Incorporation of nitrogen into rumen bacterial fractions of steers given protein- and urea-containing diets. Ammonia assimilation into intracellular bacterial amino acids.

Abstract

Experiments were carried out in vivo to investigate the pathways of ammonia incorporation into rumen bacteria, bacterial fractions and free amino acids within the bacteria. Steers were alternately given two isoenergetic, isonitrogenous diets containing the nitrogen mainly as either urea or decorticated groundnut meal (DCGM). At the end of each period on a given diet, a solution of 15NH4Cl was infused into the rumen and samples of rumen contents were removed at 2, 10, 20 and 90 min and 5, 10 and 24 h afterwards. Concentrations of ammonia and its 15N enrichment were determined and samples of mixed rumen bacteria were prepared. Bacteria were disrupted ultrasonically and separated into bacterial protein, cell wall and protein-free cell supernatant fractions. Amino acids were separated after hydrolysis and their 15N contents determined. A rumen fluid circulation pump was developed so that representative samples could be taken at very short time intervals after the introduction of the 15N label. Rumen pH changes, rumen fluid dilution rates and patterns of rumen ammonia concentrations were consistent with normal rumen metabolism. Net bacterial synthesis (as calculated from the net outflow of bacteria from the rumen) was significantly (P less than 0.05) greater with the DCGM diet (12.4 g bacterial N/d) than with the urea diet (9.24 g bacterial N/d). With both diets the 15N label rapidly left the rumen ammonia pool and entered the rumen bacteria. Analysis of the bacterial fractions indicated that the label appeared rapidly in the protein-free cell supernatant fraction and more slowly in the bacterial protein and cell wall fractions. With the DCGM diet bacteria apparently utilized intracellular label less efficiently than with the urea diet. The proportion of N in the protein-free cell supernatant was higher with the DCGM diet, suggesting increased levels of intracellular amino acids and peptides, following extracellular protein degradation. Levels of enrichment of the amino acids alanine and glutamate in the protein-free cell supernatant fraction suggested that the enzymes alanine dehydrogenase (EC 1.4.1.1) and glutamate dehydrogenase (EC 1.4.1.2 and 1.4.1.4) may be the major enzymes for assimilating ammonia when concentrations of soluble carbohydrate and rumen ammonia are high in the rumen. The high levels of intracellular alanine are discussed with reference to published work on the excretion of alanine by rumen bacteria.