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

Abstract 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 15N-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 ?40?°C for 45?days. Substrates utilised in measuring PA included 15N-labelled casein (produced by infusing (15NH)2SO4 into the rumen of a lactating dairy cow), 14C-labelled casein and azocasein. Incubations were conducted in 0.2?M phosphate buffer (pH 6.8) for 20?min at 39?°C. In the 15N-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 14C-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?