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

Abstract

Hristov, A. N., McAllister, T. A., Xu, Z., Newbold, C. J. (2002). Proteolytic activity in ruminal fluid from cattle fed two levels of barley grain: a comparison of three methods of determination. Journal of the Science of Food and Agriculture, 82 (15), 1886-1893.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.Peer reviewe

Fresh or frozen rumen contents from slaughtered cattle to estimatein vitro degradation of two contrasting feeds

“…The effect of freezing on the rumen microbes can be attributed to the mechanical damage caused by the ice particles during freezing (Furchtenicht and Broderick, 1987) which can disrupt cell membranes and release intracellular contents as a result of heightened osmotic pressure (Scopes, 1988;Hristov et al, 2002).…”

“…Scopes (1988) attributed the reduced deaminative activity of frozen rumen inoculum to enzyme inactivation that was not compensated even by the release of intracellular enzymes. Hristov et al (2002) suggested that susceptibility of an inoculum to a preservation treatment depended on the principal enzyme be- ing involved in the ruminal fermentation process. In contrast, rumen amylases are more resistant to freezing than cellulases or especially xylanases and higher amylase activity has been observed in previously frozen versus fresh samples from even fistulated cattle (Hristov et al, 2002).…”

“…Hristov et al (2002) suggested that susceptibility of an inoculum to a preservation treatment depended on the principal enzyme be- ing involved in the ruminal fermentation process. In contrast, rumen amylases are more resistant to freezing than cellulases or especially xylanases and higher amylase activity has been observed in previously frozen versus fresh samples from even fistulated cattle (Hristov et al, 2002). These apparently contradictory results may represent some compensation for the loss of enzyme activity by the release of intracellular enzymes (Hervas et al, 2005).…”

Effects of essential oils on proteolytic, deaminative and methanogenic activities of mixed ruminal bacteria

“…The proteolytic activity of ruminal bacteria was determined using 15 N-labelled casein as a substrate (Hristov et al 2002). A 5% (wt/vol) solution of 15 N-casein solution was prepared in 0.1 M NaOH for the incubations.…”

“…The resulting TCA supernatant, and the bacteria and soluble protein pellets were stored at (208C, freeze-dried, and analyzed for their DM concentration by gravimetry and for total N and 15 N enrichment using a nitrogen autoanalyzer connected to a stable isotope ratio mass spectrometry (Model Optima, GV Instruments, Middlewich, UK). Total proteolytic activity was estimated according to Hristov et al (2002), but expressed as mg of N released from 15 Ncasein per mg of bacterial N per min.…”