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 conditions predict feed quality protein, fiber microbial ecology simulate 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 flask simple to excruciatingly complex medium buffer, substrate, other nutrients gas phase

flask Glass is best Hard plastic Not red rubber, silicone tubing

buffers Variations on a theme Bicarbonate, phosphate Reducing agents Weller & Pilgrim, Burroughs, Goering & Van Soest, Menke, McDougall etc. Bicarbonate, phosphate pH 6.7 to 6.8 ?? Reducing agents

Anaerobiosis redox potential, analogous to pH O2 soluble in water Eh in rumen = -300 to 350 mV 10-56 molecules O2/L Copper column O2 soluble in water Boiling, bubbling with O2 free gas Oxidized redox cmpds are toxic Resazurin 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 F Decline Negative

Microbial growth lag phase log phase stationary phase variable with inoculum size, growth phase, media log phase highly reproducible, no substrate limitation stationary phase unbalanced growth, no DNA or net RNA synthesis, smaller cells

Batch culture pure culture studies prediction of feed digestibility Tilley & terry Goering & van Soest Menke, gas production

Tilley & Terry (1966) McDougall’s buffer 2 stage process DM digestion 48 h rumen liquor, 48 h pepsin DM digestion

Goering & Van Soest (1970) Modified Tilley & Terry 2 step More complete medium Reducing agent 2 step “true digestibility”

Gas production Abou Akkada, Menke,Pell, European groups, Iwaasa Gas production is proportional to fermentation Dependent on pH Vent 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 rate growth 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