In vitro determination of ruminal protein degradation using freeze-stored ruminal microorganisms

Mixed ruminal microorganisms were harvested from a lactating dairy cow and preserved frozen or lyophilized. Fermentation characteristics of freshly strained ruminal fluid, frozen microorganisms, or lyophilized microorganisms were evaluated during a 24-h pre-incubation and a 4-h incubation with test proteins. Differences observed during the first 4 to 6 h in total amino acid concentration, optical density, pH and VFA concentrations, acetate:propionate ratio, and lactate concentration largely disappeared later in the pre-incubation. Protein degradation rates determined for expeller and solvent meals were .015 and .092 h-1, .015 and .101 h-1, and .005 and .019 h-1, with fresh ruminal fluid, frozen microorganisms, and lyophilized microorganisms, respectively. Regression of degradation rates obtained with fresh ruminal fluid on those obtained with pre-incubated, frozen microorganisms indicated the two methods were well correlated (r2 = .98 and .94 in two experiments). Mean in vitro degradability obtained for 17 feeds using pre-incubated, frozen microbes was 89% of that obtained using the in situ method; however, in situ degradation rates for these same feeds averaged only 67% of those obtained using frozen microorganisms. Ruminal undegraded protein values for nine samples of heated soybeans and soybean meal, determined using frozen microbes, were overestimated relative to in vivo values (in vivo = 1.1 + .8 in vitro; r2 = .77). These results indicated that ruminal microorganisms can be preserved by freezing and used as the inoculum for in vitro determination of ruminal protein degradation after overnight pre-incubation.