In vitro techniques

In vivo Techniques

In vivo= in life

Fistula = a hole

Cannula = a device

Ruminant cannula in: esophagus,rumen, abomasum, duodenum, ileum, cecum

Non ruminant cannula in: duodenum, ileum, cecum

Why do you want to use an in vitro technique ?

count bacteria

microbial metabolism and growth

simulate rumen conditionspredict feed quality

protein, fibermicrobial ecologysimulate rumen digestion

Rumen in vitro techniques

The use of an artificial system to mimic a natural dynamic microbial ecosystem

Always a trade-off between simplicity and precision of mimicry

Types of in vitro systems

batch culture

fed batch culture

semi-continuous culture

continuous culture

In vitro system components

flasksimple to excruciatingly complex

mediumbuffer, substrate, other nutrients

gas phase

flask

Glass is best

Hard plastic

Not red rubber, silicone tubing

buffers

Variations on a themeWeller & Pilgrim, Burroughs, Goering &

Van Soest, Menke, McDougall etc.

Bicarbonate, phosphatepH 6.7 to 6.8 ??

Reducing agents

Anaerobiosis

redox potential, analogous to pHEh in rumen = -300 to 350 mV10-56 molecules O2/LCopper column

O2 soluble in waterBoiling, bubbling with O2 free gas

Oxidized redox cmpds are toxicResazurin at 0.00001%

Reducing agents

Resazurin (blue) resorfol (pink) resorfol (pink) resorfol (clear), -.042 mV

cysteine-HCl cystine, -340 mV

dithiothreitol, -330 mV

sulfide s, -571 mV

titanium citrate, -430 mV

ascorbic acid, -320 mV

Microbial growth

Growth & death of microbes

Section Phase Growth rate

A Lag Zero

B Acceleration Increasing

C Exponential Constant

D Retardation Decreasing

E Maximum stationary Zero

F Decline Negative

Microbial growth

lag phasevariable with inoculum size, growth phase,

media

log phasehighly reproducible, no substrate limitation

stationary phaseunbalanced growth, no DNA or net RNA

synthesis, smaller cells

Batch culture

pure culture studies

prediction of feed digestibilityTilley & terryGoering & van SoestMenke, gas production

Tilley & Terry (1966)

McDougall’s buffer

2 stage process48 h rumen liquor, 48 h pepsin

DM digestion

Goering & Van Soest (1970)

Modified Tilley & TerryMore complete mediumReducing agent

2 step “true digestibility”

Gas production

Abou Akkada, Menke,Pell, European groups, IwaasaGas production is proportional to

fermentationDependent on pHVent or no-vent ?

In Vitro Gas System – Pressure Transducer

Fed batch

not commonly used

keep organism at or near logarithmic growth for extended periods

particularly good for slow growing organisms, co-cultures

Continuous culture

maintain bacteria at exponential growth for extended periods

growth rate proportional to limiting nutrient addition rate flow rategrowth rate proportional to dilution rate

until critical dilution rate

Semi-continuous culture

more rumen-like than continuous

solid substrates

kinetics more complicated

substitute for cannulated cows

Nakimura & Kurihara

system for protozoa

dialysis membrane

2.3 l volume

90 g/d

Nakimura & Kurihara

Slyter et al.

system for ruminal digestion

simple

500 ml volume

Up to 2.5 volumes/d

40 g/d

Slyter et al.

Rusitec

feed in two bags

1000 ml volume

0.8 to 1.5 volumes/d

24 g dm/d

Rusitec

Hoover et al.

differential flow rates

500 ml volume

up to 3.2 volumes/d

80 to 160 g/d

Hoover et al.

Teather & Sauer

700 ml volume

1.6 volumes/d

30 g DM/d

Designed to maintain protozoa, study rumen ecology

Continuous culture kinetics

Logarithmic growth