Dynamics of protozoa in the rumen of sheep

Protozoa were labelled by incubating 100 ml rumen fluid with [14C]choline for 1 h. The protozoa were concentrated by centrifugation and then washed with rumen fluid. This reduced residual 14C in the fluid medium to insignificant amounts while still retaining the viability of the labelled protozoa. Washing procedures using formal saline (40 g formaldehyde/1 saline (9 g sodium chloride/1)) and saline were developed to isolate protozoa for estimation of specific radioactivity. 2. The protozoal pool in freshly-collected rumen fluid incubated in vitro retained 90% of the radioactivity for up to 6 h following addition of 14C-labelled protozoa produced as indicated previously. The specific radioactivity of protozoa did not change during the incubation period. 3. Protozoa labelled with [14C]choline and then stored until they died rapidly lost 14C to methane when they were incubated in rumen fluid or were injected into the rumen. Some [14C]choline was salvaged under these conditions by the live protozoa present as they apparently incorporated up to 13% of the label from the dead protozoa. However, protozoal debris from the injected solution could also have been present in the isolated protozoa. 4. The in vitro results suggested that the protozoal preparations were viable, and that the incorporated choline did not have a turnover in excess of the turnover of nitrogen (i.e. specific radioactivity remained constant with time in vitro) suggesting that the dilution of specific radioactivity of protozoa following mixing of a 14C-labelled dose of protozoa represented the rate of irreversible loss and also replacement of protozoa in the rumen. 5. 14C-labelled protozoa had a half-life in the rumen which was greater than that of rumen fluid and in six animals the protozoal replacement rate was 1-4.1 mg N/min. 6. Losses of 14C from labelled protozoa in the rumen in methane or via abomasal digesta were 65 and 35% respectively. 7. The results suggest that protozoal growth may be as high as 32% of the total microbial protein synthesis in the rumen but that 65% of the protozoa die and are degraded in the rumen.