THE EFFECT OF A HINDGUT FERMENTATION ON UREA METABOLISM IN SHEEP NOURISHED BY INTRAGASTRIC INFUSION

Transcription

1 Experimental Physiology (1990), 75, Printed in Great Britain THE EFFECT OF A HINDGUT FERMENTATION ON UREA METABOLISM IN SHEEP NOURISHED BY INTRAGASTRIC INFUSION A. ONCUER*, J. S. MILNE AND F. G. WHITELAWt Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB (MANUSCRIPT RECEIVED 10 NOVEMBER 1989, ACCEPTED 26 MARCH 1990) SUMMARY Four female sheep nourished wholly by infusions of volatile fatty acids, buffer and minerals into the rumen and casein into the abomasum were given, in addition, infusions of fermentable carbohydrates into the terminal ileum. The ileal infusions consisted of (1) water alone, (2) 25 g starch + 50 g cellulose, and (3) 50 g starch + 50 g cellulose. Measurement of nutrient digestibility and nitrogen retention were made over 5 days and the kinetics of urea metabolism were measured over 24 h by means of a single injection of ['4C]urea. Endogenous urinary nitrogen excretion was measured over a subsequent 5 days when casein was omitted from the infusion mixtures. Increases in hindgut fermentation resulted in a significant increase in faecal nitrogen excretion (P < 0 01) and a corresponding reduction in urinary urea nitrogen excretion (P < 0 05). The ileal infusions did not significantly affect urea irreversible loss rate or urea pool size and were also without effect on plasma urea or rumen ammonia concentrations. Urea degradation in the gastrointestinal tract increased by about 2 g/day in progressing from lowest to highest level of hindgut infusion but differences between treatments were not statistically significant. Endogenous urinary nitrogen excretion was not affected by hindgut fermentation and averaged 206 mg N/(kg0 75 day) over the three treatment groups. Faecal nitrogen excretion however increased progressively with increase in ileal infusions (P < 0 05) and was similar to that seen when nitrogen input was adequate. It is concluded that changes in hindgut fermentation can alter the partition of nitrogen excretion between faeces and urine but the quantities involved are small relative to the total exchange of urea across the digestive tract. The rumen appears to be an important site of urea degradation even when microbial fermentation is absent. INTRODUCTION The process of urea recycling is usually considered to be more highly developed in ruminants and other herbivores than in simple stomached species. However, the precise mechanism of the exchange of urea between blood and the gastrointestinal (GI) tract and the relative contributions of different parts of the tract are not yet known with any certainty (Harmeyer & Martens, 1980; Kennedy & Milligan, 1980; Egan, Boda & Varady, 1986). A major problem in resolving these questions is that of identifying those factors which are components of host-animal metabolism and those which reflect specific attributes of the diet or of the rumen or intestinal microbial populations. In looking anew at these topics the system of intragastric nutrition developed by 0rskov and his colleagues (0rskov, Grubb, Wenham & Corrigall, 1979) appeared to offer some advantages since it allows ruminants to be sustained for long periods on pure nutrients and without the complications which ensue from the presence of a fermenting biomass in the forestomachs. In an initial * Present address: Lalahan Nuikleer Arastirma Enstitusu, Lalahan, Ankara, Turkey. t For reprints.

2 690 A. ONCUER, J. S. MILNE AND F. G. WHITELAW investigation Whitelaw, Milne, Orskov, Stansfield & Franklin (1990) compared the kinetics of urea production and recycling in sheep given normal feeds and in the same sheep nourished by intragastric infusion. This trial showed that there were no major differences in urea metabolism between the two methods of feeding. Despite the absence of an active rumen microbial population in animals nourished by infusion, the quantity of urea recycled to the GI tract was similar for the two feeding systems. There was however an indication that the sites of urea degradation within the tract might differ between the two treatments. The work reported here was an extention of these studies and was designed to examine the role of the hindgut in the process of urea recycling. 0rskov, Fraser, Mason & Mann (1970) and Thornton, Bird, Somers & Moir (1970) have shown that increases in hindgut fermentation achieved by ileal infusion of nutrients can result in a transfer of urea from blood to the lower digestive tract, but the extent of this transfer in relation to whole body urea metabolism does not appear to have been examined previously. The opportunity was taken in the present work to investigate also the effect of hindgut fermentation on endogenous urinary nitrogen (EUN) excretion and on the partition of nitrogen (N) between urine and faeces when N-free infusions were given. METHODS Animals and management Four female 7-month-old Suffolk x Scottish Blackface lambs of about 37 kg were used. Each had been fitted with a rumen cannula, an abomasal infusion catheter and an ileal infusion catheter about 4 weeks before the start of the experiment (0rskov et al. 1979). The ileal catheter was inserted about 25 cm anterior to the ileo-caecal junction. One of the lambs was also fitted with a cannula in the caecum (MacRae, Reid, Dellow & Wyburn, 1973), but digesta was not collected from it in this experiment and there were no significant effects attributable to this animal. The lambs were housed indoors in metabolism cages and were adapted to intragastric nutrition over a period of 6 days. Thereafter they were maintained wholly by intragastric infusion as described by Whitelaw et al. (1990). The volatile fatty acid (VFA) solution infused into the rumen contained acetic, propionic and n-butyric acids in the molar proportions 0-65, 0 25 and 0O10 respectively. The quantities of VFA and casein infused daily were adjusted according to liveweight at the start of each period to provide energy and N to meet the requirements for maintenance, taken to be 450 kj/(kg bodyweight075 day) and 350 mg N/(kg bodyweight075 day) respectively (Hovell, 0rskov, Grubb & MacLeod, 1983). Rumen ph and osmotic pressure (OP) were measured twice daily to assess rumen conditions. In addition to the standard infusion solutions, animals were given infusions of starch and cellulose into the terminal ileum to induce fermentation in the hindgut. The starch was raw maize starch and the cellulose was a commercial spray-dried product containing 85 % microcrystalline cellulose and 15% sodium carboxymethylcellulose (Avicel CL6 1; FMC Corporation, Philadelphia, USA). The mixtures of starch and cellulose were gelatinous and required continuous stirring with an electric stirrer during infusion (Cipenco, type LC9; Park Products Ltd, Blackburn, UK). Design and treatments The treatments were intended to create different levels of fermentation in the hindgut and involved the daily infusion at the terminal ileum of either (1) water alone, (2) 25 g starch + 50 g cellulose or (3) 50 g starch +50 g cellulose. These quantities of starch and cellulose were based on estimates of what might be expected to reach the terminal ileum daily in sheep given conventional diets (0rskov, Fraser & Kay, 1969; Orskov et al. 1970). On each treatment the daily allowance was infused over 23 h in a total volume of 2 1. To help establish the initial fermentation an inoculum of rumen fluid (approx. 50 ml) from a cow was given into the hindgut at the start of each period. Animals were allocated to treatments according to a randomized block design, with experimental periods of 3 weeks duration. In each period, animals were adjusted to the prescribed level of hindgut infusion over the first 7 days and days 8-12 inclusive (5 days) were used for digestibility and N balance

3 UREA METABOLISM IN SHEEP measurements. A catheter was inserted in one jugular vein on day 13 and an intravenous injection of ['4C]urea for the measurement of urea kinetics was given on day 14. Days (5 days) constituted an N-free period, in which casein infusion was discontinued and faeces and urine were collected to establish endogenous N excretion. Body nitrogen stores were then repleted over days 20 and 21, and days 1, 2 and 3 of the next treatment period by increasing the casein allowance on successive days. Daily N input returned to the standard level of I 0 x maintenance from day 4 of the next period. The change-over of ileal infusion treatments took place on day I of each period. Measurements and sampling procedures The methods used to estimate the kinetics of urea metabolism by means of a single intravenous injection of [14C]urea were identical to those described by Whitelaw et al. (1990) except that the number of blood samples taken over the 24 h following injection was reduced to fifteen. Samples of rumen contents were taken at intervals over 24 h for the measurement of ph, OP and ammonia (NH3) concentrations. Urease activity in rumen fluid was measured in pre-injection samples only and the molar proportions of VFA in rumen fluid were measured in period one only. Urea pool size and irreversible loss rate (ILR) from the plasma urea pool were estimated from the parameters of the double exponential curve relating the decline in plasma urea SA to time (Whitelaw et al. 1990). Urea degradation was taken to be the difference between urea ILR and the rate of excretion of urea in urine. Urea space was calculated as urea pool size (mg)/plasma urea concentration (mg/i) and expressed as a proportion of bodyweight (1/kg). For the digestibility and N balance measurements, faeces were bulked over 5 days and analysed for dry matter (DM), ash, N, starch and acid detergent fibre (ADF). The ADF excreted in faeces was assumed to represent cellulose. Urine was analysed on a daily basis for N and urea. Samples of fresh faeces for microbiological examination were taken from the rectum of each sheep on day 13 of each treatment period. Daily collections of urine and faeces were also made over the 5 days when N-free infusions were given. Urine was analysed daily; faeces were bulked over days 1-3 but were analysed separately on days 4 and 5. Analytical methods The estimation of urea in urine and plasma, of total N in faeces and urine and of VFA proportions and NH3 in rumen fluid were as described by Whitelaw et al. (1990). Urease (EC ) in rumen contents was determined as described by Cook (1976). Faeces were analysed for starch as described by Bergmeyer (1963) and for ADF by the methods of AOAC (1975). Total viable bacteria in faeces samples were counted as described by Hobson (1969) using M8 roll tubes and cellulose roll tubes. Aerobic bacteria were counted using plate count agar (Leininger, 1976). The preparation of radioactive materials and assay of radioactivity in plasma was as described previously (Whitelaw et al. 1990). Statistical methods The experimental design was a randomized block in which the three treatments were unequally represented in each period. The twelve observations for each variable were subjected to an analysis of variance which allowed treatment means to be adjusted for animal and period effects. All statistical analyses were performed using GENSTAT (1982). 691 RESULTS Animal health The health of the animals remained good throughout the experiment. One sheep however showed symptoms of severe rumen distension shortly after its first introduction to ileal infusion and radiological examination indicated ileal blockage. This was treated by discontinuing the infusion and allowing access to chopped dried grass for about 1 week. A second animal showed similar symptoms on the second day of N-free infusions in period three; this animal failed to respond to a change of diet and was discarded from the experiment.

4 692 A. ONCUER, J. S. MILNE AND F. G. WHITELAW Table 1. Daily nutrient inputs, faecal excretions, apparent digestibility coefficients and bacterial counts in faeces of sheep nourished by intragastric infusion and given supplements of starch and cellulose by infusion into the terminal ileum. Mean values for each variable, adjusted for differences between animals Treatmentt S.E.D. Pt No. of animals Liveweight (kg) Nutrient inputs Energy (MJ/day) Nitrogen (g/day) Organic matter (g/day) Starch (g DM/day) Cellulose (g DM/day) Faecal excretion Dry matter (g/day) ** Organic matter (g/day) ** Nitrogen (g/day) ** Starch (g/day) n.s. Cellulose (g/day) ** Apparent digestibility coefficients Organic matter ** Starch n.s. Cellulose n.s. Faecal bacteria Aerobic (log counts/g) n.s. Anaerobic (log counts/g) n.s. t Treatments 1, 2 and 3 refer to the levels of starch and cellulose infused: 1 = nil; 2 = 25 g starch +50 g cellulose; 3 = 50 g starch + 50 g cellulose (weights expressed on air-dry basis). t P = statistical significance: n.s., non-significant; **, P < Estimated as acid-detergent fibre (ADF). S.E.D. = standard error of differences; DM = dry matter. Rumen conditions Adjustments to the volume of buffer solution infused made it possible to control rumen ph and OP within fairly narrow limits; in samples taken on the days of 14C injections, rumen ph ranged between 6-3 and 6-8 in different animals with a mean of 6 5 and OP ranged between 204 and 245, with a mean of 224 mosmol/kg. The mean molar proportions of VFA (± S.E.M.) in the rumen fluid of the four animals examined in period one were acetic acid 067+0±004, propionic acid and n-butyric acid Nutrient inputs and apparent digestibility Mean daily inputs and faecal excretions of the major nutrients are given in Table 1 together with the calculated apparent digestibility coefficients for organic matter (OM), starch and cellulose. Although there were significant increases in OM input between each of the dietary treatments, the excretion of DM and OM in faeces differed only between the zero level of hindgut infusion (treatment one) and the other two treatments (P < 0-01). Only small quantities of starch appeared in faeces on all three treatments whereas considerable amounts of cellulose were present in faeces when cellulose was infused at the terminal ileum. This was reflected in low digestibility values for cellulose (Table 1). Although there was clearly a progressive increase in numbers of both aerobic and

5 UREA METABOLISM IN SHEEP Table 2. Daily intakes, excretions and retention ofnitrogen and urinary excretions ofurea N in sheep nourished by intragastric infusion and given supplements of starch and cellulose by infusion into the terminal ileum. Mean values for each variable, adjusted for differences between animals Treatmentt S.E.D. Pt No. of animals Liveweight (kg075) Nitrogen intake (mg/(kg075 day) n.s. Urine nitrogen (mg/(kg075 day) n.s. Urine urea N (mg/(kg0'5 day) * Faecal nitrogen (mg/(kg075 day) ** Nitrogen retention (mg/(kg075 day) n.s. See Table 1 for description of treatments. t P = statistical significance: n.s., non-significant; *, P < 005; **, P < S.E.D. = standard error of differences. Table 3. Urea metabolism ofsheep nourished by intragastric infusion and given supplements of starch and cellulose by infusion into the terminal ileum. Mean values for each variable, adjusted for differences between animals Treatmentt S.E.D. Pt No. of animals Plasma urea concentration (mg/100 ml) n.s. Rumen ammonia concentration (mg/l00 ml) n.s. Rumen urease activity (,umol NH3/(min ml)) n.s. Urea metabolism Irreversible loss rate (g/day) n.s. Urea in urine (g/day) * Urea degradation (g/day) n.s. Urea pool size (g) n.s. Urea space (1/kg liveweight) n.s. t See Table 1 for description of treatments. t P = statistical significance: n.s., non-significant; * P < 0O05. S.E.D. = standard error of differences. anaerobic bacteria in faeces with increase in nutrient supply to the hindgut, differences between treatments were not signiticant. The poor digestibility coefficient recorded for cellulose was confirmed by a virtual absence of cellulolytic bacteria in faecal material; of the twelve samples examined, a significant count of cellulolytic organisms (3 5 x 108/g) was found in only one. Nitrogen metabolism The mean daily input, excretion and retention of total N and urinary excretion of urea N on each dietary treatment are given in Table 2. N intake was constant across treatment groups with an overall mean value of 453 mg/(kg075 day) (6-8 g N/day). Uninary N excretion did not differ significantly between treatment groups. Faecal N excretion however 693

6 694 A. ONCUER, J. S. MILNE AND F. G. WHITELAW showed a progressive increase with each increase in the level of hindgut infusion (P < 0-01) and this was reflected in decreases in daily N retention in progressing from treatment one to treatment three. Urea N made up a high proportion of total urinary N excretion ( ) and the total quantity of urea N excreted was significantly lower on the high level of starch infusion than on the other two treatments (P < 0-05, Table 2). Urea metabolism The mean concentration of urea in plasma and of NH3 in rumen fluid on the day of ["4C]urea injection did not differ significantly between treatments (Table 3). The values recorded for urease activity in rumen fluid were highly variable and did not differ significantly between treatments. Urea ILR did not differ between treatments and showed a mean overall value (+ S.E.M.) of g/day (Table 3). Excretion of urea in urine however was significantly lower (P < 0 05) on treatment three than on the other two treatments. Urea degradation in the GI tract increased by over 2 g/day (0-98 g N/day) in progressing from the control treatment to the highest level of hindgut infusion but again the differences were not significant. When calculated as a proportion of daily ILR, urea degradation values were 0-41, 0 45 and 0 53 for treatments one, two and three respectively. Urea pool size did not differ between treatments and was on average 2-27 g urea, equivalent to a mean theoretical urea 'space' amounting to 0-56 of bodyweight. The various indices of urea metabolism were examined by regression analysis for evidence of interrelationships and the more important of these are summarized in Table 4. A highly significant linear relationship (P < 0-001) was seen between urea pool size (mg/kg075) and plasma urea concentration (mg/100 ml), similar to that previously reported by Whitelaw et al. (1990). Significant linear relationships (P < 0-05) also existed between plasma urea concentration on the one hand and ILR, urinary urea excretion and rumen NH3 concentration on the other. Examination of the relationships between urea degradation rate and the variables thought likely to have an influence on this parameter showed that degradation rate was most closely related to ILR (P < 0-001). Urea degradation was also related to rumen NH3 concentration (P < 0-05) but was not related to either plasma urea concentration or rumen urease activity. Endogenous N excretions On all three treatments urinary N excretion declined over the first 3 days of N-free input and then remained constant at about 200 mg N/(kg075 day). This compares with a mean value of about 350 mg N/(kg075 day) when adequate N was included in the infusates (Table 2). Statistical analysis of the changes in N excretion between day 3 and day 5 of N-free infusion showed that these did not differ significantly from zero and the mean excretions over days 3-5 inclusive were therefore examined for the presence of treatment effects. Urinary N excretion on N-free input (the EUN excretion) did not differ between the three treatment groups and had an overall mean value of 206 mg N/(kg075 day) (Table 5). Urine urea N excretion however was consistently lower on treatment three than on the other two treatments throughout the 5 day period. Faecal N excretion in contrast increased progressively from treatment one to treatment three and was significantly higher in animals given starch and cellulose into the hindgut than in those given the control treatment (P < 0 05). In consequence total N excretion was also higher in the animals given infusions into the hindgut (P < 0-1). Faecal N excretion on each treatment during N-free infusions was identical to that observed when adequate N was given (Table 2).

7 UREA METABOLISM IN SHEEP ** * t~~~~~~~~d (ON C>(oNo )(-0.1 g ~~ oooooo -5 :1 0~+.0_0 en L. X et00.w q's~ ~~~r e s > ~~~~~D > C> C >.E -D -~~~~~ V~~~~ o col~ ~ ~ ~ $} E E E E,, ++ > - >~I b Do~~M O OO n ce zzs Y Y E++i 0 > U U U Q ce M

8 696 A. ONCUER, J. S. MILNE AND F. G. WHITELAW Table 5. Mean daily excretion ofnitrogen during days 3-5 inclusive of a 5 day N-free infusion period in sheep nourished by intragastric infusion and given supplements ofstarch and cellulose by infusion into the terminal ileum. Mean values for each variable, adjusted for differences between animals Treatmentt S.E.D. Pt No. of animals Nitrogen excretion (mg/(kg075 day) Urine n.s. Faeces * Total n.s. Urine urea N n.s. See Table I for description of treatments. t P = statistical significance: n.s., non-significant; *, P < 0O05. S.E.D. = standard error of differences. DISCUSSION Hindgut fermentation The extensive disappearance of starch between ileum and rectum provided clear evidence that bacterial fermentation was satisfactorily established in the caecum-colon in response to the infusion of nutrients at the terminal ileum. The infused cellulose was poorly degraded in the hindgut but this was obviously due to the absence of an adequate population of cellulolytic bacteria. Mann & Orskov (1973) also found that the number of cellulolytic organisms in caecal contents did not increase in sheep given cellulose suspensions by bottle and attributed this to an excessive rate of passage of cellulose relative to the rate of bacterial degradation. The infusions of starch and cellulose at the terminal ileum resulted in increases in faecal N excretion of 0 50 and 0-78 g/day at the low and high levels of infusion respectively (Table 1). This is equivalent to a daily excretion of about 1V7 g N for each 100 g DM fermented in the hindgut and hence is somewhat higher than values reported by others. Thus 0rskov et al. (1970) infused starch into the caecum of sheep given dried grass diets and calculated that 100 g of starch fermented in the large intestine was associated with an increase in the faecal excretion of 'bacterial and endogenous debris-n' of about 1 g/day. Similarly Thornton et al. (1970) infused up to 90 g glucose daily to the ileum of sheep receiving a diet of oat hulls and lucerne chaff and showed an increase in faecal N of 1 g/day at the highest level of glucose infusion. 0rskov & Grubb (1978) also observed an increase in faecal N excretion of 05 g/day when 50 g of a mixture of glucose and methyl cellulose was infused at the terminal ileum of a single sheep nourished by intragastric infusion. In each of the above studies (0rskov et al. 1970; Thornton et al. 1970; 0rskov & Grubb, 1978) the increased excretion of N in faeces was accompanied by a decrease in the excretion of N in urine and it was suggested that this inverse relationship indicated a transfer of urea N from the blood to the hindgut digesta. A similar conclusion can be drawn in the present work, since a decrease in the excretion of both total N and urea N in urine was seen at the higher level of hindgut infusion (Table 2). Estimates of urea degradation obtained from '4C kinetic measurements (Table 3) also indicated a progressive increase in urea transfer to the GI tract with increasing levels of hindgut fermentation. Although differences between

9 UREA METABOLISM IN SHEEP 697 treatments were not significant, the overall change in degradation between treatment one and treatment three was 2 1 g urea (0-98 g N) and was thus of a similar order to the 0-78 g increase in faecal N excretion noted above. The difference between these two values, equivalent to 0-43 g urea, presumably represents the portion of degraded urea which was not incorporated into microbial protein but simply reabsorbed as NH3 from the hindgut. Since the excretion of N in faeces was identical whether or not casein was infused at the abomasum (Tables 2 and 5) it must be assumed that casein is wholly digested and that the N eliminated in faeces on this system of feeding is entirely of endogenous origin. Sites of urea degradation The estimated passage of urea into the hindgut in this experiment represented a relatively small proportion of total urea degradation in the entire GI tract. Even when the hindgut fermentation was artificially enhanced the apparent transfer of urea to this part of the intestine accounted for only 22% of the total and this is presumably a higher value than that likely to be encountered under normal feeding conditions (0rskov et al. 1970). If excretion of N in faeces is taken as a guide, transfer of urea to the hindgut amounted to no more than 10 % of total transfer at the basal level of infusion (treatment one). Engelhardt & Hinderer (1976) reported that transfer of urea into the colon of goats was only 8 % of transfer to the total digestive tract when a hay diet was fed and reached a maximum of 14% when food was withheld for 48 h. Dixon & Nolan (1983) and Dixon & Milligan (1984) also showed that the large intestine and caecum are not of major importance in endogenous urea degradation in sheep given normal diets. We suggested in our earlier work that intragastric infusion systems and the absence of a ruminal microbial population might be associated with a shift in the site of urea degradation away from the rumen and towards the lower digestive tract (Whitelaw et al. 1990). The present work shows, however, that only a modest proportion of total degradation is associated with the large intestine. By implication, therefore, any shift in degradation site must be between rumen and small intestine. Substantial quantities of urea are reported to enter the small intestine both by diffusion and as a constituent of digestive secretions (Boda, Varady, Havassy & Egan, 1986) but the extent to which this is hydrolysed and contributes to measured urea degradation is not certain. Although an adherent epithelial population of bacteria has been identified at a variety of sites within the small intestine (Cheng, McCowan & Costerton, 1979), urease activity is reported to be absent from the duodenum and jejunum and to be low in ileal contents (Michnova, Boda, Thomas & Havassy, 1979). Egan et al. (1986) have suggested that much of the urea entering this part of the tract may be reabsorbed intact. Moreover, digestive secretions are probably much reduced under intragastric infusion conditions on account of the small quantities of dry matter passing through the digestive tract; if this is so, it is likely that urea entry to this part of the tract will be correspondingly low. Although the relative importance of the rumen vis-a-vis the small intestine as a site of urea degradation cannot be ascertained in the present work there is evidence that the rumen is probably the more important of the two. Thus rumen NH3 concentrations were positively related both to plasma urea concentration and to total urea degradation (Table 4), suggesting that the major portion of NH3 in rumen contents arises by transfer of urea through the rumen wall. Urease activity in the rumen epithelial tissue of animals nourished by infusion is reported to be similar to that seen in conventionally fed animals (Wallace, Cheng, Dinsdale & 0rskov, 1979) and although highly variable in the present work the activities recorded in the rumen fluid were probably adequate to effect complete hydrolysis

10 698 A. ONCUER, J. S. MILNE AND F. G. WHITELAW of the urea presented. The absence of any significant relationship between urease activity and total urea degradation probably indicates that urease activity was not a major constraint to urea transfer to the rumen. If the rumen is indeed the major site of urea degradation in infusion sheep, the absence of a microbial population suggests that the NH3 produced must simply be reabsorbed as such and converted once more to urea in the liver. In this regard it is important to note that although urea ILR appears to be the most important determinant of urea degradation as judged by the relationship presented in Table 4, conclusions based on this relationship must be treated with caution. This is so, firstly, because ILR is used in the calculation of degradation and hence the two variables are not truly independent and secondly, because [14C]urea on hydrolysis gives rise to unlabelled NH3 and this, if not incorporated into bacterial proteins or other large molecules, will contribute anew to the measured ILR. Thus urea degradation will always be positively related to ILR when '4C is used as the marker and the relationship will be closer the greater the extent of urea degradation and the greater the proportion of hydrolysed urea which is reabsorbed as NH3. Endogenous nitrogen excretion The responses noted here to N-free conditions confirm the suggestions made by 0rskov & McLeod (1982) and Hovell et al. (1983) that the sum of urinary and faecal losses must be considered in arriving at estimates of total endogenous N (TEN) losses in ruminants. The mean EUN in this experiment was (S.E.M.) mg N/(kg075 day) and the mean TEN (S.E.M.) mg N/(kg075 day), both considerably lower than the value of 350 mg N/(kg075 day) adopted by the Agricultural Research Council in formulating recommendations for the protein requirements of ruminant livestock (ARC, 1984). However Hovell, Kyle, Reeds & Beermann (1989) have recently reported EUN values of (S.E.M.) mg N/(kg075 day) in ten sheep and Inkster, Hovell, Kyle, Brown & Lobley (1989) observed a mean value of 258 mg N/(kg075 day) in four lambs and two mature sheep. Hovell et al. (1989) have discussed these variations in endogenous nitrogen excretion and have suggested that while age and liveweight might play a part, the lower estimates may simply relate to animals which have been held at a constant plane of nutrition close to maintenance for several weeks before measurements were made and have therefore stabilized at a lower level of metabolic activity. In the present work, the mean daily N retention on the basal infusion treatment (65 mg N/kg0 75) was consistent with an estimate of maintenance N needs based on a TEN value of 255 mg N/(kg075 day). Conclusions It can be concluded from these findings that changes in hindgut fermentation can influence the extent of urea transfer from blood to the lower GI tract and that this is reflected in alterations in the partition of N excretion between faeces and urine. This transfer of urea to the hindgut would appear however to be small relative to the normal exchange of urea across the whole digestive tract. From the evidence available it would appear that the rumen remains the major site of urea degradation even when an active microbial fermentation is absent. We are grateful to Messrs Honeywell & Stein, Ltd, Wallington, Surrey, for the gift of Avicel microcrystalline cellulose. We would also like to express our thanks to Dr M. F. Franklin for statistical analyses, to Mrs Sylvia Duncan for microbiological examinations and to Miss Maureen Annand,-Mr R. I. Smart and their respective colleagues for assistance with chemical analyses.

11 UREA METABOLISM IN SHEEP 699 REFERENCES AGRICULTURAL RESEARCH COUNCIL (1984). The Nutrient Requirements of Ruminant Livestock, suppl. 1. Commonwealth Agricultural Bureaux, Farnham Royal, Slough. AOAC (1975). Methods of Analysis. Association of Official Agricultural Chemists, Washington DC. BERGMEYER, H. U. (1963). Methods of Enzymatic Analysis, p Academic Press, New York & London. BODA, K., VARADY, J., HAVASSY, I. & EGAN, A. R. (1986). Nitrogen recycling in ruminants. Archives of Animal Nutrition, 86, CHENG, K.-J., MCCOWAN, R. P. & COSTERTON, J. W. (1979). Adherent epithelial bacteria in ruminants and their roles in digestive tract function. American Journal of Clinical Nutrition 32, COOK, A. R. (1976). Urease activity in the rumen of the sheep and the isolation of urease activity. Journal of General Microbiology 92, DIXON, R. M. & MILLIGAN, L. P. (1984). Nitrogen kinetics in the large intestine of sheep given bromegrass pellets. Canadian Journal of Animal Science 64, DIXON, R. M. & NOLAN, J. V. (1983). Studies of the large intestine of sheep. 3. Nti_ogen kinetics in sheep given chopped lucerne (Medicago sativa) hay. British Journal of Nntrition 50, EGAN, A. R., BODA, K. & VARADY, J. (1986). Regulation of nitrogen metabolism and recycling. In Control of Digestion and Metabolism in Ruminants, ed. Milligan, L. P, Grovum, W. L. & Dobson, A., pp Prentice-Hall, NJ, USA. ENGELHARDT, W. v. & HINDERER, S. (1976). Transfer of blood urea into the goat colon. In Tracer Studies on Non-protein Nitrogen for Ruminants III, pp International Atomic Energy Agency, Vienna. GENSTAT (1982). Release Lawes Agricultural Trust, Rothamsted Experimental Station, England. HARMEYER, J. & MARTENS, H. (1980). Aspects of urea metabolism in ruminants with reference to the goat. Journal of Diary Science 63, HOBSON, P. N. (1969). Rumen bacteria. Methods in Microbiology 3B, HOVELL, F. D. DEB., KYLE, D. J., REEDS, P. J. & BEERMANN, D. H. (1989). The effect of clenbuterol and cimaterol on the endogenous nitrogen loss of sheep. Nutrition Reports International 39, HOVELL, F. D. DEB., 0RSKOV, E. R., GRUBB, D. A. & MAcLEOD, N. A. (1983). Basal urinary nitrogen excretion and growth response to supplemental protein by lambs close to energy equilibrium. British Journal of Nutrition 50, INKSTER, J. E., HOVELL, F. D. DEB., KYLE, D. J., BROWN, D. S. & LOBLEY, G. E. (1989). The effect of clenbuterol on basal protein turnover and endogenous nitrogen loss of sheep. British Journal of Nutrition 62, KENNEDY, P. M. & MILLIGAN, L. P. (1980). The degradation and utilization of endogenous urea in the gastrointestinal tract of ruminants: a review. Canadian Journal ofanimal Science 60, LEININGER, H. V. (1976). Equipment, media, reagents, routine tests and stains. In Compendium ofmethodsfor the Microbiological Examination offoods, ed. Speck, M. L., p. 57. American Public Health Association. MAcRAE, J. C., REED, C. S. W., DELLOW, D. W. & WYBURN, R. S. (1973). Caecal cannulation in the sheep. Research in Veterinary Science 14, MANN, S. 0. & 0RSKOV, E. R. (1973). The effect of rumen and post-rumen feeding of carbohydrates on the caecal microflora of sheep. Journal of Applied Bacteriology 36, MYCHNOVA, E., BODA, K., THOMAS, J. & HAVASSY, I. (1979). Urease activity in the contents and tissues of sheep, pig and chicken gastrointestinal apparatus. Physiologia bohemoslovaca 28, RSKOV, E. R., FRASER, C. & KAY, R. N. B. (1969). Dietary factors influencing the digestion of starch in the rumen and small and large intestine of early-weaned lambs. British Journal of Nutriton 23, RSKOV, E. R., FRASER, C., MASON, V. C. & MANN, S. 0. (1970). Influence of starch digestion in the large intestine of sheep on caecal fermentation, caecal microflora and faecal nitrogen excretion. British Journal of Nutrition 24, RSKOV, E. R. & GRUBB, D. A. (1978). The minimal nitrogen metabolism of lambs. Proceedings of the Nutrition Society 38, 24A.

12 700 A. ONCUER, J. S. MILNE AND F. G. WHITELAW 0RSKOV, E. R., GRUBB, D. A., WENHAM, G. & CORRIGALL, W. (1979). The sustenance of growing and fattening ruminants by intragastric infusion of volatile fatty acids and protein. British Journal of Nutrition 41, RSKOV, E. R. & MAcLEOD, N. A. (1982). The determination of the minimal nitrogen excretion in steers and dairy cows and its physiological and practical implications. British Journal of Nutrition 47, THORNTON, R. F., BIRD, P. R., SOMERS, M. & MOIR, R. J. (1970). Urea excretion in ruminants III. The role of the hind-gut (caecum and colon). Australian Journal of Agricultural Research 21, WALLACE, R. J., CHENG, K.-J., DINSDALE, D. & 0RSKOV, E. R. (1979). An independent microbial flora of the epithelium and its role in the ecomicrobiology of the rumen. Nature 279, WHITELAW, F. G., MILNE, J. S., ORSKOV, E. R., STANSFIELD, R. & FRANKLIN, M. F. (1990). Urea metabolism in sheep given conventional feeds or nourished by intragastric infusion. Quarterly Journal of Experimental Physiology 75,