Determination of protein degradation rates using a rumen in vitro system containing inhibitors of microbial nitrogen metabolism.

1. A previously reported rumen in vitro system (Broderick, 1978) was modified to include chloramphenicol (CAP) with hydrazine sulphate (HS) to give quantitative recovery of protein breakdown products. Degradation rates were determined by regression v. time of log proportion remaining undegraded (computed by subtracting from added nitrogen the amount of N recovered as ammonia and amino acids). Concentrations of reagents giving optimal N recoveries and estimated degradation rates for casein and expeller soya-bean-meal (SBM) were: 1.0 mM-HS, 30 micrograms CAP/ml, 2.0 mM-mercaptoethanol, 3.3 mg maltose/ml, when protein was added at 0.125 mg N/ml. 2. Digestion of azo-casein and azo-albumin, solubilization of radioactivity from 14C-labelled casein, ovalbumin and bovine serum albumin (BSA), and hydrolysis of benzoyl-L-tyrosine p-nitroanilide and benzoyl-L-arginine p-nitroanilide were not significantly decreased by HS and CAP. This suggests that the inhibitors did not reduce microbial proteolysis. 3. Mean fractional degradation rates (/h) were: 0.395 casein, 0.135 BSA, 0.159 solvent-SBM, 0.045 expeller-SBM, 0.061 meat meal, 0.070 lucerne (Medicago sativa) hay. Extents of protein escape, estimated assuming rumen passage of 0.06/h, were 13, 28, 56 and 40% for casein, solvent-SBM, expeller-SBM and lucerne hay respectively. This method appears more reliable for assessing rumen degradability than buffer N solubility and protein digestibility with ficin protease. 4. Azo-dye treatment slowed the rate of casein degradation, measured by ammonia plus amino acid release, but did not alter digestion of BSA. 5. The validity of the inhibitor in vitro method for estimating protein degradability, as well as potential problems in its application, are discussed. The complete procedure may be limited to laboratories with automated analytical equipment, but a simplified version of the method may be more generally applicable.