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United States Department of Agriculture

Agricultural Research Service

Title: Preservation of Ruminal Microorganisms for in Vitro Determination of Ruminal Protein Degradation

Authors
item Luchini, Nestor - UNIV OF WISCONSIN
item Broderick, Glen
item Combs, David - UNIV OF WISCONSIN

Submitted to: Journal of Animal Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 20, 1995
Publication Date: N/A

Interpretive Summary: Dairy cows obtain their protein needs both from synthesis by the microbes living in the rumen (the first compartment of the cow's stomach) plus feed protein that escapes breakdown by these same microbes. Dietary protein escaping rumen breakdown is very important because the rumen microbes often reduce the value of feed proteins by degrading more protein then they resynthesize. Therefore, it is important to know how much protein in common feedstuffs will be degraded in, and escape from, the rumen when fed to the cow. We developed an in vitro method--where tests are conducted in test tubes rather than in live animals--to estimate the amount of protein breakdown occurring inside the rumen of the living cow. The method consisted of collecting rumen contents, which contain the protein degrading microbes, from cows equipped with rumen fistulas (holes made into the rumen using surgical procedures), then harvesting rumen microorganisms from those econtents. Once harvested, the rumen microorganisms were preserved frozen in a glycerol solution until used as the inoculum for the in vitro test for rumen protein breakdown. In vitro methods are much faster and cheaper to run than tests run using live animals. By preserving the rumen microbes, the need for fistulated cows is reduced and the day to day variation in the test results was cut in half. Pre-incubation of the preserved microorganisms was required before performing the incubations with feed proteins. Pre-incubation was beneficial since it increased the microbial activity of the inoculum, giving rates and extents of protein breakdown that were comparable to those obtained using fresh rumen fluid.

Technical Abstract: Ruminal microorganisms, preserved either lyophilized or frozen, were compared with strained ruminal fluid for proteolytic activity and as inoculum source for in vitro determination of ruminal protein degradation. Dialysis and glycerol addition had no effect on the proteolytic activity of the preserved microorganisms. Net release from protein of NH3 and total amino acids using the fluid plus particle associated microorganisms was higher than that found using the fluid associated microorganisms alone. Method of inoculum preservation altered total proteolytic activity of the pellet but harvesting bacteria using centrifugal force greater than 5000 x g did not increase proteolytic activity of the pellet. The proposed method for harvesting and preserving microorganisms consisted of centrifuging strained ruminal fluid at 5000 x g (30 min at 4 deg C), stirring the pellet in a 50:50 (vol/vol) solution of glycerol : McDougall's buffer for 15 min and, then storing at -20 deg C. Protein degradation rates in incubations with preserved microorganisms were three to six times slower than when using fresh ruminal fluid; however, the feed proteins were ranked similarly for degradation rate. Preincubating the preserved microorganisms reduced background concentrations of NH3 N and total amino acid N in blanks and increased the protein degradative activity of the preserved inoculum. Degradation rates with preincubated, preserved inocula were similar to those obtained using fresh ruminal fluid. These results indicated that mixed ruminal microorganisms can be preserved by freezing and, after a preincubation period, used as the inoculum source for in vitro estimation of ruminal protein degradation.

Last Modified: 4/20/2014
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