S U M M A R YTwo sheep were reared together and then kept on a standard diet over a period of 9 months. The rumen of one sheep contained a mixed population of ciliate protozoal species ; the other was initially unfaunated, then faunated with Entodinium caudatum and then with Entodinium and Polyplastron multivesiculatum. For each sheep diurnal and day-to-day variations in total concentration, and concentration of particular types, of bacteria, in viable counts of bacteria on non-selective and differential media and in concentrations of the different protozoal species, were determined. Measurements were also made of in vitro rates of fermentation, rumen pH and ammonia and volatile fatty-acid concentrations. The presence of protozoa decreased rumen bacterial concentrations and increased ammonia and volatile fatty-acid concentrations. However, these changes were not always completely clear-cut since the protozoa changed the patterns of diurnal variation not only of bacterial numbers but of some metabolic activities and metabolites and also altered the balance of bacterial types. These results are discussed in the context of interactions between bacteria and protozoa and between protozoal species. The main effects of faunation of the rumen seem to be caused by non-selective ingestion of small bacteria by the protozoa.

I. Lambs were removed from their dams at z d of age, and at 5 weeks of age eight of one group were inoculated with a mixed rumen ciliate population and seven in a second group were maintained ciliate-free throughout the 61 weeks of the experiment. Performance of the groups was compared when given a z : I r0ughage:concentrate diet as a set ration and with ad lib. roughage.2. Mixed ciliate populations developed in all faunated lambs and average-sized populations were maintained. Large numbers of flagellate protozoa developed in the ciliate-free animals after flagellates had been introduced into the building with the ciliate inoculum.3. Higher numbers of rumen bacteria were found in the ciliate-free group. 4. Only between the 14th and zIst weeks was there a significant difference between groups in weight gain and this was in favour of the faunated group. The only significant difference in body measurements was a greater girth in the ciliate-free lambs.5 . Only minor differences were found between the groups in calorimetric trials, digestibility and nitrogen balances. There were no differences between groups in concentration of total protein N and soluble sugar in the rumen. The ammonia concentration was significantly higher in the faunated group.6. The concentrations of total rumen volatile fatty acids (VFA) were higher in the faunated group. Differences, between groups, in proportions of VFA were attributed to the activity of the rumen bacteria rather than the ciliates per se.7. No differences between groups were found in the concentrations of blood sugar and haemoglobin.8. It was concluded that the changes due to the presence of rumen ciliates were not great enough to be reflected in animal performance under the conditions of this experiment.

SUMMARY: When metabolizing glucose or other fermentable sugar in vitro at a temperature 8-12" below the normal rumen temperature, three species of rumen holotrich ciliates were liable to exhibit highly abnormal appearances which were not seen a t 35-38'. These abnormal appearances consisted essentially in a contraction and fusing together of the bulk of the storage polysaccharide (amylopectin) granules into a narrow central zone in the endoplasm. The outer clearer endoplasmic region, however, still contained numerous discrete granules in Isotricha prostoma and I . intestinalis but not in Dasytricha ruminantiurn. The abnormality was not quickly lethal to the organisms and appeared not to involve any alteration in shape or position of the macro-nucleus. The presence of nunen liquor, with all its soluble constituents but not necessarily its bacteria, is required for a high incidence of abnormality and the organisms should also initially contain but little storage polysaccharide. Evidence is presented in favour of the view that the abnormalities are connected with the utilization (auto-fermentation) of storage polysaccharide rather than with its synthesis. Nevertheless, the abnormalities cannot be induced in Isotricha when starch grains (vegetable or protozoan) have first been ingested.When harvesting in quantity from sheep rumen contents the holotrich ciliates (i.e. a mixture of Isotricha prostoma, I. intestinalis and Dasytricha ruminantiurn) by a labour-saving and apparently quite reproducible modification of the glucose-fermentation methods of Masson & Oxford (1951) and Heald & Oxford (1958), we noted in our washed suspensions what seemed to be the sporadic and unpredictable appearance of a relatively few highly abnormal but still living ciliates. Furthermore, the kind of abnormal appearance in the Isotricha organisms was not exactly the same as in the Dasytricha organisms, although a marked contraction of internal contents was a feature in each. This paper records our observations on: (a) how to prevent these abnormalities from occurring; (b) how to ensure their appearance in a large percentage of living ciliates in the culture; (c) the nature of the functional derangement which can bring about such abnormal appearances in still living organisms. METHODS

1. Three steers, each fitted with a rumen cannula and a re-entrant cannula in the proximal duodenum, were offered diets consisting of a barley-based concentrate and chopped hay at a daily intake of 61 g/kg live ~e i g h t o '~ given in three. equal meals. The ratio, concentrate: hay was changed from 50 : 50 to 90 : 10 and then to 100 :O in successive periods of 12-18 weeks and the flow and composition of digesta at the duodenum was measured over 48-h periods on each dietary treatment. 2.Samples of bacteria and protozoa were separated from rumen contents and the proportions of bacterial and protozoal nitrogen (N) in duodenal digesta were estimated using 2,ddiaminopimelic acid (DAPA) and 2-aminoethylphosphonic acid (AEP) as markers. On separate occasions, radioactive sulphur (%) was infused into the rumen for 48 h and digesta collected over the final 24 h; the specific radioactivity of S in microbial and digesta fractions was used to estimate the proportions of microbial N.gave reproducible and apparently reliable estimates of microbial protein formation: the proportion of microbial N in digesta was significantly higher (P < 0.05) for the 50:50 diet than for the other treatments but the energetic efficiency of microbial protein formation did not differ significantly between diets. 4. Estimates of bacterial N based on DAPA concentrations were highly variable and frequently impossibly high. It is suggested that many of the anomalous values were the result of non-representative sampling of the rumen microbial population and that this is particularly likely to occur when conditions within the rumen are unstable. AEP was found to be unsuitable as a marker for rumen protozoa as considerable concentrations of this substance were found also in rumen bacteria. 3.

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