In vitro cumulative gas production techniques: History, methodological considerations and challenges
C. Rymer a,∗ , J.A. Huntington b , B.A. Williams c , D.I. Givens a
Animal Science Research Group, School of Agriculture, Policy and Development, University of Reading, P. O. Box 237, Reading RG6 6AR, UK b Harper Adams UniversityCollege, Newport TF10 8NB, UK c Animal Nutrition Group, Wageningen University, Marijkeweg 40, 6709 PG Wageningen, The Netherlands
Abstract 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, therebyresulting 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 themedium 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
Abbreviations: DM, dry matter; GPP, gas production proﬁle; NDF, neutral detergent ﬁbre; OM, organic matter; SCFA, short chain fatty acids ∗ Corresponding author. Tel.: +44 1183786436; fax: +44 118 3786595. E-mail address: firstname.lastname@example.org (C. Rymer). 0377-8401/$ – see front matter © 2005 Published by Elsevier B.V. doi:10.1016/j.anifeedsci.2005.04.055
C. Rymer et al. / Animal Feed Science and Technology 123–124 (2005) 9–30
optimum preparation that mimics animal chewing. Inoculum is the single largest source of variation in measuring GPP, as rumen ﬂuid isvariable 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 notrelated. 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 freezedried rumen ﬂuid (which were both less active than fresh rumen ﬂuid). Use of mixtures of cell-free enzymes, or pure cultures of bacteria,may be a way of increasing GPP reproducibility, while reducing reliance on surgically modiﬁed animals. However, more research is required to develop these inocula. A number of media have been developed which buffer the incubation and provide relevant micronutrients to the microorganisms. To date, little research has been completed on relationships between the composition of the medium and...