Microbial protein synthesis and its estimation in ruminants

Protein metabolism
• Proteins provide the amino acids for vital functions,
reproduction, growth and lactation.
• Non-ruminant animals need pre-formed amino acids in their
diets, but ruminants can utilize many other nitrogen sources
because of their rare ability to synthesize amino acids and
protein from non-protein nitrogen sources.
• In addition, ruminants possess a mechanism to spare nitrogen.
When a diet is low in nitrogen, large amounts of urea (from
urine) return in the rumen where it can be used by the
microbes.
• In non-ruminants, urea is always entirely lost in the urine.
• It is possible to feed cows with diets containing non-protein
nitrogen as the only nitrogen source

PROTEIN TRANSFORMATION
IN THE RUMEN
 Feed proteins degraded by microorganisms in the rumen via
amino acids into ammonia and branched chain fatty acids .
 Non-protein nitrogen from the feed and the urea recycled
into the rumen through the saliva or the rumen wall
contribute also to the pool of ammonia in the rumen.
 Too much ammonia in the rumen leads to wastage, ammonia
toxicity, and in extreme cases, death of the animal. The
bacterial population uses ammonia in order to grow.

 use of ammonia to synthesize microbial protein is dependent upon the
availability of energy generated by the fermentation of carbohydrates.
 On the average, 20 grams of bacterial protein is synthesized per 100 grams
of organic matter fermented in the rumen.
 Bacterial protein synthesis may range from less than 400 g/day to about
1500 g/day depending primarily on the digestibility of the diet.
 The percentage of protein in bacteria varies from 38 to 55% .
 A portion of the dietary protein resists ruminal degradation and passes un
degraded to the small intestine.
 Forage proteins degraded 60 to 80% ,Concentrates or industrial by-
products degraded 20 to 60%.Major part of the bacterial protein flows to
the abomasum attached to feed particles.
 The strong acids secreted by the abomasum stop all microbial activity and
the digestive enzymes start breaking down the protein into amino acids.

• Amino acids produced about 60% from bacterial protein, 40% from un
degraded dietary protein.
• The amino acid composition of bacterial protein is relatively constant
regardless of the composition of dietary protein.
• All amino acids, including the essential ones are present in bacterial protein in
proportion that is fairly close to the proportion of amino acids required by the
mammary gland for milk synthesis.
• The conversion of dietary protein to bacterial protein is usually a beneficial
process. The exception occurs when high quality protein is fed

PROTEIN IN FECES
1. Undigested protein about 80% of the protein reaching the
small intestine is digested, 20% goes into the feces.
2. Digestive enzymes secreted into the intestine also major source
of nitrogen in the feces
3. Fecal metabolic protein the rapid replacement of intestinal cells
(On the average, for every increment of1 kg of dry matter
ingested by the cow, there is an increase of 33 g of body protein
lost in the intestine and excreted in the feces.
Ruminant feces rich in nitrogen (2.2 to 2.6% N) as compared
to the feces of non-ruminant animals.

LIVER METABOLISM AND
UREARECYCLING
• Not all the ammonia produced in the rumen may be converted to
microbial protein.
• Excess ammonia cross the ruminal wall and is transported to the
liver.
• The liver converts the ammonia to urea which is released in the
blood. Urea in the blood can follow two routes:
1) Return to the rumen through the saliva or through the rumen
wall.
2) Excreted into the urine by the kidneys.
• When urea returns to the rumen, it is converted back to ammonia
and can serve as a nitrogen source for bacterial growth.

• Urea excreted in the urine is lost to the animal. With rations
low in crude protein, most of the urea is recycled and little is
lost in the urine.
• However, as crude protein increases in the ration, less urea is
recycled and more is excreted in the urine.

MILK PROTEIN SYNTHESIS
• The mammary gland needs large amounts of amino acids to
synthesize milk protein.
• The metabolism of amino acids in the mammary gland is
extremely complex.
• Amino acids may be converted into other amino acids or
oxidized to produce energy.
• Most of the amino acids absorbed by the mammary gland are
used to synthesize milk proteins.
• Milk contains about 30 g of protein per kg, but vary between
cows /breed and among breeds.

Milk Protein Composition
• About 90% of the protein in milk is casein.
• There are many types of casein and they contribute to the high nutritive
value of many dairy products.
• Whey proteins are also synthesized from amino acids in the mammary
gland.
• The enzyme alpha-Lactalbumin is essential for the synthesis of lactose
and alpha−lactoglobulin is important in curd formation during cheese
production.
• Some proteins found in the milk (immunoglobulin) play a role in
immunity of newborn calf.
• The immunoglobulin's absorbed directly from the blood and not
synthesized within the mammary gland, so their concentration in the
colostrum is high.
• Milk also contains non-protein nitrogen compounds in very small
amount (e.g., urea: 0.08 g/kg).

Methods of estimating
microbial protein (MP)
synthesis

Importance
• Microbes allow ruminants to use diet fiber & NPN
• The amino acid profile of MP is better than several
dietary protein sources
• 50 –75% of the amino acid synthesised absorbed by
ruminants are from MP (AFRC, 1992)
• Hence, MP synthesis determines extent of forage CP
utilization by animal

Microbial yield expression
• Usually expressed as amount of microbial mass produced / amount of
substrate fermented / digested
• NRC
– Bacterial CP=130 g/kg TDN intake
• AFRC (1984)
– Microbial CP = 32 g /kg organic matter digested in the rumen
• AFRC (1999)
– Microbial CP = 9 to11g /MJ Fermentable metabolizable energy

MP estimation
Ideal methods must determine :
1. Post-ruminal protein flow
2.Proportion of post ruminal protein flow that is
microbial i.e. must separate microbial N from:
• Dietary UDP
• Endogenous N

Estimating MP synthesis in vivo
• Involves collecting duodenal fluid from fistulated cows & quantifying MP in the fluid
• However
– Opening cannulas affects digesta flow & fistulation limits replication
– Representative sampling can be challenging
– Method is difficult due to need for correction for dietary & endogenous N;
– Using purified or urea-based diets simplifies things

Using purified diets
1. Feeding protein-free diets
– Duodenal protein flows assumed to be microbial
2. Feeding urea + purified diets
– If urea is the only N source in the diet,
– Duodenal true protein / aa flow assumed to be microbial
Problems
– OK for ‘proof of principle’ studies but
expensive and practically unrealistic

Estimating MP synthesis in vitro
After in vitro fermentation / digestion, microbes are
harvested as shown on previous slide
Pros & cons
– Fistulation also required to supply rumen fluid
– Easier replication & representative sampling
– Some in vitro methods don’t account for ruminal
outflow

Methods for quantifying MP
1. Trichloro acetic acid (TCA) precipitation
2. Marker studies

1. Measuring MP with TCA
Principle
– TCA precipitates protein & small peptides in fluids
Procedure
1. Macerate feed with distilled water to release CP
2. Add TCA to precipitate CP, filter & analyze CP (= Sample TCA
CP)
3. Add TCA to buffered, rumen fluid with no sample,filter &
analyze CP (= Media TCA CP)

4. Incubate feed sample in buffered rumen fluid,
5. Add TCA to digestion residue + residual fluid
6. Centrifuge to separate solids from liquids & filter
7. Analyze CP in residue (= Residue TCA CP)
MP = TCA CP in residue
(TCA CP in sample + TCA CP in media)

Problems with the TCA method
 TCA will also precipitate dietary protein
 Can’t differentiate between dietary and microbial
protein

2. Measuring MP with markers
Internal markers- can be either
– Natural microbial component that is unique to
bacteria, protozoa or fungi e.g. DAPA, D Alanine
External markers
– A stable or radioactive isotope (N, C, P, S) that is
incorporated into microbial matter during growth.

Markers used for estimating
rumen microbial synthesis

DAPA (Diaminopimelic acid)
 DAPA = An amino acid in the peptidoglycan layer of the bacterial cell wall
 Rumen bugs have relatively constant DAPA:protein ratio
 Hence, duodenal MP flow can be estimated by comparing DAPA: protein
ratio in microbes to that in duodenal digesta
Assumes
– all DAPA flowing from rumen is bacterial in origin (not dietary or protozoal)
– The DAPA : protein ratio is constant

Problems with the DAPA method
 DAPA:protein ratio affected by:
– Identity, size & shape of bacteria
– Digesta component (higher in liquid associated bacteria)
– Time after feeding
 DAPA is highly metabolized & absent in some bacteria
 DAPA is present in soybean & silages
 Bacterial lysis may release DAPA from cell walls such that
>50% of DAPA is not cell bound

Nucleic acids
Principle
– High RNA conc. found in bacteria, hence RNA:total N ratio of
duodenal digesta can indicate MP synthesis
Cons
1. The ratio varies with:
• Bacterial growth rate
• Digesta component (higher in liquid-assoc. bacteria)
2. Requires cannulation, measuring digesta flow
3. Nucleic acids are present in feeds & tissues
4. Microbial nucleic acids can be degraded ruminally

Purine bases / derivatives
• Purine bases (adenine/guanine) are usually minimal / absent
in feeds but relatively abundant in microbes
• Hence can function as internal markers for MP synthesis
• Associated assumptions:
– Dietary purines are degraded by rumen bugs
– The bacterial purine: total N ratio is constant.

• Purine bases in the duodenal fluid are assumed to be
microbial in origin
• But purine bases may be degraded . in the SI,
• Hence conc. of purine derivatives or purine excretory
products (e.g. allantoin) are used instead of purines to
estimate MP synthesis.
• Using purine excretory products is most beneficial since it
avoids the need for fistulated animals

General problems with markers for
estimating MP synthesis
• Different markers give different results
• No standard, hence accuracy difficult to decide
• Lack of knowledge about proportions of different bacterial
types in doudenal digesta
• require cannulation, measuring digesta flow etc
• Representative sampling of bacterial protein is problematic in
vivo

,
References
• Dewhurst R, J, Davies D. R. and Merry, R J. 2000.
Microbial protein supply from the rumen. Animal Feed
Science and Technology. 85: 1-21
• Tamminga and Chen (2000). Animal-based techniques for
the estimation of the protein value of forages. In: Givens et
al., 2000. Forage Evaluation in Ruminant Nutrition. Pp.
215-235
• Broderick, G A and Merchen, N R., 1992. Markers for
quantifying microbial protein synthesis in the rumen. J.
Dairy Sci. 75:2618-2632
• Hespell and Bryant 1979 Efficiency of rumen microbial
growth : influence of some theoretical and experimental
factors on YATP J.Anim Sci 49:1640-59
• The Rumen Microbial Ecosystem (1997). Hobson and
Stewart, Blackie A and P, London
• Van Soest (1994) Nutritional ecology of the ruminant
• Introduction to rumen studies (1986) Czerkawski.

Microbial protein synthesis and its estimation in ruminants

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