A Procedure for Measuring Peptides in Rumen Fluid and Evidence that Peptide Uptake Can Be a Rate-Limiting Step in Ruminal Protein Degradation

Abstract

A relatively simple and sensitive procedure was developed to measure the concentration of peptides in rumen fluid. Feed particles and microorganisms were removed by centrifugation, and the supernatant was treated with perchloric acid (5% final concentration). Perchloric acid precipitated macromolecules that included protein, RNA, and DNA. Perchlorate was subsequently removed by precipitation with an excess of potassium carbonate. Ammonia was removed by boiling the alkaline sample. Supernatant samples were then analyzed for ninhydrin reactive material before and after HCl hydrolysis. Because ninhydrin reaction was 3 to 7.5 times greater after HCl hydrolysis, peptides rather than amino acids were the primary source of nonprotein, nonammonia nitrogen. Rumen fluid from a cow fed timothy hay and concentrate supplement (16% crude protein) contained more than 1200 mg peptides/L (192 mg N/L), 1 h after feeding, and this value declined the prefeeding value of 400 mg/L (64 mg N/L) by 8 h after feeding. Comparison of ninhydrin reactivity with and without HCl hydrolysis indicated that peptides present before feeding contained more peptide bonds than the peptides soon after feeding. High pressure liquid chromatography revealed a variety of peaks soon after feeding and fewer dominant peaks 8 h later. The data suggest that peptide uptake into rumen microorganisms can be a rate-limiting step in ruminal protein degradation.