1. A method is described whereby a large quantity of rumen microbial dry matter of high purity was isolated from whole rumen contents obtained from abattoirs, by means of a continuous process of one filtration through four sieves followed by three differential centrifugations.2. The contents of ash, carbohydrate, lipid, nitrogen, RNA, DNA and individual amino acids of the three centrifugal fractions are given and compared with values summarized from more than sixty published reports on the chemical composition of rumen micro-organisms isolated from both whole rumen contents and pure cultures.3. The amino acid composition of isolated rumen micro-organisms, in particular that of the bacteria, was found to be remarkably constant.

1.Four experiments were conducted with eighteen lambs sustained entirely by intragastric nutrition at gross energy inputs varying from 430 to 860 kJ/kg live ~eightO'?~ (Wo79. Isolated rumen micro-organisms (RMO) were infused into the abomasum in quantities varying from 0 to 2 g digestible N/kg W07S to assess the increase in N balance as a result of increasing RMO input when N was limiting.

1. An experiment was conducted with three sheep maintained entirely by intragastric nutrition to estimate the digestibility of isolated individual constituents and amino acids (AA) of rumen micro-organisms (RMO) in the small intestine.2. Five levels of RMO were infused into the abomasum. The apparent and true disappearance of the individual components were measured by regression of abomasal input on the passage at the ileum. In order to proceed further to state the AA requirement of ruminants precisely it is also necessary to estimate the digestibility of each AA from RMO since this is the main protein source for ruminants. The present paper describes the estimation by regression analysis of the true digestibilities of total N, total AA-N, RNA, DNA and nineteen individual AA of RMO and the endogenous losses of these substances in the small intestine of three sheep sustained entirely by a constant intraruminal infusion of volatile fatty acids (VFA) and minerals and abomasal infusion of five different amounts of RMO. A preliminary account of some of this work has been given previously (Storm & Orskov, 1982). MATERIALS A N D METHODS Animals.Three Suffolk x (Finnish Landrace x Dorset Horn) wether lambs, weighing between 27 and 36 kg, were used.Each animal was surgically prepared with a permanent rumen cannula and an abomasal catheter, as previously described by Orskov et al. (1979). Furthermore, the animals were fitted with a simple T-piece cannula in the distal part of the ileum, 15-20 mm anterior to the ileo-caecal junction. The open end of the ileal cannula was exteriorized midway up on the right flank of the animal and a plastic loop arranged on each side of the cannulas according to Hecker (1974) so that total collection of ileal fluid was possible.Infusion treatment. After the lambs had been fitted with the cannulas they were kept in individual metabolism cages for 2 weeks before intragastric infusion was commenced. The infusion regimen was then gradually introduced over 10-12 d. Clean drinking water was freely available at all times. 16-2

.Four experiments were carried out to identify and quantify the limiting amino acids (AA) in rumen microbial protein (RMP).2. A method was developed which involved first, an assessment of the efficiency of utilization of absorbed AA-nitrogen (v) of RMP, defined as the retention of AA-N from RMP absorbed from the small intestine, and second, addition of a mixture of AA similar to the absorbed AA profile in a quantity defined by the U of RMP and equal to (1 -v)/U. Third, it involved removal of each AA in turn and measurement of the resultant N retention. Using this approach it was possible to calculate both the order and extent of AA limitations in RMP.3. Apart from methionine which was found to be the most limiting AA, only lysine, arginine and histidine reduced N retention when omitted, and accordingly only these AA were limiting in RMP.4. The method is discussed in detail and the amount of supplementary AA required to utilize RMP fully is calculated.Although ruminants usually absorb a mixture of dietary, microbial and endogenous amino acids (AA) simultaneously, the microbial protein usually accounts for the largest proportion of the total AA-nitrogen entering the small intestine of ruminants. It is therefore meaningful to determine the limiting AA of the rumen micro-organisms (RMO) separately because the AA composition of microbial protein is relatively constant (Storm 8z 0rskov, 1983).Unfortunately, AA requirements are not easily measured in large animals such as ruminants, and progress towards the elucidation of the limiting AA in this important class of livestock has been slow and laborious. Although an indication of the limiting AA may be obtained by comparing the animal's AA composition with that of the absorbed AA profile, such estimates frequently relate to only a few specific conditions (Williams & Hewitt, 1979). Furthermore, even when AA profiles are available for RMO, the difficulty of measuring AA absorption and its varying extent make comparisons based on AA profiles alone (Buttery & Cole, 1977) uncertain.Given the complex AA and N metabolism of the ruminant and its microbes, it is difficult to identify and quantify the limiting AA in any particular diet. The standard approach of identifying the first limiting AA and then determining its optimum concentration in a diet suffers from serious disadvantages which make the precise level of requirement of any particular AA difficult to define.Experiments to identify those AA which are limiting, other than the major AA, are not easy to design. Harper (1959) suggested a method whereby individual AA were omitted in turn. This method has been useful in giving basic qualitative information on limiting AA, and on problems of imbalance. In the present paper we describe a method to determine quantitatively the order of limitation of essential AA in RMO or, more precisely, the limiting AA in the AA absorbed from the small intestine, in sheep nourished by infusions of volatile fatty acids and given RMO as the only source of protein. The non-AA-N in the isolated RM...