Criteria for Establishing the Validity of in vitro Studies with Rumen Micro-organisms in so-called Artificial Rumen Systems

Abstract

SUMMARY: Several criteria of normal rumen function which can be applied to in witro studies with the whole rumen microbial population are suggested. These include: the maintenance of numbers and normal appearance of the bacteria, selenomonads and protozoa of the rumen ; the maintenance of normal rates of digestion of cellulose, starch and protein, and of normal interactions between these ; the ability to predict quantitative results in vivo. An 'artificial rumen' was constructed, consisting of a cellophan sac containing rumen liquor and substrate dialysing against a complex mineral solution whose composition was based on that found in rumen liquor, the whole being incubated at 39' in an atmosphere of nitrogen and carbon dioxide. This system was shown to meet the criteria which are suggested, with reasonable success for periods of about 8 hr.; over longer periods an increasing failure to meet the biological criteria was seen. For the microbial population to remain normal in numbers and activity it was shown to be necessary to use as test substrate in vitro only substances similar to the diet fed to the animal from which the rumen liquor inoculum was taken.Of recent years there has been an increasing interest in the biochemistry and microbiology of the rumen. Because of the complexity of the rumen environment, many in vitro techniques have been applied including the use of: pure cultures of bacteria isolated from the rumen; washed suspensions of the whole or part of the rumen microbial population ; cell-free enzyme preparations ; whole rumen liquor under conditions which permit or encourage microbial multiplication. Under these last conditions, sometimes known as the ' artificial rumen ' technique, it is essential to ensure that the microbial multiplication which takes place should proceed in a fashion similar to that found in the rumen of the living animal. There is normally present in the rumen a very large number of microbial species of widely differing physiological needs and capabilities, some found apparently only in the rumen and some found widespread in foodstuffs, soil, etc.; some of these microbial species may be expected to be able to multiply in almost any environment, and to metabolize almost any substrate that is likely to be tested. When, however, the environment or substrate is not that found in the rumen of the animal from which the rumen liquor inoculum was taken, then, in time, the results of this multiplication and metabolism may bear little or no relation to events in the rumen in vivo, since the organisms which multiply may be simply those most suited to the experimental conditions, and may well be only chance contaminants originally present in the rumen

Proteolysis by Rumen Micro-organisms

“…All the casein and just under half the ardein was digested in 46 hr. ; this faster rate of attack on casein as compared with ardein was noted in several experiments with different amounts of substrate and other preparations of groundnut meal protein, and is in contrast to the nearly equal rate of attack in the rumen or by whole rumen liquor in vitro (Warner, 1956) where crude groundnut meal was used; no explanation can be offered for this discrepancy. When digestion neared completion, about half the non-protein nitrogen which appeared was in the form of free amino acids (carboxyl-N).…”

“…Groundnut meal, herring meal, Paisley meal and casein were as described by Anriison et 01. (1954), starch and cellulose as described by Warner (1956). Ardein was a spray-dried groundnut protein (Imperial Chemical Industries Ltd., Nobel House, Stevenston); solutions of it were prepared by shaking with that volume of c. 0.2 N-NaOH which, when diluted to the required volume, would be c. 0.05 N. Solutions of casein were made in a similar way, but neutralized with ic-H3P04 before making to volume.…”

“…Experiments with the artijicial rumen apparatus Warner (1956) showed that casein was digested in an artificial runien apparatus at a rate of about 0-3 g. N/1. rumen liquor/hr.…”

“…Starch has been shown to lower ammonia production by rumen microorganisms in vitro as in viva (Warner, 1956). This effect, as Table 4 shows, is shared by other polysaccharicles and polysaccharide-rich foodstuffs.…”

Incubation inside the bovine rumen

“…Experiments in vitro involve collection of samples of rumen contents from the rumens of slaughtered animals or from live animals provided with rumen cannulas, and the subsequent incubation of these contents in the laboratory with suitable substrates under conditions which approximate to those in the intact rumen (Warner, 1956). This approach is suitable for theoretical studies (e.g.…”