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 15 N‐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 15 N‐labelled casein (produced by infusing ( 15 NH) 2 SO 4 into the rumen of a lactating dairy cow), 14 C‐labelled casein and azocasein. Incubations were conducted in 0.2 M phosphate buffer (pH 6.8) for 20 min at 39 °C. In the 15 N‐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 14 C‐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 14 C‐casein. The lowest activity was recorded using the azocasein method. Within the 15 N‐ and 14 C‐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 15 N‐casein method yielded higher proteolytic activity values than the 14 C‐casein method.