Determining the Suitability of the Radial Diffusion Assay for Quantifying the Interaction Between Tannins and Ergot Alkaloids.

Tannins produced by many forage species are beneficial to ruminants due to their ability to improve the utilization of dietary nitrogen. Due to similarities in their structure, tannins may also interact with ergot alkaloids produced by an endophyte of tall fescue [Lolium arundinaceum (Schreb.) S.J. Darbyshire], which would provide an alternative method of minimizing the negative impact alkaloids may have on livestock production. The radial diffusion assay utilizes the interaction between tannins and proteins to quantify tannin concentrations as a function of the size of the precipitate ring formed. However, it may be possible to modify this assay to determine if tannins have the potential to bind ergot alkaloids. A preliminary study using the radial diffusion assay demonstrated an interaction between ergot alkaloids and tannins, but results from subsequent studies have been inconsistent. The objectives of this study were to evaluate several possibilities for this inconsistency in order to improve the effectiveness of this analytical tool. Several experiments were conducted to determine if the causes of the issues were due to the gel assay itself, the tall fescue seed extracts, the tannin extracts, or human error. While the individual making the measurements, gel pH, and seed extracts were all shown to have an effect on the size of the precipitate ring formed, they did not appear to be the source of the variability. Variation in gel density of each plate appeared to be the largest source of error and a revision of the gel-making protocol to account for this resulted in a more consistent assay. In addition, plant maturity also affected tannin interaction with ergot alkaloids as only tannins extracted from vegetative sainfoin [Onobrychis viciifolia Scop.] bound ergovaline. Future studies will address the effect of plant maturity at time of harvest in relation to tannin chemistry and binding activity.