Technical note: effect of removal of microbial cells by centrifugation on peptide and alpha-amino nitrogen concentrations in ruminal fluid
2002
Ives, S.E. | Titgemeyer, E.C. | Nagaraja, T.G.
We evaluated the effect of centrifuging rumen fluid prior to analysis on concentrations of alpha-amino N (AAN) and peptides. Rumen fluid was collected from steers fed grain-based diets at either various times after feeding or after dosing the rumens with solubilized casein. Fluid was either directly processed for peptide analysis by acidifying 10 ml of rumen fluid with 0.5 ml of 70% (wt/wt) perchloric acid, or first centrifuged at 500 x g for 20 min to remove protozoa and then at 30,000 x g for 15 min to remove bacterial cells prior to further processing. By removing microbial cells, intracellular AAN and peptides were not included in subsequent analyses. Concentrations of AAN were determined using an automated trinitrobenzene sulfonic acid assay, and peptides were determined as the increase in AAN following acid hydrolysis of the samples. When casein was not dosed, removal of microbial cells prior to analysis decreased concentrations of both AAN and peptides, and the decrease was greater for AAN (2.2 mM) than for peptides (1.2 mM). Dosing with casein led to much higher concentrations of ruminal peptides and AAN. After casein dosing, decreases in AAN and peptide concentrations due to prior centrifugation (2.1 mM and 1.0 mM for AAN and peptides, respectively) were similar to the decreases observed before the casein dosing. Results suggest that the contribution of intracellular AAN and peptides to the concentrations in ruminal fluid are relatively constant across broad ranges of dietary protein supply for cattle fed corn-based diets.
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