In vitro gas methods for evaluation of feeds containing phytochemicals

Measurement of gas, a reflection of short chain fatty acids (SCFA), by in vitro gas methods provides information on effects of phytochemicals on rumen fermentation. However, to obtain complete information, it is necessary to measure other end products of fermentation, in particular microbial mass, especially for studies involving phytochemicals or bioactive moieties. Examples using tannins, saponins and alkaloids are discussed, highlighting the limited and often misleading information that can be obtained on potential effects of these compounds by measuring only gas production. In these studies, microbial protein was determined using purines or diaminopimelic acid as a marker, or by 15N incorporation into microbes. In addition, the suitability of using the difference between apparently and truly degraded residues, as the estimate of microbial mass, for evaluation of tannin-free fibrous feeds, low-starch feeds, or when the objective is to investigate potential effects of phytochemicals other than tannins, is discussed. However for tannin-containing feeds, the presence of tannin-protein complexes in these residues, and/or solubilization of tannins from the substrate that do not contribute gas or microbial mass, produce artifacts in apparently and truly degraded values, rendering this approach invalid. For quantification of truly degraded substrate in tannin-rich samples, an indirect method based on level, and molar proportion, of SCFA and microbial mass is suggested. An approach for microbial mass determination, especially useful for tannin-rich samples, based on N balance is also discussed. An advantage of gas methods is that the fate of a phytochemical in the rumen can be investigated simultaneous with its effect on rumen fermentation. Such studies, using condensed tannins, alkaloids and saponins are presented. The in vitro gas method is a relatively simple and inexpensive tool to study potential effects, mechanisms of action and fates of phytochemicals in the rumen. The method could also be used to screen for novel bioactive moieties such as those having antimethanogenic, antiproteolytic and antiprotozoal activity.