Determining the Legume‐Fraction of a Grass‐Legume Mixture by Pinitol Analysis 1

The majority of the methods, presently used for determining the botanical composition of mixed forages, are complicated, time consuming, and seldom accurate. The increasing interest in research on grass/legume forage mixtures gives rise to the need for accurately surveying the botanical composition available to the grazing animals. The purpose of this report is to describe a simple, accurate, and rapid method for determining the legume foliage composition in a grasslegume mixture. Pinitol (l‐D‐3‐0‐methyl‐ chiro ‐inositol) was isolated from forage legumes, grown in the greenhouse and field plots, and determined to be unique to the Leguminosae family. Since pinitol occurs only in the legume fraction of a grass‐legume forage mixture, the legume composition is directly proportional to the pinitol concentration in the 80% ethanolic extract of the dried ground foliage mixture. Contrary to carbohydrates, pinitol concentration in legume foliage has been determined to be relatively stable over a normal daily range of environmental conditions and each stage of plant growth. The procedure was evaluated by determining the alfalfa ( Medicago sativa L.) component of a greenhouse grown alfalfa‐fescue ( Festuca arundinacea Schreb.) mixture over a range of concentrations and found to accurately estimate the actual legume composition (r 2 = 0.989). This method could be easily adapted for use in quantifying the legume composition in animal diets collected by esophageal fistulated animals.