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

Alexander N. Hristov, Timothy A. McAllister, Zhong Xu, C. Jamie Newbold

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23 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)1886-1893
Number of pages8
JournalJournal of the science of food and agriculture
Issue number15
StatePublished - Dec 2002

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Food Science
  • Agronomy and Crop Science
  • Nutrition and Dietetics


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