proteins amino acids ammonia and urea

1. NATANGA GODFREY
2200702268
2022/HD02/2268U
ANS: 7204 Rumenology
“Utilization and conversion of nitrogenous
materials in the rumen”

2. PRESENTATION TOPICS
1. Utilisation and conversion of Proteins, ammonia, urea,
2. Rumen synthesis of amino acids,
3. Nature of microbial proteins,
4. Quantities of microbial N available to the host,
5. Thermodynamic limitations on protoplasmic synthesis
imposed by anaerobiosis,
6. N Pollution of the environment.
2

3. Utilisation and conversion of nitrogenous
material in the rumen
proteins
 Proteins are extensive chains of amino acids.
 All amino acids contain four basic components
linked to a carbon skeleton. The four basic
components are a hydrogen, an amine, a
carboxyl, and a side chain, commonly referred to
as the R group.
 The specific R group determines the specific
amino acid. All amino acids contain an amine,
therefore all proteins contain nitrogen.
3

4. Proteins cont….
 Individual amino acids are linked via a peptide
bond.
 The specific amino acid sequence and subsequent
linkages between components of the amino acids,
determine the chemical and physical properties of
the protein.
 Figure illustrates the complex
structure of a protein
 each color represents a specific atom such as
nitrogen, carbon, hydrogen, and oxygen, etc. 4

5. Proteins cont….
 The primary functions of proteins are to aid in structure,
movement, digestion, metabolism, growth, and defense.
Protein digestion in the rumen
5

6. DESCRIPTION OF THE ILLUSTRATION
 Dietary or feed proteins are divided into three
categories.
 The first category is rumen inert protein.
(Rumen inert protein is also known as rumen
undegradable protein (RUP) or undegradable
intake protein (UIP).)
 Rumen inert protein passes through the rumen
unchanged. As the protein continues to flow
through the GI tract, it is exposed to
mammalian secretions and enzymes in the
abomasum and small intestine. 6

7. CONT…..
The portion of the protein digested to
amino acids will be absorbed in the
small intestine.
The undigested portion will pass
through the remainder of the GI tract
and be excreted in the feces.
7

8. RUMEN DEGRADABLE PROTEIN
 The second category is the rumen degradable
protein (RDP) or degradable intake protein
(DIP).
 Rumen degradable protein is digested in the
rumen. Rumen microorganisms metabolize the
protein to ammonia (NH3).
 The rumen microbes use the ammonia to
synthesize microbial proteins.
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9. CONT……..
 As rumen fluid and materials, including the
rumen microbes, flow out of the rumen and
continue through the GI tract, the
microbial proteins will be available to the
animal.
 Microbial proteins will be digested in the
abomasum and small intestine and the
amino acids will be absorbed in the small
intestine.
 The undigested portion of the microbial
proteins is excreted in the feces. 9

10. Non protein nitrogen compound
 The third category is nonprotein nitrogen
compounds, often abbreviated NPN
compounds. Similar to rumen degradable
protein, microbes are able to metabolize
NPN compounds to produce ammonia and
use the ammonia for microbial protein
synthesis.
 Microbial protein synthesis is dependent
upon substrate availability.
10

11. Cont…..
 The two most important substrates are
nitrogen and energy. If the availability of
nitrogen as ammonia exceeds the synthesis
of microbial proteins, the ruminant will
absorb the ammonia and recycle the
nitrogen in the body.
 If the excess exceeds the ruminant’s ability
to recycle, the excess nitrogen will be
excreted in the urine.
11

12. Summary of protein digestion and
Utilisation
12

13. Protein Utilisation
 First insoluble protein is solubilized.
 Then, peptide bond of solubilized protein is cleaved
enzymatically by a variety of endo and exo proteases to
peptides and amino acids and absorbed rapidly by bacteria
 The bacteria then ferment mixtures of amino acids to form cell
material, ammonia, carbon dioxide, and acids.
 Single Amino Acids fermented by a few bacteria while
fermentation of Mixed Amino Acids may proceed more rapidly than
fermentations of single acids.
 This is because Some amino acids are more readily reduced
(hydrogen acceptors) and others more readily oxidized (hydrogen
donors). 13

14. Not all proteins are degraded in the rumen.
 Proteins that are not degraded by rumen microbes are
called escaped, “bypassed,” or “undegradable” (rumen
undegradable protein, RUP), and have a low rumen
degradation rates (e.g. proteins in corn).
RUP enters the abomasum and small intestine of the
ruminant animal for digestion and absorption.
 Proteins reaching the small intestine could be RUP or
those from microbial sources. The amino acid needs of the
host animal are met by RUP and microbial proteins.
 Both ruminants and monogastrics require the essential
amino acids in their diet, and amino acids cannot be
stored within the body, so a constant dietary supply is
necessary.
14

15. Bypass” Potential of Protein
Supplements:
 Among the cereal grains, corn has the highest bypass
potential.
 However, it should be noted that corn is deficient in
essential amino acids such as lysine and methionine.
 Animal protein sources such as fish meal and meat
meal have high bypass potential.
 Drying forages and heat treatment increases bypass
potential.
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16.  Feed processing methods, such as pelleting,
steam rolling. or flaking, tend to denature
the feed protein due to the generation of
heat, thereby “protecting” the protein
from lysis in the rumen.
 Rumen protected protein sources (through
formaldehyde treatment) that remain
intact in the rumen and dissolve in the
abomasum are commercially available.
16

17. Examples of protein bypass
17

18. Note that..
 Concentrations of soluble protein and amino acids in the
rumen are always very low
 Often undetectable except immediately after a meal
which indicates that soluble protein is degraded rapidly
 Some proteolytic bacteria cannot use amino acids but,
instead only use ammonia as a source of N.
 For such microbes, protein serves only as a source of
carbon and energy
18

19. 2. Ammonia Utilisation
 Ammonia in the rumen is a pool with several inputs and exits, and
with Levels varying from 0 to 130 mg/100 ml. It is derived from;
 Degradation of dietary protein
 Degradation of dietary NPN
 Hydrolysis of urea recycled to the rumen
 Degradation of MCP.
 Ammonia disappears from the rumen pool due to;
 Uptake by microbes
 Absorption through the rumen wall
 Flushing to the omasum
19

20. 20

21.  Ammonia fixation
 Ammonia -N is fixed to carbon by ruminal bacteria by two
enzymes, glutamine synthetase (GS) and glutamate
dehydrogenase (GDH).
 GS concentration is highest when extracellular ammonia N is low
while GDH is a constitutive enzyme which does not vary in
concentration
21

22.  At a higher ammonia concentration, uptake is primary via the
GDH system and at low ammonia concentration, the GS
pathway has a higher affinity for ammonia.
 GS requires one mole of ATP for each mole of ammonium ion
fixed while no ATP is used with GDH action.
 Hence, if ammonia concentration is low, efficiency of microbial
growth is reduced because ATP is diverted from growth to the
process of ammonia uptake.
Note:
Absorption of ammonia from the rumen is dependent on ammonia
N concentration and pH.
22

23.  Because the non-ionized ammonia is absorbed while the
ammonium ion is not, a lower ruminal pH automatically decreases
ruminal absorption of ammonia N.
 Feeding animals with a urea feed generally increases ruminal pH
which in turn increases ammonia absorption.
 Inhibit ammonia absorption by lowering rumen pH, for example a
vinegar dench is one treatment of ammonia toxicity but such action
is only useful prior to the onset of tetany.
23

24. 3. Utilization of Urea
 Urea is a non-protein N containing compound used by ruminants as an
ammonia source, obtained from feed and endogenous sources. It is hydrolyzed
by ureases from rumen bacteria to produce NH3 which is used for microbial
protein synthesis.
 Plasma urea enters the rumen by two routes; with saliva and by diffusion
through the ruminal wall
 Diffusing urea meets urease from adherent ruminal bacteria in the ruminal
epithelium, and is hydrolyzed to ammonia and CO2
 Urease, an enzyme that catalyzes the hydrolysis of urea, forming ammonia
and carbon dioxide
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25. Cont….
 Thus, splitting of urea to ammonia would be of value to an
organism only to supply ammonia to be used in growth
 Although urea in the blood stream is harmless, hydrolysis
yields ammonia which at high levels is toxic to all
mammals
 Ruminants first convert urea to ammonia and eventually
to microbial protein by those ureolytic bacteria.
 35% of rumen bacteria belong to the ureolytic species.
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26. Urea recycling
 It is significantly related to ammonia production and absorbed in the
gastro intestinal tract of ruminants.
 All ammonia absorbed from the rumen epithelium, small intestine
mucosa, and large intestinal mucosa travels via the portal vein to
the liver.
 Body tissue ammonia also enters the liver.
 Liver metabolism has a central role in the integration of body N
metabolism.
 Ammonia in the liver is detoxified by conversion to urea.
 Urea can be recycled directly into the rumen, small intestines or
large intestines
 It can then diffuse into the rumen through the rumen epithelia,
deposited in the saliva, excreted by the kidney or secreted in milk.
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27. 4. Rumen synthesis of amino acids
 Ruminants obtain amino acids (AA) from microbial protein
synthesized in the rumen and from feed proteins that escape
ruminal degradation.
 Synthesis of microbial protein provides a mechanism for
obtaining AA from NPN. Effectiveness of NPN utilization
depends upon production and utilization of ammonia by rumen
microbes.
 Organisms vary in their ability to synthesize the 20 common
amino acids. Most bacteria and plants can synthesize all 20.
Some simple parasites, such as the bacteria Mycoplasma
pneumoniae, lack all amino acid synthesis and take their amino
acids directly from their hosts. All amino acids are synthesized
from intermediates in glycolysis, the citric acid cycle, or the
pentose phosphate pathway. 27

28. Cont……
 Nitrogen is provided by glutamate and glutamine.
 Amino acid synthesis depends on the formation of the appropriate
alpha-keto acid, which is then transaminated to form an amino
acid.
 Amino acids are made into proteins by being joined together in a
chain by peptide bonds. Each different protein has a unique
sequence of amino acid residues.
 Proteins are made from amino acids that have been activated by
attachment to a transfer RNA molecule through an ester bond.
 Of the 22 amino acids naturally incorporated into proteins, 20 are
encoded by the universal genetic code and the remaining two,
selenocysteine and pyrrolysine, are incorporated into proteins by
unique synthetic mechanisms.
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29. Note
 pyrrolysine: An amino acid found in
methanogenic bacteria.
 selenocysteine: A naturally-occurring amino acid,
present in several enzymes, whose structure is
that of cysteine but with the sulfur atom replaced
by one of selenium.
 genetic code: The set of rules by which the
sequence of bases in DNA are translated into the
amino acid sequence of proteins
29

30. 5.Nature of the Microbial Protein
 Ruminal microbes generally contain between 20 and 60% of their dry
matter as crude protein.
 The rumen bacteria content varies averaging to 50% (+-5) crude protein.
 The nitrogen content of the protozoa is low as compared to the bacteria,
because of a higher polysaccharide content averaging 40% crude protein
with a range of 20 to 60%
 Considering that amino acids make up approximately 80% of the MCP
,
estimates of BV of MCP suggest that BV of the true proteins present in MCP
is near 100.
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31. Microbial protein
31

32.  Protozoa protein high in , lysine, leucine, isoleucine, and
phenylalanine
 Methionine and valine are slightly greater in the bacterial
protein
 Tyrosine, threonine, and histidine compose about the
same proportion in both.
 Overall, microbial protein tends to be high in lysine and
threonine and marginal in methionine relative to
requirements by animals for maintenance and growth
32

33. 6. Quantities of microbial N available to the
host
 Direct analyses indicate that more than half of the rumen nitrogen is in the
form of microbial cells , N is used in synthesis of all key constituents of the cell
such as AA, pyrimidines and purines, NAD, and amino sugars.
 Microbial N comprises about 40% of the non- ammonia N entering the small
intestine with high dietary protein levels,
 Some 60% with low protein diets and 100% with purified NPN supplemented
diets
 With lower protein diets or with more extensively degraded dietary sources,
the percentage of protein coming from MCP usually is limited by the amount
of some nutrient or energy (ATP) available for microbial growth
33

34. NOTE:
 Though protozoa and fungi are active in the
rumen, both MCP synthesis and outflow depend
primary upon bacteria
 Yet indeed, half of the MCP in the rumen can be
protozoal protein, yet as a proportion of the MCP
leaving the rumen, protozoal protein is usually
under 10%
34

35. 7. Thermodynamic limitations on protoplasmic
synthesis imposed by anaerobiosis
 Radiant energy converted to chemical energy during photosynthesis exists in
the form of synthesized carbohydrates
 And released only if oxygen is available to combine with the carbon of the
carbohydrate
35

36.  The energy released is essential to perform biological work more
importantly synthesis of new protoplasm
 food is rearranged into protoplasm, and part of the energy in the
food is stored in the chemical constituents of protoplasm, the
proteins, carbohydrates, lipids, and nucleic acids
 The amount of protoplasm synthesized depends on ATP and the
amount and nature of the food derivatives which can be built into
cells
 The utilization of feed constituents to form protoplasm poses
much the same problem for aerobes as for anaerobes
36

37.  Except that the availability of oxygen makes the energy
derivable from a carbohydrate substrate much greater
 Aerobic microorganisms can synthesize into protoplasm as
much as 60-70% of carbohydrate substrate,
 Whereas for most anaerobic bacteria the quantity is
usually of the order of 10%, rarely exceeding 20%.
 Anaerobiosis limits the extent to which food can be
synthesized into cell material
 Since the rumen is anaerobic, microbial synthesis is
significantly less than that possible under aerobic
conditions
37

38.  Because the ruminant is aerobic, the greater energy
supply gives it the potential for greater synthesis of
protein,
 But its digestive arrangement makes it dependent on the
protein synthesized in the anaerobic microbial metabolism
 Insofar as protein is concerned, the ruminant lacks the
alimentary features necessary to give the conversion
efficiencies characteristic of non ruminants
 The rumen anaerobiosis imposes a thermodynamic limit on
the extent of host protein synthesis
38

39. 8. N Pollution of the environment
 Livestock manure contains a high concentration of nitrogen
Around 80% of all nitrogen consumed by animals is excreted in
manure
 Methanogens ferment feed in the rumen and produce methane
gas
 Released through eructation, normal respiration, and small
quantities as flatus
 Methane is a greenhouse gas with a global warming potential 25
times higher than that of carbon dioxide over a 100-year time
horizon
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40. Thank you for your time…….
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