In vitro techniques

In vivo TechniquesIn vivo= in lifeFistula = a holeCannula = a deviceRuminant cannula in: esophagus,rumen, abomasum, duodenum, ileum, cecumNon ruminant cannula in: duodenum, ileum, cecum

Why do you want to use an in vitro technique ?

count bacteriamicrobial metabolism and growthsimulate 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 systemsbatch culturefed batch culturesemi-continuous culturecontinuous culture

In vitro system componentsflasksimple to excruciatingly complex

mediumbuffer, substrate, other nutrients

gas phase

flaskGlass is bestHard plasticNot red rubber, silicone tubing

buffersVariations on a themeWeller & Pilgrim, Burroughs, Goering &

Van Soest, Menke, McDougall etc.

Bicarbonate, phosphatepH 6.7 to 6.8 ??

Reducing agents

Anaerobiosisredox 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 agentsResazurin (blue) resorfol (pink) resorfol (pink) resorfol (clear), -.042 mV

cysteine-HCl cystine, -340 mVdithiothreitol, -330 mVsulfide s, -571 mVtitanium citrate, -430 mVascorbic acid, -320 mV

Microbial growth

Growth & death of microbesSection Phase Growth rate

A Lag Zero

B Acceleration Increasing

C Exponential Constant

D Retardation Decreasing

E Maximum stationary Zero

F Decline Negative

Microbial growthlag phasevariable with inoculum size, growth phase,

medialog phasehighly reproducible, no substrate limitation

stationary phaseunbalanced growth, no DNA or net RNA

synthesis, smaller cells

Batch culturepure culture studiesprediction of feed digestibilityTilley & terryGoering & van SoestMenke, gas production

Tilley & Terry (1966)McDougall’s buffer2 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 productionAbou Akkada, Menke,Pell, European groups, IwaasaGas production is proportional to

fermentationDependent on pHVent or no-vent ?

In Vitro Gas System – Pressure Transducer

Fed batchnot commonly usedkeep organism at or near logarithmic growth for extended periodsparticularly good for slow growing organisms, co-cultures

Continuous culturemaintain bacteria at exponential growth for extended periodsgrowth rate proportional to limiting nutrient addition rate flow rategrowth rate proportional to dilution rate

until critical dilution rate

Semi-continuous culturemore rumen-like than continuoussolid substrateskinetics more complicatedsubstitute for cannulated cows

Nakimura & Kuriharasystem for protozoadialysis membrane2.3 l volume90 g/d

Nakimura & Kurihara

Slyter et al.system for ruminal digestionsimple500 ml volumeUp to 2.5 volumes/d40 g/d

Slyter et al.

Rusitecfeed in two bags1000 ml volume0.8 to 1.5 volumes/d24 g dm/d

Rusitec

Hoover et al.differential flow rates500 ml volumeup to 3.2 volumes/d80 to 160 g/d

Hoover et al.

Teather & Sauer700 ml volume1.6 volumes/d30 g DM/d Designed to maintain protozoa, study rumen ecology

Continuous culture kinetics

Logarithmic growth