Development of an in vitro procedure to determine ruminal Development of an in vitro procedure to determine ruminal availability of protein availability of protein

A series of in vitro experiments was placed into nylon bags and suspended in the conducted to determine the ruminal availabilrumen for digestion. Although somewhat ity of protein from grains. Procedures were expensive and labor-intensive, it assesses the based on assumptions that 1) ruminal availprotein availability for a variety of feedstuffs. ability of protein is first-limiting to microbial However, questions have been raised about growth, 2) accumulation of microbial cells the in situ procedure for feedstuffs that are accurately predicts ruminal protein availabillow in protein. Our objective was to deity, 3) cytosine can be used to accurately velop a method for measuring ruminally estimate microbial cell mass, and 4) cytosine degradable protein that would be appropriate is present in microorganisms but not in feeds. for all feedstuffs, regardless of protein level. Cytosine content of in vitro cultures was measured by high performance liquid chromatography. Early experiments determined that adding 0.75 g soluble starch provided enough Samples of various feedstuffs (0.5 g) energy that culture growth depended on were measured into 50-ml centrifuge tubes. available protein. In the final experiment, A 30-ml aliquot of a mixture of rumen microbial cytosine was measured for several fluid/McDougall’s buffer was added to each processed grains and for graded levels of tube. The tubes were flushed with carbon sodium caseinate (as a standard for comparidioxide, sealed with one-way valve rubber son). Cytosine increased as sodium caseinate stoppers, and allowed to incubate for 12 levels increased. Heat-processed grains hours at 39 C. At the end of the incubation yielded less cytosine than grains processed period, the microbial fermentation was without heat. Cytosine accumulation during stopped by adding 0.1 ml formalin and refrigin vitro fermentation provides a useful meaerating the tubes. Solids were harvested by sure of ruminal protein availability. centrifuging for 15 minutes at 30,000 x g. The resulting pellet was dried in a 55 C (


Introduction
The proportion of dietary protein that is Experiment 1. Dry-rolled wheat, grain ruminally degradable vs. undegradable is a sorghum, and corn were prepared by crackcomponent of modern ration formulation ing whole kernels and then grinding them systems.Protein degradability can be mea-through a 1-mm mesh screen.Steam-flaked sured in numerous ways; the most common is the in situ procedure, in which feeds are

Experimental Procedures
o o performance liquid chromatography was used to measure cytosine.All feed samples were run in quadruplicate.
corn was simulated by autoclaving corn for Experiment 1. Microbial growth was 45 minutes under dry steam, then grinding higher (P<.05) for samples containing soluble through a 1-mm mesh screen.High-moisture starch, indicating that energy, not protein, corn was produced by reconstituting cracked was first-limiting.Microbial growth reached corn, allowing 4 weeks for fermentation, and a maximum at 12 hours of fermentation, so then grinding through a 1-mm mesh screen this incubation period was used.using dry ice.Dry samples were kept in sealed bags at room temperature.Wet sam-Experiment 2. As the level of starch ples were kept frozen in sealed bags.Pure supplementation increased, cytosine content soluble starch was used as a control (an increased in a linear manner (P<.01), indicatenergy source without protein).Incubations ing that microbial growth was stimulated. of 3, 6, 9, 12, and 24 hours were tested to Cytosine amounts also increased (P<.01) determine appropriate fermentation times for when sodium caseinate was added to fermenmicrobial growth.
tation samples.No interactive effects Experiment 2. Previously ground wheat added in combination.and grain sorghum samples were combined with 0.25 or 0.5 g soluble starch to determine Experiment 3. Adding soluble starch at if the fermentation system was energy defi-levels above 0.5 grams had no effect on cient.Grain samples also were supple-microbial growth (P>.7),so energy was no mented with sodium caseinate (0.05 g) to longer limiting above the level.Cytosine determine the effect of degradable protein on microbial growth.Samples without additives served as controls.
Experiment 3. The ground wheat and grain sorghum used in experiments 1 and 2 were supplemented with identical caseinate levels.In this experiment, soluble starch was added at 0.5, 0.75, or 1.0 g to evaluate effects of energy addition on microbial growth.
Experiment 4. Samples of wheat and corn were autoclaved to simulate flaking.Samples of autoclaved wheat and corn were compared to the same grains dry rolled without heat.Samples of soybean meal and nonenzymatically browned soybean meal (a high escape protein source) also were tested.Sodium caseinate, a protein source that is completely rumen-degradable, was used to develop a standard curve.Starch was added to all incubations at 0.75 g per tube to ensure that energy was not limiting.

Results and Discussion
occurred when caseinate and starch were increased (P<.01) when sodium caseinate was added to grain samples, indicating that microbial growth was responding to added degradable protein.
Experiment 4. Microbial growth increased (P<.05) with additions of sodium caseinate up to levels of about 0.03 g of protein (Fig. 1).Thus, the availability of protein appeared to be limiting microbial growth.Cytosine concentrations resulting from fermenting dry-rolled wheat and soybean meal were significantly higher (P<.01) than those from autoclaved wheat or the nonenzymatically browned soybean meal, indicating that heat processing in those feeds decreased the availability of protein to rumen microbes.Cytosine contents were numerically, but not significantly, higher for dryrolled than autoclaved corn.This in vitro procedure currently is being applied to a wider range of feedstuffs for further validation.

Figure 1 .
Figure 1.Average Cytosine Contents after Fermentation of Processed Feeds andAdditions of Graded Levels of Sodium Caseinate.