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 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 < 0.001) proteolytic activity was measured by the SPR method, followed by TCAMICR, TCASN and ¹⁴C-casein. The lowest activity was recorded using the azocasein method. Within the ¹⁵N- and ¹⁴C-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 ruminai fluid. The proposed ¹⁵N-casein method yielded higher proteolytic activity values than the ¹⁴C-casein method.