I . A procedure for sampling digesta from within the omasal canal of sheep given a variety of roughage diets was used to enable comparison to be made of the composition of effluent from the reticulo-rumen with that of rumen fluid.2. Concentrations of protozoa in effluents, relative to a soluble marker continuously infused intraruminally, were usually less than 20 yo of corresponding rumen fluid concentrations. It was estimated that the amount of protozoal nitrogen leaving the rumen represented less than 2% of dietary N.3. Passage of volatile fatty acids (VFA) from the rumen in effluent was less than 75% of that indicated by rumen concentrations.4. A continuous, in vitro fermentation system was developed, in which outputs of protozoa were comparable with in vivo outputs.

1. The extent to which ammonia-N (NH3-N) serves as a starting point for synthesis of microbial nitrogenous compounds was assessed when 15N as (15NH4)2SO4 was continuously infused into the rumen of a sheep for periods of 78–98 h. Steady states were reached in the composition of the rumen contents because the animal was fed equal parts of its ration at hourly intervals. Concentrations of 15N in bacterial-N, protozoal-N and rumen NH3-N were compared.2. In two trials with a low-N diet consisting largely of wheaten hay the 15N concentration in bacterial N was 76 and 78% of that in the NH3-N. For protozoa the values were more variable —64 and 43%.In two trials with a higher-N diet (lucerne hay), the corresponding values were lower—bacterial-N 62 and 64%, protozoal-N 41 and 35%.It was concluded that synthesis of microbial protein was more dependent on ammonia as a starting point with the low-N diet than with the higher-N diet.3. Entry and exit rates for ammonia into and out of the rumen system were determined, and the results, in combination with those obtained for bacterial-N in the first part of the work, allowed calculations to be made of the production of microbial-N/d formed from NH3-N, and this in turn allowed calculation of minimal values for conversion of plant-N to microbial-N in the lumen. Minimal extent of conversion was 68% for the low-N diet and 53–55% for the higher-N diet.4. Total production of microbial-N in relation to the amount of N given was also calculated by using previously reported values for the relative proportions of protozoal-N and bacterial-N in sheep given diets similar to those used here. These values for extent of conversion were 73% for the low-N diet and 58–59% for the higher-N diet.

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1) In sheep fed at 12-hr intervals on roughage diets (lucerne and wheaten hay) total volatile fatty acid production in the rumen has been measured by an isotope dilution procedure based on the infusion of a single 14C-labelled volatile fatty acid.(2) Total and individual acid production has been measured through the infusion of a mixture of 14C-labelled acids in which the proportions of individual acids were such that transfers of 14C by interconversions between the acids were balanced.(3) The findings support the view that the mixture of acids produced in the rumen is similar in composition to that present in the rumen fluid throughout the feeding cycle. Consequently it was possible to determine the production of individual acids by a relatively simple procedure ~equiring only the infusion of a single 14Clabelled acid and measurement of the concentration of 14C in a composite sample of the acids in the rumen fluid collected throughout the feeding cycle. This procedure is considered suitable for routine use; automatic sampling of rumen fluid still further reduces the work involved.(4) The infusion of certain mixtures of 14C-labelled fatty acids showed that the composition of the acid initially formed in the rumen was acetic 77-83%, propionic 15-18 %, and butyric 1-7 % according to the diet given and the time elapsed after feeding. From 50 to 80% of the butyric acid in the final volatile acid product was formed from acetic acid. Degradation of 14C-labelled butyrate formed from [I-14C] acetate in the rumen showed 93 % of the 14C to be nearly evenly divided between carbon atoms C, and C,.In previous experiments (Gray et al. 1966) production of total volatile fatty acid (VFA) in the rumen of a sheep was measured by an isotope dilution procedure based on continuous infusion of 14C-labelled VFA into the rumen throughout a feeding cycle. The mixture of acids infused was one in which the 14C was distributed between acetic, propionic, and butyric acids in proportions similar to those in which the corresponding unlabelled acids occurred in the rumen fluid.The present work shows that:(1) Any mixture of the labelled acids, or any one of them alone, can be used to determine the total acid produced during a feeding cycle.(2) Infusion of a suitable mixture, based on the observed behaviour of each labelled acid in the rumen, makes it possible to determine the quantity of each acid formed.