Submitted to: Journal of Dairy Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/30/2003
Publication Date: 5/1/2004
Citation: Broderick, G.A., Murphy, M., Uden, P. 2004. Effect of inhibitor concentration and end-product accumulation on estimates of ruminal in vitro protein degradation. Journal of Dairy Science. 87:1360-1371. Interpretive Summary: Dairy cows obtain an about a third of their protein needs from dietary protein that escapes digestion by the microbes living in the rumen (the first compartment of the cow's stomach). Rumen microbes often degrade large amounts of high quality protein, such as that in legume forages and oilseed meals, reducing the efficiency with which it is used for milk production. The amount of feed protein escaping the rumen, referred to by farmers as 'bypass protein', determines much of the value of feeds. Bypass protein varies greatly from feed to feed and there are no reliable methods available to commercial labs for its determination. We developed a technique to measure bypass protein involving collecting microbes from the cow's rumen and incubating them with the feeds being tested. In this method, the microbes are incubated with two metabolic inhibitors so that the rate of release of protein digestion products serves as a true indicator of protein breakdown in the rumen of the animal. The technique has proven to be accurate, but one of the inhibitors is potentially toxic to people using this method. The objective of this research was see if the toxic inhibitor could be replaced without sacrificing accuracy and to test new mathematical methods for more accurately calculating protein bypass. Replacing the toxic inhibitor by adding as much as 10 times more of the second inhibitor did not result in reliable measurement of bypass protein in several common feeds. It was also found that, after an extensive re-evaluation, the inhibitor concentrations adopted gave accurate results for bypass protein. An additional finding was that, for common U.S. dairy feeds, the simple mathematical procedure used by most commercial labs to calculate bypass protein was not as reliable as a more complex computation that we developed. Although further work may identify an effective replacement for the toxic inhibitor, this research confirmed the effectiveness of the inhibitors currently used in our technique to measure bypass protein. This research also yielded a more precise method for calculating bypass protein. This means that the bypass protein value of dairy feeds can be determined with increased accuracy by laboratories adopting our technique or using other approaches.
Technical Abstract: Effects of varying the concentrations of hydrazine sulfate (HS) and chloramphenicol (CAP), inhibitors of microbial N uptake and protein synthesis, on rates of protein degradation estimated from net appearance of NH3 and total amino acids (TAA) were studied in a ruminal in vitro system. Without inhibitors, recoveries of N added as NH3 and TAA were 4 and 6% after 4-h incubations and apparent degradation rates estimated for casein, solvent soybean meal (SSBM), and expeller soybean meal (ESBM) approached 0. Increasing inhibitor concentrations from the standard amounts of 1 mM HS plus 30 mg CAP/L to 2 mM HS plus 90 mg CAP/L gave rise to numerically, but not significantly, greater N recoveries and degradation rates. Use of 2 mM HS, without CAP, yielded similar recoveries and rates but 30 or 90 mg CAP/L, without HS, was not satisfactory. Versus that with 1 mM HS plus 30 mg CAP/L, media containing 2 mM HS plus 90 mg CAP/L gave increased TAA recoveries and higher rates for casein, but not SSBM, in the presence of added starch. Faster degradation rates were obtained for casein, but slower rates for SSBM and ESBM, in Sweden versus Wisconsin using inocula from cows fed different diets but with similar CP and energy contents. Differences in microbial catabolism of peptides may account for differences in degradation rates observed between Sweden and Wisconsin. Adding NH3 plus free and peptide bound amino acids to the inoculum reduced apparent degradation rates, possibly via end-product inhibition. Analysis of data from multiple time-point incubations indicated that casein degradation followed simple, first-order kinetics while a bi-exponential model fitted degradation patterns for both SSBM and ESBM.