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In vitro techniques. In vivo Techniques In vivo= in life Fistula = a hole Cannula = a device Ruminant cannula in: esophagus,rumen, abomasum, duodenum,

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Presentation on theme: "In vitro techniques. In vivo Techniques In vivo= in life Fistula = a hole Cannula = a device Ruminant cannula in: esophagus,rumen, abomasum, duodenum,"— Presentation transcript:

1 In vitro techniques

2 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

3 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

4 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

5 Types of in vitro systems batch culture fed batch culture semi-continuous culture continuous culture

6 In vitro system components flask simple to excruciatingly complex medium buffer, substrate, other nutrients gas phase

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

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

9 Anaerobiosis redox potential, analogous to pH E h in rumen = -300 to 350 mV molecules O 2 /L Copper column O 2 soluble in water Boiling, bubbling with O 2 free gas Oxidized redox cmpds are toxic Resazurin at %

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12 Reducing agents Resazurin (blue) resorfol (pink) resorfol (pink) resorfol (clear), mV cysteine-HCl cystine, -340 mV dithiothreitol, -330 mV sulfide s, -571 mV titanium citrate, -430 mV ascorbic acid, -320 mV

13 Microbial growth

14 Growth & death of microbes SectionPhaseGrowth rate ALagZero BAccelerationIncreasing CExponentialConstant DRetardationDecreasing EMaximum stationaryZero FDeclineNegative

15 Microbial growth lag 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

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

17 Tilley & Terry (1966) McDougalls buffer 2 stage process 48 h rumen liquor, 48 h pepsin DM digestion

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

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23 Gas production Abou Akkada, Menke,Pell, European groups, Iwaasa Gas production is proportional to fermentation Dependent on pH Vent or no-vent ?

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26 In Vitro Gas System – Pressure Transducer

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31 Fed batch not commonly used keep organism at or near logarithmic growth for extended periods particularly good for slow growing organisms, co-cultures

32 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

33 Semi-continuous culture more rumen-like than continuous solid substrates kinetics more complicated substitute for cannulated cows

34 Nakimura & Kurihara system for protozoa dialysis membrane 2.3 l volume 90 g/d

35 Nakimura & Kurihara

36 Slyter et al. system for ruminal digestion simple 500 ml volume Up to 2.5 volumes/d 40 g/d

37 Slyter et al.

38 Rusitec feed in two bags 1000 ml volume 0.8 to 1.5 volumes/d 24 g dm/d

39 Rusitec

40 Hoover et al. differential flow rates 500 ml volume up to 3.2 volumes/d 80 to 160 g/d

41 Hoover et al.

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43 Teather & Sauer 700 ml volume 1.6 volumes/d 30 g DM/d Designed to maintain protozoa, study rumen ecology

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45 Continuous culture kinetics

46 Logarithmic growth


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