Anaerobic in vitro rumen incubation technique to simulate rumen function

Scope of the method

The Method relates to
  • Animal health
The Method is situated in
  • Basic Research
  • Translational - Applied Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Other (e.g. bacteria)
Rumen microbial inoculum


Method keywords
  • Rumen inoculum
  • rumen microbes
  • anaerobic fermentation
  • gas production
  • digestion
Scientific area keywords
  • Feed degradation
  • Fermentation (kinetics)
  • Enteric methane (mitigation)
  • Rumen degradable/undegradable protein
  • Biohydrogenation of unsaturated fatty acid
  • Rumen microbiology
  • Rumen health
Method description

The in vitro rumen fermentation technique is used to simulate the rumen function/fermentation. The rumen is the first compartment of the digestive tract of ruminants, in which feed - consumed by ruminants - is fermented by rumen microbes. For optimal fermentation, the rumen provides an anaerobic environment at constant temperature and pH, and ensures a good mixing. We mimic the above-mentioned rumen conditions externally in an incubation flask. Briefly, the test products and the simulated ruminant feed are weighed into the flasks, which are sealed and flushed with CO2 to create the anaerobic conditions. Afterwards, bicarbonate/phosphate-buffered rumen fluid is added into the flasks which are incubated at 39 °C in a shaking incubator for a prescribed duration (mostly for 24 h). The rumen inocula/microbes are obtained from cannulated or non-cannulated live ruminants. Gas and incubated liquid samples are collected at the end of the incubation period to determine gas composition and volatile fatty acids. Additionally, samples are collected for microbial analysis. The anaerobic in vitro rumen incubation technique is well established in our lab, and we perform various experiments using this method which can be customized according to the research question. This includes kinetics of feed fermentation, protein and fat degradation in the rumen, biohydrogenation of unsaturated fatty acids, enteric methane mitigating ability of feed/additive, etc.

Lab equipment
  • - Incubator;
  • - CO2 flushing system;
  • - Water bath;
  • - pH meter;
  • - Pressure transducer;
  • - Gas chromatography to analyze the gas composition and (volatile) fatty acid profile;
  • - Equipment related to feed (residue) characterization;
  • - Equipment needed for microbial analysis (qPCR machine, bead beater, etc.).
Method status
  • Published in peer reviewed journal

Pros, cons & Future potential

  • - Resembles in vivo rumen condition;
  • - Versatile applications;
  • - Low cost and simultaneous screening of a large number of treatments;
  • - Does not need a large number of live animals (only rumen inoculum donor animals);
  • - Ethically superior;
  • - Less time-consuming.
  • - End products and wastes accumulate in the bottle which may influence rumen function (particularly for incubation periods exceeding 72h);
  • - Absorption of nutrients through rumen wall cannot be mimicked;
  • - Interaction with host is not mimicked.

- Preservation of rumen inocula for future use is currently under investigation

References, associated documents and other information


Gadeyne, F., De Ruyck, K., Van Ranst, G., De Neve, N., Vlaeminck, B., & Fievez, V. 2016. Effect of changes in lipid classes during wilting and ensiling of red clover using two silage additives on in vitro ruminal biohydrogenation. Journal of Agricultural Science, 154, 553–566.

Associated documents

Contact person

Veerle Fievez


Ghent University (UGent)
Animal sciences and Aquatic Ecology