Detailed protein metabolism in ruminants by rumen microbes

2.  Rumen has evolved as their first digestive organ
potentially affords ruminants an efficiency of protein
nutrition that is not available to non ruminants .
 Protein is synthesized in the gut in the form of rumen
microorganism
 Necessary energy is derived from plant polysaccharides
such as cellulose and the nitrogen is derived from
ammonia and amino acids in the rumen
 This microbial protein is directly available for digestion
and absorption by the host animal and principle source of
amino acids

3.  Cow can maintain unimpaired milk production on diets
lacking protein and with cellulose as principle carbon
source and urea as the main nitrogenous nutrient
 Yet with intensive production system the nitrogen
metabolism is usually regarded as being insufficient
 Dietary protein is broken down much rapidly relative to
breakdown of the energy containing plant fibre results in
excess of ammonia production

4.  To capitalize on the microbial capacity to form protein
from ammonia by feeding non protein nitrogen
 To minimize protein breakdown in rumen and thereby
increase the escape dietary protein

5.  Bulk of ruminant feed are composed of as vast array of
nitrogenous compounds along with nucleic acid in
association with protein and nitrate and ammonia may be
present
 Nitrogenous input to the rumen fermentation are salivary
mucoprotein and urea, the latter enters both in saliva and
by diffusion through the rumen wall

7.  All interconversions are generally considered to occur by
the action of microbial enzymes
 Microbial protein the most abundant form of protein
leaving the rumen
 Endogenous urea will only be of quantative significance
for microbial protein synthesis when dietary protein is
low
 Ammonia is major product of catabolism and also main
substrate for microbial protein synthesis
 Ammonia overflow leads to inefficient nitrogen retention

8.  Protein degradation : soluble protein are broken down more
slowly than insoluble proteins depending on degree of secondary
and tertiary structure and cleavage of disulphide bonds enhances
the breakdown and similarly heavily cross linked molecules
 Introduction of artificial cross linking into proteins inhibiting their
hydrolysis
 Heating and formaldehyde treatments affecting both solubility
and cross linking thereby provide bypass protein to the lower tract
 However these treatments may impair the subsequent availability
of some amino acids notably lysine , cysteine and tyrosine

9.  Nature of the diet has a major influence on the
proteolytic activity of rumen content
 Fresh herbage promotes an activity up to nine times than
that of dry rations , higher soluble protein content of
herbage enriching for proteolytic bacteria
 Cereal diet also yield higher proteolytic activity than dry
forage because proteolytic rumen microorganism tends
to be amylolytic rather than cellulolytic
 Rumen proteolysis probably varies from protein to
protein and with the other constituents of the diet

10.  Holotrichs undergoes rapid endogenous protein
breakdown in the absence of an exogenous source of
nitrogen and ingest and digest rumen bacteria
 Many of entodenium including E.caudatum
Polyplastron multivesiculatum are also proteolytic
 Mixed protozoa had a much higher aminopeptidase
activity than rumen bacteria and a slight higher trypsin
like activity

11.  Main role of the protozoa is likely to be in hydrolysis
of particulate proteins of an appropriate particle
 Predatory activity of protozoa against rumen bacteria
is of enormous significance to bacterial protein
turnover in rumen
 In absence of protozoa bacterial protein turnover
varied from 0.3-2.7%, in presence the rate increased to
2.4-3.7% per hour
 Main role of protozoa is not to hydrolysis exogenous
protein , but in metabolizing bacterial protein with its
lysozyme activity

12.  Major proteolytic organism namely Ruminobacter
amylophilus, B. fibrisolvens and P. ruminocola
R.amylophilus does not require peptides or amino acids
for growth even if available, ammonia remains the
principle nitrogen source , with only a small proportion
of cell nitrogen derived from labelled protein , peptides

13.  Major role of these to break down the structural proteins
within cereals particles thereby exposing starch granules
to amylolytic attacks
 Most proteolytic activity is cell associated indeed, the
soluble activity that can be found in the cell free
supernatant fluid may have been largely displaced from
the main site of enzyme activity, namely exocellular
polysaccharides capsular material.

14.  Cell associated proteases can be liberated by gentle
shaking, this cell surface location results in a
mechanism of proteolysis whereby the substrate
protein adsorbs rapidly and irreversibly to rumen
bacteria as an integral part of the process.
 The activity of both P. ruminicola and Ruminobacter
amylophilus remain almost entirely cell associated
during growth and are released into medium largely as
the result of autolysis in stationary phase

15.  Hydrolysis of protein by rumen microbial enzymes
releases oligopeptides which further breakdown into
smaller peptides and finally into amino acids
 Peptides breakdown to amino acids must occur for the
amino acids to be incorporated into microbial protein, and
when there is sufficient energy available to fuel
biosynthesis
 When energy is unavailable or when the rate of peptide
breakdown exceeds the rate at which it is assimilated,
peptide catabolism contributes to excessive ammonia
production and poor nitrogen retention

16.  Most reports of the uptake of 14c labelled amino acids
and peptides until recently indicated that the mixed
microbial population preferentially incorporates
peptide rather than free amino acids
 Different protein are broken down at different rates
by the mixed ruminal population and hydrophobicity
of peptides determines their rate of breakdown

17.  The main features of amino acids sequences that
dispose some peptides to be more resistant than other
to degradation appears to be GLY-GLY, pro- X
residues at the N-terminus
 Peptides which survives for a prolonged time in
rumen fluid tend to be enriched in these amino acids
 Majority of peptidase activity in the rumen is amino
peptidase a property that confers on N-terminally
blocked peptides a high degree of resistance to
ruminal degradation

18.  Some of these peptides may survive degradation
because they are naturally N-formulated or acetylated
 Predominated aminopeptidase activity found in
mixed ruminal bacterial cleaves dipeptidase rather
than single amino acids from the peptide chain,
enzyme of this nature are classified as dipeptidyl
peptidasas

19.  Peptide breakdown in rumen fluid is a two step
process , dipeptidyl dipeptidases releasing dipeptides
from oligopeptides, followed by separate dipeptidases
which cleave the resulting dipeptides to amino acids
 Ciliates had a particularly high dipeptidase activity
and this may be their main contribution to peptides
metabolism

20.  Slowing peptide breakdown would help to decrease
the flux of protein nitrogen to ammonia in the rumen
and help to increase the efficiency of nitrogen
retention by the ruminant animal
 Ionophores such as monensin have been found to
cause peptides to accumulates in rumen fluid
 It is possible that the species present in rumen
microbial population alter in response to ionophores
causing a change in peptide metabolism

21.  The low carboxypeptidase activity in the rumen
means that peptides can be blocked effectively from
degradation by treatments with acetic anhydrides
 Acetylation could therefore convert rapidly degraded
peptides in food to undegraded dietary protein

22.  Metabolism of amino acids themselves is the next
stage in the metabolism of most of the constituents of
dietary protein
 Rate of uptake of peptides or amino acids into cells
might limit the rate of ammonia production
 Amino acids essential to the animals, lysine
phenylalanine, leucine and isoleucine, are broken
down at 0.2-0.3mmol per hour and valine and
methionine are most stable and protection may not be
necessary to deliver substantial amounts of
undegraded methionine to the abomasum

23.  The carbon skeletons arising from deamination give
rise to a variety of volatile fatty acids products which
contributes only to the maintenance energy of the
bacteria via coupled oxidation and reduction
 The dispose of reducing equivalents is usually
achieved by methanogenesis
 Carbon monoxide inhibit bacterial hydrogenase
results in inhibition of methane formation and
fermentation stoichiometry switched to a higher
propionate production

24.  Ammonia production also declines as a consequence of
inhibition of branched chain amino acids fermentation
 In cell extracts of rumen bacteria, the NADH/NAD+ ratio
was an important effector of branched chain amino acids
fermentation, with NAD+ being essential as an electron
acceptor
 When hydogenases was inhibited by carbon monoxide
the NADH/NAD+ ratio increased and amino acids
deamination declined

25.  The feed ionophores, monensin and lasalocid also
inhibited both methanogenesis and deamination
 Ciliate protozoa seem to have a significant role in
deamination and amino acids and ammonia being
produced mainly from glutamine, asparagine,
citrulline, arginine and ornithine
 Main products of amino acids catabolism by protozoa
are similar to those of bacteria i.e. short and branched
chain VFAs

26.  The inhibition of amino acids degradation is an
obvious objective for manipulation
 If the amino acids were not to pass undegraded from
the rumen, if they were to be incorporated directly
into microbial protein rather than be degraded to
ammonia and then resynthesized, the energy cost
would be saved
 Nutritional management to ensure the simultaneous
availability of energy and amino acids to rumen
fermentation is an obvious means of improving
nutrient utilization