In vitro Techniques

• Simulation of the rumen fermentation

• Alternative to time consuming and expensive in vivo trials

Q tifi ti f th d d t f f t ti d/• Quantification of the end products of fermentation and/or rumen dry matter digestibility

End products of fermentation

• Gases (CO and CH )• Gases (CO2 and CH4)

• Short chain fatty acids (acetate, propionate, butyrate, valerate, isobutyrate, isovalerate)

• Ammonia

• Microbial protein

In vitro Incubation Systems

Short time in vitro batch culture systems- Tilley and Terry System (determination of digestibility)- Hohenheim Gas Test (determination of gas production)- Reading Pressure Technique (determination of gas

production)

Continuous culture systems- Rumen Simulation Technique (RUSITEC)- Dual Flow Continuous Culture System (Hoover, 1964)

H h h i S t (Si l Fl C ti C lt- Hohenheim System (Single Flow Continuous Culture System)

Collection of Rumen Fluid

f d t

gas phase

feed mat

liquid phase

The Tilley and Terry Method

PepsinIn vitroi b ti Pepsin

digestion37°C20.000 g

4°C, 10 min

incubation48 h

A t di tibilit

True digestibility

Apparent digestibility

The Hohenheim Gas Test (HFT)

hours

In vitro incubation

0 2418126

RumenRumenfluid

Gas production

F dFeedCorrect for Blank and for Standard GP

ME (MJ/kg) =2.2 * 0.136GP + 0.057CP + 0.0029CL2

Digestibility (%DOM) = 14.88 * 0,146GP + 0.45CP +0.65CA

Determination of in vitro Digestibility from the HFT

I itNeutral

DetergentSolution

Filtering50 µm

In vitroincubation

Solution100°C

~ 50 µm pore size

20.000 g4°C, 10 min

Apparent digestibilityMicrobial biomass

True digestibilityMicrobial biomass

End Product Quantification

Volume & Gas chromatographyGases

Neutral detergent

Microbial markerMicrobial biomass

Neutral detergent solution

Undigested feed

G h t hShort chain Gas chromatographyShort chain fatty acids

Relationship between SCFA and Gas Production

Gases

Glucose = 2 acetate + 2 CO2 + 8 H

Glucose = 1 butyrate + 2 CO2 + 4 Hy 2

Glucose + 4 H = 2 propionate

Short chain

CO2 + 8 H = CH4 + 2 H2O

fatty acids

The Concept of the Partitioning of Nutrients

Gases

Microbial biomass

Undigested feed

Feed 1 Feed 2Short chain f tt idfatty acids

Microbial Biomass Marker

Internal Marker External Marker

Cell components Stable or radioactive Isotopes

• Amino acids

Isotopes

• 15 NH4+

• Nucleic acids

• Phospholipids

• 32 PO42-

• 35 SO42-

Microbial marker

15N UreaNucleic acids

0.7

0.8

12

14

0.5

0.6

8

10--PEG

0.3

0.4mg

--4

6

8

mg

0 0

0.1

0.2 --PEG

0

2

4

0.00 6 12 18 24 30 36 42 48

time (h)

00 6 12 18 24 30 36 42 48

time (h)

Reading Pressure Technique (RPT)

Pressure Transducer

Receiver

G hGas phasePressure:

0 – 700 mb

Signal-converter

Incubation medium

Rate of Gas Production

30

25

ml/h

)

15

20

uctio

n (m

arabinose

10

as p

rodu D-glucose

maize starchll l

0

5G cellulose

0 12 24 36 48 60 72 84 96time (h)© University of Reading, Dr. F. Mould

Fermentation of Individual Feed Components

20TMR 1 TMR 2

Components g/kg g/kg

15l/h)

Components g/kg g/kggrass silage 496 499maize silage 367 369

10

15

ctio

n (m

l gmilled wheat 40 65rapeseed meal 37 37

10

s pr

oduc soyabean meal 30 30

cane molasses 30 -

5

Gas

00 12 24 36 48 60 72 84 96

© University of Reading, Dr. F. Mould time (h)

Kinetics of Gas Production

Gas production is described by: Gas prod. = A + B*(1-exp(-c*t))

120,0

Barley straw:

Gas = -13.7 + 127.6 *(1-exp(-0.0428*t))80,0

100,0

( p )

Sesbania sesban leaves40,0

60,0ml

Barley strawSesbania sesban

Gas = -12.3 + 104.3 *(1-exp(-0.0955*t))0,0

20,0

0 12 24 36 48 60 72 84 96

time (h)

Production of SCFA and Microbial Activity

SCFA concentration RNA concentration

60 0

70,0

SCFA concentration

200,0

RNA concentration

40,0

50,0

60,0

ml 120,0

160,0

ml

20,0

30,0

,

µM /

Barley strawS. sesban 40 0

80,0µg /

0,0

10,0

0 12 24 36 480,0

40,0

0 12 24 36 480 12 24 36 48time (h)

0 12 24 36 48time (h)

In vitro Quantification of the Effects of Tannins

Gas production

Feed Feed PEG(Polyethylene(Polyethylene

glycol)

Tannin Effect on Gas Production

160

120

140

160

80

100

120

ml

40

60

80m

--

0

20

40PEG

0 6 12 18 24 30 36 42 48

time (h)( )

Corrections to be made

Blank In all batch culture systems a blank is needed to account f th b t t th t l ith th fl idfor the substrate that comes along with the rumen fluidBlank gas production is subtracted from substrate gas production

Standard Hohenheim Gas TestHay mixture with a known 24h gas productionHay mixture with a known 24h gas production (calibrated)

The ratio of calibrated to measured gas production ofThe ratio of calibrated to measured gas production of the substrate is used to correct the gas production from the test substrates of the respective incubation for differences in microbial activityfor differences in microbial activity

Implications

+ Short time in vitro incubation systems offer the possibility to quantify the kinetics and absolute amounts of end product q y pformation

+ Partitioning of nutrients for the host animal can be determined

+ Rate and extent of the substrate degradation provides information about the potential intake of the substrate examined

+ They also can be used to assess the antinutritional effects

Influence of host animal missing !- Influence of host animal missing !

- No adaptation of the microbial community to the tested feeds !

Continuous in vitro Systems

Buffercontinuous buffer inflow simulates the saliva flow

substrate administration

39°C

Rumen fluid

Overflow

overflow is collected for analysis of end products of fermentationOverflow

RUSITEC

B ff

RUmen SImulation TEChnique

Buffer • Substrate administration in nylon bags with defined pore sizes

• Substrate is completely removed and replaced at different time intervals

Semi contin o s in itro fermentation s stem

39°C

⇒ Semi continuous in vitro fermentation system

Feed - no access of protozoa to Feedfeed particles

Stirrer

Overflow

Single flow continuous in vitro system

B ffBuffer

MotorF d

39°CMotor

FeederControl unit

pH

Gasbag

Stirrer

pH

O fl

St e

• Substrate added directly to rumen fluid- no selective absorption

of SCFAOverflow• Substrate added directly to rumen fluid

• Formation of a feed mat

• Full access of larger microorganisms (protozoa) to the substrate

of SCFA

Single flow continuous in vitro system

Single flow continuous in vitro system

pH-value fermenter 1-6 F1F2

77.25

7.57.75

F2F3F4F5F6

66.25

6.56.75

7

5.255.5

5.756

4.755

25.N

ov

26.N

ov

27.N

ov

28.N

ov

29.N

ov

30.N

ov

01.D

ec

02.D

ec

03.D

ec

04.D

ec

05.D

ec

06.D

ec

07.D

ec

08.D

ec

09.D

ec

10.D

ec

11.D

ec

• No selective absorption of SCFA• Lack of the bacterial flora adherent to the rumen wall• No method to mimic rumination• No selective removal of small particles as in the rumen

Summary

Sh t ti i it i b ti t i i thShort time in vitro incubations systems can mimic the rumen fermentation to a certain extent and are therefore a good

supplement to the estimation of feeding value by substrate compositioncomposition.

Interpretation of data however has to be done very carefully because of the influence of the incubation time on the results.because of the influence of the incubation time on the results.

Continuous in vitro incubation systems can be run on a steady state and allow the examination of adaptation of microbial communities

to feeds. But they are time consuming and laborious.

In vivo trials are the only reliable method to determine the feeding value of different substrates, but they are laborious and expensive.y

In vivo trials

Parameters for the nutritive value

Animal performance

Growth

Substrate parameter

Digestibility • Growth

• Milk, wool or egg production

• Reproduction

• Digestibility

• Energy content• Reproduction

• Animal health

In situ digestibility trial: The Nylon Bag Technique

Feed 1 Feed 2

+ Feeding more than one feed to an animal at the same time

+ conditions close to in vivo

- Fistulated animals required- Influence of pore size:

access of protozoa and bacteria, access o p oto oa a d bacte a,small particles escape

- Postrumen degradability

In vivo Production Trial In vivo digestibility trial: Monogastrics

Glucose, Lipids GasEndogenousGlucose, Lipids

& Amino acidsEndogenouscomponents

Enzymaticdigestion

Microbialfermentation

SCFAMicroorganisms

Apparent digestibilityby mass difference

Nitrogen flows in monogastric animals

A i idAmino acids Urea-N

Dietary-N Undig. Diet-NMicrobial-N

Di t N U f Di t N

Urine-N

Dietary-N Unferm. Diet-NMicrobial-N

In vivo Digestibility Trial: RuminantsGas

Endogenous components

Mi bi lGas

Endogenous components

Microbial fermentation

Microbial fermentationEnzymatic

digestion

SCFASCFA Glucose, Lipids & Amino acids

Microorganisms

Apparent digestibilityby mass difference

Balance Studies

CO & CH CO2 & CH4Measure Heat Energy

CO2 & CH4 2 4

Basic metabolic rateDigestion, absorption,

i ( i fexcretion (maintenance of body functions)

Measure Faeces & UrineMeasure Feed Intake& Energy Content

Measure Faeces & UrineExcretion & Energy Content

Balance Studies in Metabolic Cages

Urine collection

Faeces collection