Methods of estimating

microbial protein (MP)

synthesis

Dr A T Adesogan

Department of Animal Sciences,

University of Florida

Importance

Microbes allow ruminants to use diet fiber & NPN

The aa profile of MP is better than several dietary

protein sources

50 –75% of the aas 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 (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

Harvesting microbes

Rumen Fluid

Crude bacterial fraction

Pellet = protozoa +

heavy feed particles

Slow spin(1000g x 30 min)

(Davies, 2000)

The CP content of the bacterial

fraction is the MP

Pure bacterial

fraction

Fast spin

(10000g x 20 min)

Light feed particles

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)

Measuring MP with TCA

Procedure continued

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

– A fingerprint pattern in terms of amino acid

composition, DNA, lipid etc.

External markers

– A stable or radioactive isotope (N, C, P, S) that is

incorporated into microbial matter during growth.

(Merry, 2000)

Markers used for estimating

rumen microbial synthesis

RNA,DNA

Amino or fatty acid profiles

Phospholipids

Tritiated leucine

ATP

Sulphur 35

Phosphorus 32

Carbon 14

Nitrogen 15

(After, Merry, 2000)

haz

ard

ous?

Cost

ly D-alanine

Purine bases

DAPA/AEPA

Com

ple

x

Mo

st

com

monly

use

d

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

DAPA concentration changes

(Broderick & Merchen, 1992)

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

High post-ruminal flow of dissociated DAPA

overestimates MP estimates

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 / derivatives

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

Pu

rin

e c

ata

bo

lism AMP

IMP

Inosine

Hypoxanthine

Xanthine

Uric acid

Allantoin

Guanosine

AdenineGuanine

Adenosine

Purine

derivatives

Purine excretion product(Chen & Gomes, ’92)

Microbial RNA

Xanthine oxidase

Xanthine oxidase

Purine conc. (/g N) in certain

rumen microbes & feeds

Liquid assoc. bacteria 0.80

Particle assoc. bacteria 0.78

Mixed ruminal bact 0.90

Mixed ruminal protozoa 0.44

Soybean meal 0.17

Corn gluten meal 0.17

Blood meal 0.03

Fishmeal 0.67

(Broderick & Merchen, ’92)

Purine –derivative method – pros

Purine excretory products (allantion) in the urine (or

milk) are assumed to be of microbial origin

Allantoin can be estimated instead of using purines, or

nucleic acids, provided milk / urine can be

representatively sampled.

Benefits

1. Cheaper & less invasive than measuring purines

2. Less complicated than other marker methods

3. Can be analyzed with common equipment

(HPLC, spectrophotometer or autoanalyzer)

Purine method - Cons

1. Fishmeal purines not completely degraded in the rumen

2. Total urine collection problems vs. spot sampling

3. Species specific predictions of MP synthesis.

– Xanthine oxidase activity (high in cows but low in

sheep) determines whether hypoxanthine is degraded

into allantoin or salvaged into nucleic acids

4. Must account for

– Partitioning of allantoin b/w excretion in urine and

recycling (into milk, saliva etc)

– ‘Endogenous secretion’ of purine derivatives / excretory

products from tissue nucleic acids

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

In vivo marker methods (except purine derivatives)

require cannulation, measuring digesta flow etc

Representative sampling of bacterial protein is

problematic in vivo

Calculating microbial N flow and

efficiency

1. Bacterial N in effluent (g/d) = 15N residue ÷ 15N bacteria x effluent volume

2. OM digested (g/d) =

OM in feed (g/d) - OM in residue(g/d)

OM in feed

3. g N/ Kg OM Truly (or apparently) digested

(Davies, 2000)

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.

See chen reference for purine method on website

or in ans 6452 folder