I . The results of over 500 determinations of the heat of combustion of the urine produced by cattle and sheep have been analysed statistically. 2. The analytical errors for nitrogen, carbon and heat of combustion were k 0.54, f 1'4 and ? 2.2 %. The error attached to an estimate of the heat of combustion of the urine produced by an individual sheep in 4 days was & 10 %. 3. At the maintenance level of feeding, the heat of combustion of the urine ( U kcal/ 100 kcal food) was related to the crude protein content of the diet (P%) by the equation U = 0.25P+ 1.6, with a residual standard deviation of To.88 kcal/Ioo kcal. 4. Regression analysis of the relation between the heat of combustion of urine and its N content showed significant differences with diet. The heat of combustion of the urine of sheep was 9.7 kcal/g C and of cattle 10.3 kcal/g C, and did not vary with diet. 5. It is shown that the variation in the heat of combustion of urine/g N and its relative constancy/g C arises largely from variation, from diet to diet, in the proportion of the N excreted as hippurate. The metabolizable energy of a feed is its heat of combustion less the heat of combustion of the faeces, urine and combustible gas produced when it is eaten. In many trials with ruminants designed to provide an estimate of the nutritive value of feeds, the measurements made are limited to determinations of the heat of combustion of the feed and the faeces and neither methane nor urine energy are determined. In some trials, however, the nitrogen content of the urine is determined. Methods are available which enable the methane produced when different diets are given to ruminants in different amounts to be predicted with reasonable precision from knowledge of the faecal loss of energy (Blaxter & Clapperton, 1965) and, if methods were available for estimating the loss of urine energy from commonly measured attributes of the feed, then metabolizable energy could be estimated from the results of trials more simple than complete calorimetric ones.Understandably many attempts have been made to estimate the heat of combustion of ruminant urine from its more easily measured N content. The earliest of these consisted of assigning a constant calorific value to the urine/g N although most of the workers concerned realized that this ratio varied with diet (see Armsby, 1908). More recently the heat of combustion of urine has been related to its N content by linear regression methods, which, since they rarely have intercepts of zero, implies that the heat of combustion of urine changes with its N content. Thus Paladines, Reid, Van Niekerk & Bensadoun (1964) Other less satisfactory approaches have been made. Thus both Elliot & Loosli (1959) with cattle and Street, Butcher & Harris (1964) with sheep and cattle have related the heat of combustion of urine/unit weight to its N content/unit weight. Both found positive intercepts for their regressions. This approach implies that the heat of combustion of the urine produced by an animal in a day is greater when it is s...

Many of the diets given to ruminants contain more protein than is required to meet the protein needs of maintenance and growth. Thus cattle fattening on very highquality pasture receive on occasions nearly five times as much protein as they need as a source of amino acids. I n winter, larger amounts of nitrogen are usually given than minimal requirements dictate because the appetite for diets low but adequate in N content is usually poor. Rations containing an excess of N are more readily eaten.T h e value to be placed on protein as a source of energy for productive purposes is thus of importance in any assessment of the nutritive value of diets. showed that when protein supplements were given they were digested to the extent of 86 and 93%, and that for every IOO kcal of protein energy given in addition to a maintenance ration 33 and 35 kcal were retained in the body by cattle and sheep respectively. Similar experiments with pigs by Fingerling, Kohler & Reinhardt (1912-13) and with rats by Kriss, Forbes & Miller (1934) showed protein supplements to be better digested by these simple-stomached species than by ruminants and that pigs and rats retained respectively 57 and 55 kcal energy/Ioo kcal protein energy given.The net availability of the metabolizable energy of protein in the two sets of experiments was 46 and 48 yo with cattle and sheep respectively and 7 4 and 69 % with pigs and rats respectively. Ruminant animals thus produce about 60% more heat than do non-ruminants when the metabolizable energy of protein is used to synthesize fat.The experiments reported here, which have already been briefly summarized (Martin & Blaxter, 1961) were undertaken to find whether the low utilization of protein by ruminants for fat synthesis is contingent upon its fermentation in the rumen or whether it reflects some difference in the way ruminants obtain free energy from the dissimilation of protein when they are compared with species with simple stomachs. E X P E R I M E N T A LAnimals. Two sheep (B and P) were fitted with cannulas into the rumen and two (E and F) with cannulas into the abomasum. The abomasal cannulas were inserted 2 cm distant from the pylorus in each sheep. The sheep were of mixed breeding and weighed between 63 and 72 kg.

1. The extent to which phenolic derivatives of benzoic acid (seven); of phenylacetic acid (one); of 3-phenylpropionic acid (one) and of cinnamic acid (six) served as precursors of the urinary benzoic acid excreted by sheep was determined after administration as continuous drips via rumen or abomasal cannulas.2. Phenolic derivatives of benzoic or of phenylacetic acid were not dehydroxylated to yield aromatic acids following administration via either route.3. Rumen infusion of phenolic derivatives of both 3-phenylpropionic and cinnamic acids gave enhanced rumen concentrations of 3-phenylpropionic acid'with negligible amounts of benzoic acid. Between 63 and 106% of the 2-, 3-or 4-hydroxy acids, of the 3,4-dihydroxy acids or of the 3-rnethoxy, 4-hydroxy acids infused were excreted in the urine as benzoic acid and a variable proportion, characteristic of the individual animal, of up to 20% of the dose as cinnamic acid.4. Abomasal infusion of monohydroxy 3-phenylpropionic and cinnamic acids did not yield urinary benzoic acid increments. However, between 1 1 and 34% of abomasally-infused disubstituted phenolic cinnamic acids infused were excreted in the urine as benzoic acid due, it is postulated, to entero-hepatic circulation and microbial metabolism of the infused acids in the large intestine.5. It is concluded that rumen microbial metabolism of dietary phenolic cinnamic acids to 3-phenylpropionic acid followed by its absorption and oxidation in the body tissues is responsible for the greater part of the benzoic and cinnamic acids found in ruminant urine.

I . The contribution of dietary constituents to the large urinary output of benzoic acid characteristic of ruminants and some herbivores is not well understood.2. Methods for the analysis of quinic, cyclohexanecarboxylic, benzoic, phenylacetic, 3-phenylpropionic and cinnamic acids in urine and in rumen Huids were developed.3. The urinary output ofaromatic acids by sheep given seven rations was determined: benzoic acid output varied between 2.8 and 7.8 g/d; phenylacetic acid output between 0.16 and 1.3 g/d; cinnamic acid between 0.08 and 0.25 g/d and small amounts of 3-phenylpropionic acid were found in some samples. 4.Increments in urinary aromatic acid excretion were determined when the acids listed in paragraph 2 were infused via rumen or abomasal cannulas.5. When cyclohexanecarboxylic acid was infused 40% of the dose was excreted as urinary benzoic acid after either route of infusion. Quinic acid was completely metabolized in the rumen; following rumen infusion between 16 and 53% of the infused acid was recovered as urinary benzoic acid; none was so recovered after abomasal infusion.6. Urinary recoveries of rumen-and abomasally-infused aromatic acids were: benzoic acid 90 and 88% respectively as benzoic acid, phenylacetic acid 78 and 83% respectively as phenylacetic acid, 3-phenylpropionic acid 96 and l050/, respectively as benzoic acid and cinnamic acid, 70 and 70% respectively as benzoic acid.7. The concentration of aromatic acids in rumen fluid varied with time after feeding: cyclohexanecarboxylic acid was maximal (7 mg/l) 1 h after feeding, benzoic acid was always a minor component (0.5k0.5 mg/l), phenylacetic acid varied between 0 and 35 mg/l and 3-phenylpropionic acid between 25 and 47 mg/l. Cinnamic acid was not found in rumen fluid but on rumen infusion of this acid the concentration of 3-phenylpropionic acid in rumen Huid increased by 10 mg/l rumen fluid per g infused per d.8. The incomplete metabolism of quinic and cyclohexanecarboxylic acids to urinary benzoic acid is discussed. It IS concluded that the principal dietary precursors of urinary benzoic acid in ruminants are compounds yielding 3-phenylpropionic acid on microbial fermentation in the rumen. The small amount of cinnamic acid characteristic of ruminant urine arises as an intermediate in the /3-oxidation of 3-phenylpropionic acid in the body tissues.

I. Two adult wether sheep were maintained on a diet of hay and two on a diet of dried grass for 3 weeks before starvation for a period of 10 d. Urinary excretion of the following acids w-as determined when the animals were fed and when they were fasted: total diethyl ethersoluble acids of hydrolysed and unhydrolysed urine, hippuric acid, benzoic acid and phenylacetic acid. By the 5th day of fasting, urinary output of all acids had attained stable levels that did not change during the remaining starvation period. The output of all urine fractions except phenylacetic acid declined rapidly during the first 4 d of fasting: phenylacetic acid output by all sheep increased to a maximum during the first 4 d of fast and then declined to stable values (0'42-0.73 g/24 h) which were greater than those observed when the sheep were fed. It is concluded that prolonged retention of food and microbial residues in the digestive tract is responsible for the large ouptut of phenylacetic acid in the urine of fasted sheep. 2.Solutions of casein which supplied between 6.3 and 26-5 g nitrogen/zq. h were i n b e d into the rumens (fifteen experiments) or abomasums (sixteen experiments) of cight adult wether sheep. Ruminal infusions of casein caused increments in the urintlry excretion of diethyl ethersoluble acids and phenylacetic acid. Both these increments were described by linear regression equations ( P < 0.001)~ the coefficients of which showed that 284 f44 and zzo +ZI mg benzoic acid equivalent were excreted as diethyl ether-soluble acids and phenylacetic acid respectively per g casein N infused. The phcnylacctic acid excreted was equivalent to 95 o//b of the phenylalanine of the infused casein. No increments in urinary benzoic acid were observed. One sheep scoured when it was given an abomasal infusion of casein. This was the only animal to show any increment in urinary aromatic acids when casein was infused into the abomasum.3. When four sheep were given two rations containing an excess of carhohydratc as sugarbeet pulp or rolled barley, I I and 16 : ( , respectively of their phenylalanine intakes were excreted in the urine as phenylacetic acid. When the same sheep were given two rations containing an excess of N as linseed meal or field beans, 51 and j9 74 respectively of their phenylalanine intakes appeared in the urine as phenylacetic acid.4. Methods for the determination of creatinine, and of benzoic, phenylacetic, g-phenylpropionic, cinnamic, hippuric and phenaceturic acids are described. 5.It is suggested that the amount of phenylacetic acid excreted in the urine is a measure of the equilibrium occurring in the rumen between catabolism of phenylalanine and reutilization of the products of catabolism for phenylalanine synthesis.