Proteolytic activity in ruminal fluid from cattle fed two levels of barley grain: a comparison of three methods of determination

The effects of proportion of concentrate in the ruminant diet and the effects of freezing ruminal content prior to assay on proteolytic activity in ruminal inoculum were evaluated using three analytical techniques. A novel approach for determining proteolytic activity (PA) of ruminal fluid utilising N-15-labelled casein was compared with two published procedures. In a crossover experiment, four heifers were fed two isonitrogenous diets containing (dry matter basis) 50% barley silage, 45% rolled barley grain and 4% soybean meal (medium-grain diet, MG) or 8% barley silage, 89% rolled barley grain and 2% soybean meal (high-grain diet, HG). Ruminal fluid was analysed either fresh or after having been frozen at -40degreesC for 45 days. Substrates utilised in measuring PA included N-15-labelled casein (produced by infusing ((NH)-N-15)(2)SO4 into the rumen of a lactating dairy cow), C-14-labelled casein and azocasein. Incubations were conducted in 0.2m phosphate buffer (pH 6.8) for 20 min at 39degreesC. In the N-15-casein incubations, PA was estimated as (i) N soluble in 5% trichloroacetic acid (TCASN), (ii) N soluble in 5% TCA corrected for microbial N uptake (TCAMICR) and (iii) N depleted from the soluble protein N pool (SPR). In the C-14-casein incubations, PA was measured as TCA-soluble radioactivity; in the azocasein method it was measured as dye released during incubation. Across treatments the highest (P <0.001) proteolytic activity was measured by the SPR method, followed by TCAMICR, TCASN and C-14-casein. The lowest activity was recorded using the azocasein method. Within the N-15- and C-14-casein methods, PA in previously frozen ruminal fluid was higher (P <0.05) with the HG diet than with the MG diet, and higher (P <0.05) in previously frozen fluid than in inoculum processed fresh. This study demonstrates that increasing the proportion of grain in the diets of cattle and freezing the ruminal inoculum both increase proteolytic activity measured in ruminal fluid. The proposed N-15-casein method yielded higher proteolytic activity values than the C-14-casein method. Copyright (C) 2002 Society of Chemical Industry. Contributions of AN Hristov, TA McAllister and Z Xu. (C) Minister of Public Works and Government Services, Canada 2002.