinal di- and tripeptidases
(cell membrane and cytosol)
Amino
acids
Protein absorption
Small intestine

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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

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



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
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.
Feed
Undegradable in rumen
Indigestible
Rumen
Digestible
Peptides
amino
acids
Degradable
in rumen NPN
NH3
Energy
Plasma urea
Microbial N
Small intestine
Peptides
amino
acids
NH3
Endog N
Energy
Tissues
Maintenance
Growth
Conceptus
Lactation
Wool
NH3
Large intestine
Microbial N
Feces
Endog N
Urine
Meeting the protein requirements of
ruminant animals
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.

This is important to the dairy
producer because...
 You
are looking for rations that will support
higher milk production levels.
 You are being forced to place more
emphasis on milk protein production.
 With high feeding costs and low milk
prices, you are trying to meet the N needs
of ruminal fermentation and the AA
requirements with a minimum
concentration of CP in the diet.
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The goal should be...
 To
provide the appropriate quantities and
balance of AA’s to the intestine for
utilization keeping in mind both the needs
of the animal and the economics of the
situation.
i.e. Increase UIP or increase microbial
protein. Which is more economical?
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Optimizing Protein and AA
Nutrition
 Maximize
dry matter intake (DMI)
– positive correlation between DMI,
microbial protein synthesis and AA flow
to the gut
– as DMI’s increase, the rate of passage of
digesta also increases
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Optimizing Protein and AA
Nutrition
 Maximize
Microbial Protein Synthesis
– a combination of high rumen available
carbohydrate with high rumen available
protein will maximize bacterial protein
production and hence maximize rumen
microbial protein delivery to the lower
gut.
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Defaunation (protozoal removal)

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


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.
Chemistry of the Maillard reaction between reducing sugars
and lysine residues during heat treatment of proteins
Rumen Protected Amino Acids
(RPAA)
 Commercial
products now exist which
allow nutritionist to raise intestinal levels of
methionine and lysine.
 Basically “by-pass” AA’s
– Some RPAA’s are more resilient than others
and will not degrade in the rumen
– Some RPAA’s will be more resistant to
mechanical and thermal stresses at the mill
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