In vitro cumulative gas production techniques: History, methodological considerations and challenges

• Published in: Animal feed science and technology 2005, Vol.123, p.9-30
• Main Authors: Rymer, C., Huntington, J.A., Williams, B.A., Givens, D.I.
• Format: Article
• Language: English
• Subjects: energy content; feces; Feed characterisation; feeds; fermentation characteristics; Gas production; gas production (biological); history; in vitro digestion; in vitro studies; inoculum; invitro; literature reviews; measurement; methodology; nitrogen; pressure; pressure transducer; production profiles; repeatability; rumen fermentation; rumen fluids; Rumen in vitro methodology; rumen microorganisms; ruminal bacteria; ruminants; temperature dried grass; volume; water solubility
• ISSN: 0377-8401; 1873-2216
• DOI: 10.1016/j.anifeedsci.2005.04.055

Methodology used to measure in vitro gas production is reviewed to determine impacts of sources of variation on resultant gas production profiles (GPP). Current methods include measurement of gas production at constant pressure (e.g., use of gas tight syringes), a system that is inexpensive, but may be less sensitive than others thereby affecting its suitability in some situations. Automated systems that measure gas production at constant volume allow pressure to accumulate in the bottle, which is recorded at different times to produce a GPP, and may result in sufficiently high pressure that solubility of evolved gases in the medium is affected, thereby resulting in a recorded volume of gas that is lower than that predicted from stoichiometric calculations. Several other methods measure gas production at constant pressure and volume with either pressure transducers or sensors, and these may be manual, semi-automated or fully automated in operation. In these systems, gas is released as pressure increases, and vented gas is recorded. Agitating the medium does not consistently produce more gas with automated systems, and little or no effect of agitation was observed with manual systems. The apparatus affects GPP, but mathematical manipulation may enable effects of apparatus to be removed. The amount of substrate affects the volume of gas produced, but not rate of gas production, provided there is sufficient buffering capacity in the medium. Systems that use a very small amount of substrate are prone to experimental error in sample weighing. Effect of sample preparation on GPP has been found to be important, but further research is required to determine the optimum preparation that mimics animal chewing. Inoculum is the single largest source of variation in measuring GPP, as rumen fluid is variable and sampling schedules, diets fed to donor animals and ratios of rumen fluid/medium must be selected such that microbial activity is sufficiently high that it does not affect rate and extent of fermentation. Species of donor animal may also cause differences in GPP. End point measures can be mathematically manipulated to account for species differences, but rates of fermentation are not related. Other sources of inocula that have been used include caecal fluid (primarily for investigating hindgut fermentation in monogastrics), effluent from simulated rumen fermentation (e.g., ‘Rusitec’, which was as variable as rumen fluid), faeces, and frozen or freeze-dried rumen