Proteins

Proteins are the most common nitrogenous materials in forages; in nature proteins
provide the major part of the nitrogen used by rumen microbes and the host. Plant
proteins are partly soluble, but there are also relatively insoluble proteins, some
associated with the plant fiber

First step is digestion

Dietary protein is subject to degradation in the rumen and only variable proportion
will escape to the small intestines. Protein hydrolysis in the rumen is a multi-step
process. First insoluble protein is solubilized. Next, the peptide bond of solubilized
protein is cleaved enzymatically by a variety of endo and exo proteases to peptides
and amino acids are absorbed rapidly by bacteria and used as such or deaminated.
Concentrations of soluble protein and amino acids in the rumen are always very
low and often undetectable except immediately after a meal. This indicates that
soluble protein is degraded rapidly
However, 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

Second step is fermentation of amino acids

Bacteria ferment mixtures of amino acids to form cell material, ammonia, carbon
dioxide, and acids.

Single Amino Acids

A few rumen bacterial species are able to ferment single amino acids.
Mixed Amino Acids
Fermentations of mixtures of amino acids may proceed more rapidly than
fermentations of single acids. Some amino acids are more readily reduced
(hydrogen acceptors) and others more readily oxidized (hydrogen donors).

B. utilization of Ammonia

Most ruminal bacteria can use ammonia N as a source of N though some species
require additional N compounds (intact protein or carbon chains of certain amino
acids) for most efficient rapid growth. Bacteria actively absorb ammonia N while
protozoa do not.
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.
Changes in any of the above factors will alter ammonia concentration in the rumen.
Ammonia absorption increases as ruminal concentration increases. Ammonia
toxicity often occurs when ruminal ammonia concentration is 100mg/dl. No
ammonia peak concentrations will be detected with low protein diets.
Rate of digestion and feed intake are reduced with low ruminal ammonia
concentration due to starvation of ruminal bacteria for ammonia

Ammonia fixation

Ammonia -N is fixed to carbon by ruminal bacteria by two enzymes, glutamine

synthetase (GS) and glutamate dehydrogenase (GDH). The concentration of GS is
highest when extracellular ammonia N is low while GDH is a constitutive enzyme
(not subject to metabolic control) which does not vary in concentration. Hence at a
higher ammonia concentration, uptake is primary via the GDH system, but 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.
Absorption of ammonia from the rumen is dependent on ammonia N concentration
and pH. 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 urea generally increase ruminal pH which in turn increases ammonia
absorption. Inhibiting ammonia absorption by lowering rumen pH as with, for
example a vinegar dench is one treatment of ammonia toxicity but such action is
only useful prior to the onset of tetany
C. Utilization of Urea
Plasma urea enters the rumen by two routes; with saliva and by diffusion through
the ruminal wall. Transfer across the rumen wall occurs by attenuated diffusion.
When urea diffusing from the blood stream into the ruminal tissue meets urease
from adherent ruminal bacteria in the ruminal epithelium, it is hydrolyzed to
ammonia and CO2. Thus, splitting of urea to ammonia would be of value to an
organism only to supply ammonia to be used in growth.
A high concentration of urease is not necessary since this enzyme is extremely
active .10 to 15% of the bacteria which adhere to the ruminal wall produce urease.
Urease in the ruminal contents is presumably due to sloughing of epithelia cells
which carry adherent bacteria, otherwise, no urease is produced by animal tissue
Although urea in the blood stream is harmless, hydrolysis yields ammonia which at
high levels is toxic to all mammals

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
arrange of 20 to 60%

Nutritive quality of microbial protein

Considering that amino acids make up approximately80% of the MCP, estimates
of BV of MCP suggest that BV of the true proteins present in MCP is near 100.

Of the amino acids required by the ruminant, lysine, leucine, isoleucine, and
phenylalanine appear to occur in slightly greater concentration in protozoal protein.
Methionine and valine are slightly greater in the bacterial protein, and 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

F. Quantity of Microbial Nitrogen Available to the Host

Direct analyses indicate that more than half of the rumen nitrogen is in the form of
microbial cells.
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.
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%

Thermodynamic Limitations on Protoplasmic Synthesis Imposed by

Anaerobiosis

The radiant energy converted to chemical energy during photosynthesis is usually

stated as existing in the form of the synthesized carbohydrates

energy + C02 + H20 -> CH20 + 02

The energy stored in carbohydrate is released only if oxygen is available to

combine with the carbon of the carbohydrate, or more accurately speaking, with
the hydrogen which can be removed.

The energy released is essential to perform biological work, of which the synthesis
of new protoplasm is of primary importance.

In the process of growth, the 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. Part of the energy of the food is
dissipated as heat. The heat generated in metabolism represents the loss of energy
unavoidable during the conversion of substrate molecules into cell constituents.

The amount of protoplasm which can be synthesized depends on two things, ATP
and the amount and nature of the food derivatives (intermediates easily synthesized
into monomers) which can be built into cells. In many cases the energy-yielding
material and the material transposed into cells are the same. For example, the
fermentable substrates serve not only as a source of energy, but also as the source
of the chemical compounds built into the microbial cells.
The utilization of feed constituents to form protoplasm poses much the same
problem as for anaerobes, 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. 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. Since this anaerobic synthesis is small, the synthesis by the
aerobic host must be small. Insofar as protein is concerned, the ruminant lacks the
alimentary features necessary to give the conversion efficiencies characteristic of
nonruminants. The rumen anaerobiosis imposes a thermodynamic limit on the
extent of host protein synthesis. The thermodynamic limitation on the possible
extent of host protein synthesis has as its corollary an excess of energy-yielding
materials available to the host.

N pollution to the environment

Methane (CH4) is the main greenhouse gas produced by grazing systems. Ruminant
livestock have microbes in the rumen called methanogens that ferment feed in the
rumen and produce methane gas that is released through eructation, normal
respiration, and small quantities as flatus. Methane as a greenhouse gas (GHG) has
a global warming potential 25 times higher than that of carbon dioxide over a 100-
year time horizon and it is the most abundant organic gas in the earth’s
atmosphere).
Livestock manure (dung and urine) contains a high concentration of nitrogen.
Around 80% of all nitrogen consumed by animals is excreted in manure

Protein Digestion: Ruminants

Protein digestion in the ruminant animals can be divided into two phases: (1)
digestion (degradation) in the reticulorumen and (2) digestion in the abomasum
and small intestine. Therefore, in ruminant animals, dietary proteins are classified
as rumen degradable and rumen undegradable proteins.
Like monogastric animals, the main goal for protein supplementation is to provide
amino acids to the animal. However, in ruminants, proteins serve as a source of
nitrogen for rumen microbes so they can make their own microbial protein from
scratch. Microbes do not “care” where the nitrogen sources come from and can use
nonprotein nitrogenous substances such as urea for microbial protein synthesis.
Urea is 100% degradable in the rumen by microbial urease (can be toxic at higher
levels).
Protein entering the rumen may be degraded by both bacteria and protozoa, which
produce proteolytic enzymes. The rumen microbes provide proteases and
peptidases to cleave peptide bonds in polypeptides to release the free amino acids
from proteins. Several factors such as solubility and the physical structure of
protein can affect rumen degradation. These rumen-degraded amino acids release
NH3 and the C skeleton by a process called deamination. Along with volatile fatty
acids (from carbohydrates), rumen microbes synthesize their own microbial
protein, which serves as a primary source of protein to the host ruminant animals.
Microbial protein is enough for maintenance and survival but not for high-
producing animals. Ammonia absorbed from rumen is converted to urea and
secreted into the blood as blood urea nitrogen (BUN). Urea can be filtered and
recycled to the rumen via saliva or through the rumen wall. The concentration of
BUN in ruminants reflects the efficiency of protein utilization.
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.

Research on “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. 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.

For ruminants, protein nutrition is more complex. Figure 2.8 illustrates protein
nutrition in a ruminant.

As illustrated, 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. 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. 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. 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. 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. From there, the digestion of microbial protein is as stated above for
RDP. Microbial protein synthesis is dependent upon substrate availability. The two
most important substrates are nitrogen and energy. If the availability of nitrogen as
ammonia exceeds the synthesis of microbial proteins, as illustrated in Figure 2.8,
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.