H.—34.

Analytical Methods. It has become increasingly apparent during the year's work that more refined methods of analysis for the materials which comprise the soluble fraction of leaf tissue are essential, and considerable work has been done to that end. (1) Carbohydrates. —The soluble carbohydrates are of importance in that they are believed to be the end products of photosynthesis and supply the carbon chain not only for the structural carbohydrates of the cell wall, but also for the nitrogenous derivatives. A method for the estimation of sugars having advantages in both simplicity and specificity over the accepted methods has been worked out. Further work on this fraction is proceeding, with the purpose of determining the individual sugars which comprise the soluble carbohydrate fraction. (2) Amino-acids. —The ammo-acids are the units of which proteins are composed, and probably comprise the most important group in the soluble nitrogen fraction. Methods for the quantitative estimation of the amino-acids have been in process of continual improvement and depend almost entirely on the properties of the amino group. Since, however, substances other than true aminoacids cause considerable interference, there still exists doubt as to the interpretation of the values obtained for amino-nitrogen. Progress has been made in an examination of current methods and in the application of new ones, and it is believed that it will be possible to define the degree of interference fairly accurately. Peptides are composed of amino-acids and may be looked on as proteins of a low order of complexity. They contribute largely to the soluble nitrogen fraction, and methods for their determination are available. The criticisms which apply to the amino-acid fraction apply also to the estimation of the peptides. (3) Purines. —The purines are constituents of all nuclear material and form an important part of certain enzyme systems concerned with cellular oxidation, while recent work has shown that those virus proteins which have been available for intensive investigation contain adenine and guanine as part of the molecule. Methods of estimation have hitherto been tedious and of doubtful specificity, and an accurate micro method has been eminently desirable. By a modification of a method developed for the analysis of animal tissues a considerable improvement has been achieved. Analyses of clover and rye-grass indicate that purine nitrogen may account for as much as 5 per cent, of the total nitrogen, and that most of the purine occurs in combination, probably as nucleic acid. (4) Rest-nitrogen Fraction. —Of the soluble organic nitrogen fraction in pasture plants, aminoacids and peptides are undoubtedly the most important constituents, and a fair estimate of their contribution is possible. The amides, glutamine, and asparagine, together with free ammonia, can also be estimated with a fair degree of certainty. But when the nitrogen derived from all these sources is added together it is found that a considerable discrepancy still exists between the sum and the total soluble nitrogen figure. This nitrogen unaccounted for by present methods of analysis is conveniently referred to as the " rest-nitrogen " fraction, which may vary from as little as 20 per cent, to as much as 60 per cent, of the total soluble nitrogen. Comparatively little work has been done on this fraction by plant physiologists, either qualitatively or quantitatively, yet the chemical individuals of which it is comprised and their relative amounts is a matter of the greatest practical importance. The analyses which have been briefly described are of interest to the animal physiologist who wishes to have as complete a knowledge as is possible of the food ingested by the animal. But they are of little use to the pathologist who is investigating some stock ailment directly attributable to the pasture on which the stock is grazing. Amino-acids, peptides and the amides, asparagine and glutamine, are normal constituents of any animal diet and no pharmacological action can be ascribed to them. If, however, amines, alkaloids, or other nitrogenous materials with possible pharmacological activity are present in grass, and the many stock ailments which have been reported would point to that conclusion, they will appear in the rest-nitrogen fraction. The fraction is of almost equal interest to the plant physiologist who is interested in the role played by the constituent parts in the metabolism of the leaf, and who desires to know whether they are intermediary metabolites in the synthesis of protein or by-products of synthesis and breakdown. No answer can be given at the moment to this question, and it will be obvious that an investigation which sets out to answer it must necessarily be a long-term one. Certain preliminary experiments have been made in order to establish the identity of the materials present in the rest-nitrogen fraction of rye-grass, and a fraction, containing about 6 per cent, of the total soluble nitrogen has been isolated. Its examination is proceeding. Facial-eczema Investigations. The work which has been outlined above, and its extension, is the chief contribution which this Laboratory can make to the study of diseases such as facial eczema, where composition of the herbage eaten by the animal is suspected as being the primary cause of the complaint. But simultaneously with such studies it is necessary to carry out experiments which are designed as a result of observations made in the field. One of these observations relates to the high soil temperatures in seasons when the disease is prevalent, and an investigation of the effect of high soil temperatures on the growth of perennial rye-grass has been initiated in collaboration with the Grasslands Division and the Department of Agriculture. This has involved the growing of rye-grass in glasshouses and outside under conditions where soil temperatures can be controlled. As is stated in the report by the Director of the Grasslands Division, difficulties were experienced with grass grown under glass, and modifications in treatment will be tried in order to give a better sward.

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