UREA DEGRADATION IN SHEEP NOURISHED BY INTRAGASTRIC INFUSION: EFFECTS OF LEVEL AND NATURE OF ENERGY INPUTS

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1 Experimental Physiology (1991), 76, Printed in Great Britain UREA DEGRADATION IN SHEEP NOURISHED BY INTRAGASTRIC INFUSION: EFFECTS OF LEVEL AND NATURE OF ENERGY INPUTS F. G. WHITELAW AND J. S. MILNE Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB (MANUSCRIPT RECEIVED 26 MARCH 1990, ACCEPTED 6 SEPTEMBER 1990) SUMMARY Four female sheep nourished wholly by infusions of volatile fatty acids (VFA), buffer and minerals to the rumen and casein to the abomasum were given in addition infusions of supplementary energy calculated to increase the energy input to the rumen by 30 %. The design was a Latin square and the supplements given were (a) nil, (b) a standard VFA mixture similar to the basal infusion, (c) butyric acid alone and (d) glucose. Measurements were made of nitrogen retention and rumen fermentation characteristics and the kinetics of urea metabolism were measured over 24 h by means of a single injection of ['4C]urea. An active microbial fermentation was established in the rumen in response to the infusion of glucose and estimates of microbial protein synthesis derived from urinary purine excretion agreed well with those calculated from stoichiometric principles. The presence of a microbial population in the rumen resulted in a decrease in urinary urea excretion and reductions in plasma urea concentration, urea pool size and rumen ammonia (NH3) concentration. Infusion of the mixed VFA or butyric acid supplements had no effect on these indices of urea metabolism. Measurements of urea irreversible loss rate showed high variability and the mean values did not differ significantly between the four treatments. Urea degradation in the gastrointestinal tract was also highly variable but increased, on average, by 2 4 g day-1 on the high-energy treatments. Examination of regression relationships between these variables also indicated a difference between the glucose treatment and the others in the metabolic fate of the NH3 derived from urea hydrolysis. It is concluded that urea degradation increased in response to additions of energy but did not differ according to the nature of the supplements supplied. In the glucose-supplemented group, the NH3 arising from degraded urea was incorporated into microbial protein and so removed from the urea-nh3 cycle; when additions of mixed VFA or butyric acid were given, the NH3 arising in hydrolysis appeared simply to be reabsorbed as NH3 and to contribute anew to urea formation. INTRODUCTION It is well established that ruminant species can transfer significant quantities of urea from the blood to the gastrointestinal (GI) tract under a wide variety of dietary circumstances (Houpt, 1959, 1970; Cocimano & Leng, 1967). This transfer is thought to be of nutritional importance to the host animal since the degradation of urea to ammonia (NH3) can help maintain the microbial fermentation in the forestomach at times of inadequate nitrogen (N) supply. Although there is still considerable debate about the mechanisms which control this process, the balance of evidence suggests (a) that events within the reticulo-rumen play a prominant part in the control of urea degradation and (b) that the extent of urea transfer is in some way related to the digestible energy intake of the animal (see reviews by Harmeyer & Martens, 1980; Kennedy & Milligan, 1980; Egan, Boda & Varady, 1986). An association between digestible organic matter intake and urea degradation was first proposed by Thornton (1970). Later, Engelhardt, Hinderer & Wipper (1978) suggested from studies involving eight low-protein diets that the permeability of the GI tract to urea increased with increasing energy content of the diet. In a review of literature values,

2 78 F. G. WHITELAW AND J. S. MILNE Kennedy & Milligan (1980) demonstrated by multiple regression analysis that a positive relationship existed between urea transfer to the rumen and the apparent digestion of organic matter. In other studies, direct additions of fermentable energy to the rumen in the form of sucrose (Potthast, Prigge & Pfeffer, 1977; Kennedy, 1980; Kennedy, Clarke & Milligan, 1981), sugar beet pulp (Engelhardt & Hinderer, 1976) or flaked barley (Norton, Mackintosh & Armstrong, 1982 b) have almost invariably resulted in an increase in urea transfer to the rumen or in the calculated permeability of the rumen wall to urea. A recurring problem in the interpretation of these findings is that additions of energy in this way frequently give rise to changes in the rumen environment or in the concentrations or proportions of rumen fermentation end-products, such as VFA, NH3 or CO2 Studies with rumen pouch or isolated rumen preparations have shown that increases in CO2 tension in rumen fluid (Thorlacius, Dobson & Sellars, 1971; Hinderer & Engelhardt, 1976) or in butyric acid concentrations (Hinderer & Engelhardt, 1976) have the effect of increasing the permeability of the rumen wall to urea. Increases in NH3 concentration have been shown to have an opposite effect (Houpt & Houpt, 1968) and an inverse relationship between rumen NH3 concentration and the transfer of the urea to the rumen has been demonstrated in a number of practical trials (Kennedy & Milligan, 1978; Kennedy, 1980). Thus it is difficult to determine in many cases whether changes in urea degradation are due to additions of energy per se or are secondary effects resulting from the metabolic activities of the rumen microbial population. We have shown in previous work that urea metabolism in sheep maintained wholly by intragastric infusion of nutrients is similar in most respects to that of sheep given conventional feeds (Whitelaw, Milne, 0rskov, Stansfield & Franklin, 1990). Urea degradation in the GI tract did not differ between the two dietary situations and we have therefore used the sheep nourished in this way as a model for the study of dietary factors thought to be associated with the process of urea recycling (Oncuer, Milne & Whitelaw, 1990). A prime advantage of this method of feeding is that all nutrient inputs can be precisely controlled and can be varied independently of each other when required. The work reported here designed to examine the separate effects of energy supply and the presence or absence of a rumen microbial population on the extent of urea transfer to the GI tract. In addition, since there is evidence that increased concentrations of butyric acid in rumen fluid can stimulate the transfer of urea to the rumen (Hinderer & Engelhardt, 1976; Norton, Janes & Armstrong, 1982 a), we have also examined the effect of this rumen metabolite on urea degradation. METHODS Animals and management Four female, 6-month-old Suffolk x Scottish Blackface lambs of about 32 kg live weight were used. Each had been fitted with a rumen cannula and an abomasal infusion catheter and they were maintained throughout the experiment by the intragastric infusion procedures described by 0rskov, Grubb, Wenham & Corrigall (1979). A standard volatile fatty acid (VFA) solution containing acetic, propionic and n-butyric acids in the molar proportions 0 65, 0-25 and 010 respectively was infused into the rumen, together with the major minerals (MacLeod, Corrigall, Stirton & 0rskov, 1982), in a total daily volume of The appropriate quantity of buffer solution was infused separately to the rumen in a total daily volume of Lactic casein was the sole source of nitrogen (N) and was infused at the abomasum in a daily volume of The animals were housed throughout in metabolism cages designed for the separate collection of faeces and urine. Fresh water was available ad libitum at all times.

3 UREA DEGRADATION IN SHEEP 79 Design and treatments The animals were subjected to four dietary treatments according to a 4 x 4 Latin square design, with experimental periods of 14 days. The treatments involved variations in the source and level of energy-yielding nutrients infused into the rumen and consisted of: treatment 1, the standard VFA solution described above, in the amounts required to achieve an overall maintenance level of energy input; treatment 2, the same standard VFA solution, but in amounts calculated to provide 30 % more energy to the rumen; treatment 3, the standard VFA solution supplemented with butyric acid to provide the same level of energy input as treatment 2; and treatment 4, the standard VFA mixture supplemented with glucose to provide the same energy input as treatment 2. The energy required for maintenance was taken to be 450 kj (kg live weight (W)075)-1 (0rskov et al. 1979). The 30% increase in energy input was based on previous work in which this amount of additional energy (as cassava) gave a significant increase in urea degradation in sheep given a pelleted diet. (Whitelaw et al. 1990). On each treatment casein was infused in amounts sufficient for maintenance, calculated on the basis that endogenous urinary N loss was 350 mg N (kg W0O75)-l(Hovell, 0rskov, Grubb & MacLeod, 1983). Details of the nutrient inputs on each dietary treatment are given in Table 1; the quantities of VFA, casein and glucose infused daily were adjusted according to individual live weights at the start of each period. Additional amounts of buffer were given on treatments 2 and 3 to compensate for the extra VFA infused. Preliminary experiments in other animals had shown that the addition of fermentable substrates such as glucose or lactose to the normal infusion solutions had the effect of creating active rumen microbial populations in sheep nourished by intragastric infusion (F. G. Whitelaw, J. S. Milne & S. Duncan, unpublished data). These populations arose without the need for manual inoculation of rumen organisms and were presumed to develop from the facultatively anaerobic bacterial populations known to be associated with the rumen epithelium of such animals (Wallace, Cheng, Dinsdale &0rskov, 1979). No allowance was made for possible losses of energy as methane or as fermentation heat in estimating the glucose required to equate the energy inputs of treatments 2, 3 and 4. Measurements and sampling procedures In each 14 day period the first 6 days served as a preliminary period in which animals adapted to the prescribed treatments. Collections of faeces and urine for N balance measurements and for the estimation of urinary purine excretions were made over days 7-11 inclusive (5 days). In two animals an intravenous injection of ['4C]urea for the measurement of urea kinetics was given via an indwelling catheter on day 13 and this was followed on day 14 by an intraruminal injection of polyethyleneglycol (PEG) for the measurement of rumen volume and outflow rate. In the other two animals the PEG injection was given on day 12 and [14C]urea measurements were made on day 14. The methods used in estimating urea pool size and irreversible loss rate (ILR) by means of a single intravenous injection of [14C]urea and the procedures for plasma and rumen sampling were as described by Whitelaw et al The activity of urease [EC ] in rumen fluid was measured in three samples taken at approximately 2 h intervals on day 12 and rumen bacterial counts were made on single samples taken at I 1.00 h on that day. Rumen fluid volumes and outflow rates were determined as described by Hyden (1961); the daily dose of PEG (10 g) was given into the rumen as a solution in water (166-7 g kg-1) and samples of rumen fluid for PEG analysis were removed at 1 5, 3, 6, 12 and 24 h after injection. For the N balance measurements, faeces were bulked over 5 days and the composite sample analysed for dry matter (DM) and N. Urine was collected into H2SO4 (100 g kg-')and was analysed on a daily basis for N, urea, and total purines. Analytical methods The methods for the estimation of urea in urine and plasma, total N in faeces, urine and casein and osmotic pressure (OP), VFA proportions, NH3 and urease in rumen fluid were as described by Whitelaw et al. (1990) and Oncuer et al. (1990). Plasma glucose concentrations were measured by the method of Trinder (1969) and the concentrations of total purine bases in urine were assayed by the method of Chen, Mathieson, Hovell & Reeds (1990b). Viable aerobic and anaerobic bacteria in rumen contents were counted as described by Hobson (1969). The preparation of radioactive materials and assay of radioactivity in plasma was as described previously (Whitelaw et al. 1990).

4 80 F. G. WHITELAW AND J. S. MILNE Table 1. Daily allowances of nitrogen and energy for maintenance ('Control') and supplements of mixed volatile fatty acids (VFA), butyric acid or glucose required to increase energy input to the rumen by 30% Treatments 'Control' 'Mixed VFA' 'Butyric' 'Glucose' Casein nitrogen (mg N (kg W075)-1) 460t Casein energy (kj (kg W075)-l) 65t Energy from standard VFA mixture* (kj (kg W075)-1) Energy supplementst (kj (kg W075)-1) Standard VFA mixture 115 Butyric acid 115 Glucose 115 Total energy input (kj (kg W075)-1) * See text. t The energy values of the supplements were: standard VFA mix, kj g-'; butyric acid, kj g-1; glucose, kj (g DM)-'. The casein contained g N and 22-4 MJ (kg DM)-1; DM = dry matter. I Calculations and statistical methods Urea pool size and irreversible loss rate (ILR) were estimated from the parameters of the double exponential equation relating the decline in plasma urea specific radioactivity to time (Whitelaw et al. 1990). Urea degradation in the GI tract was taken as the difference between ILR and the rate of excretion of urea in urine over the 48 h following 14C administration. Urea space was calculated as described previously (Whitelaw et al. 1990). Endogenous plasma urea clearance, suggested by Engelhardt et al. 1978) as a measure of the permeability of the gastrointestinal wall to urea, was calculated as urea degradation divided by plasma urea concentration and expressed as millilitres plasma per minute. Microbial protein production in the rumen was estimated from urinary purine excretions using the relationships established by Chen, Hovell, Orskov & Brown (1990a) and factors of 0 83 for the apparent digestibility of nucleic acid N and for the proportion of purine N in the total N of rumen bacteria (Chen, 1989). The sixteen observations for each variable arising from the Latin square were subjected to an analysis of variance which allowed treatment means to be adjusted for animal and period effects. No adjustments for animal effects were made when establishing regression relationships between pairs of variables. For one pair of variables, the regression relationship was examined further by covariance analysis to determine whether the regression within each treatment set differed from the others in slope or intercept. All statistical analysis were performed using GENSTAT (1982). RESULTS Animal health All animals appeared healthy throughout the experiment. In one animal, however, unusually high values for plasma urea concentration (15 55 mg (100 ml)-') were recorded in period 4, in which the animal received the supplement of glucose. These values were about 2-0 times higher than other values for the same animal and 2 5 times higher than values for other animals on the glucose treatment. The only explanation we can offer is a possible impairment of renal function in this animal in this final period, which resulted in an elevated renal threshold for urea. Similar findings were observed previously by Whitelaw et al. (1990) in a sheep nourished by intragastric infusion and Inkster, Hovell, Kyle, Brown & Lobley (1989) have noted occasional haematuria in sheep fed in this way. In the present animal, rumen NH3 concentration, urea pool size, and endogenous urea clearance were also

5 UREA DEGRADATIONIN SHEEP 81 Table 2. Indices of rumen function in sheep nourished by intragastric infusion and given additional energy into the rumen in the form of mixed volatile fatty acids (VFA), butyric acid or glucose (mean values for four animals) Rumen bacteria counts ml-1): (logl0 Treatments 'Control' 'Mixed VFA' 'Butyric' 'Glucose' S.E.D. P Aerobic ** Anaerobic ** ph n.s. Osmotic pressure (mosmol ** (kg H2O) 1) Ammonia concentration n.s. (mg (100 ml)-,) (5 07)t (0 75) (**) Urease activity(jumol NH3 min n.s. (ml fluid)-1) Volatile fatty acids (molar proportions) Acetic Propionic * Butyric Other acids Rumen fluid volume (1) n.s. Rumen fluid outflow rates: (% h-') * (1 day-') * S.E.D. = standard error of differences; P= statistical significance: n.s. = non-significant; * P < 005; **P<0-01; *** P< t Values in parentheses indicate revised mean, S.E.D. and significance when one anomalous value is omitted from 'glucose' treatment (see text below). affected by the anomalous plasma urea values but other indices of urea metabolism appeared unchanged. All observations for this treatment period were subjected to the full statistical analysis but for the variables noted above an additional analysis was carried out in which the suspect values were omitted. Rumen conditions Mean values for the major indices of rumen function are given in Table 2. The total counts of both aerobic and anaerobic bacteria in rumen fluid increased (P < 0 01) by a factor of 102 when glucose was included in the rumen infusion mixture and reached numbers that would be considered typical of an active rumen fermentation in animals given conventional diets (Jayne-Williams, 1979). No protozoa were observed in rumen fluid. Rumen ph was constant across treatment groups whereas OP was significantly higher (P < 0 01) on the two treatments in which additional VFA, and hence additional buffer, were infused. Rumen NH3 concentrations were lower on butyric acid and glucose treatments but differences between these and the other two treatments were not significant (P = 0 052); however, omission of the high rumen NH3 value noted above gave an adjusted mean NH3 concentration of 5 07 mg (100 ml)-' for the glucose treatment and both this and the butyric acid treatment value were significantly lower than those for the other two treatments (P < 0 01). Rumen urease activity showed considerable variability between animals and differences between treatments were not significant.

6 82 F. G. WHITELAW AND JṢ. MILNE The addition of supplementary butyric acid resulted in a major change in rumen VFA proportions. On this treatment butyric acid made up 23 % of the total VFA present and was significantly higher than on any of the other treatments (P < 0-001); corresponding downward changes were noted for the proportions of acetic acid (P < 0001) and propionic acid (P < 005). The addition of glucose also had an effect on rumen VFA proportions; relative to animals given the standard VFA mixture, acetic acid decreased (P < 005) and there were significant increases in butyric acid (P <0O05) and in the proportion of branched-chain and higher acids ('other acids', P < 0-001). This latter change was due entirely to the presence of caproic acid on the glucose treatment and its absence from the others. Rumen fluid volumes did not differ significantly between treatments whereas rumen fluid outflow rates were significantly higher (P < 005) on the two treatments which involved inputs of additional VFA to the rumen. No significant relationships could be established, however, between rumen fluid outflow rate and OP. Nitrogen metabolism Mean daily inputs of energy and N on each treatment and daily excretions of N in faeces and urine during the 5 day collection periods are given in Table 3. Daily energy inputs were close to the intended levels (Table 1) and did not differ between the three supplemented treatments. Nitrogen inputs were also close to the intended level (mean 454 mg (kg W075)-1, S.D. 13 3) and did not differ between treatments. The presence of a rumen microbial population in animals given the glucose supplement resulted in a very obvious increase in the volume of faecal material excreted and this was associated with a higher faecal N excretion on this treatment. The excretion of both total N and urea N in urine was significantly lower on the glucose treatment then on the others (P < 0-01) and the ratio of urea N to total N in urine was also significantly lower in the glucose-supplemented animals (P < 005). Urinary N excretion was highest on the butyric acid treatment (P <0O05) and this was reflected in a lower value for N retention on this treatment than on the others. The daily excretion of purine derivatives in urine was approximately 28 % greater on the glucose treatment than on the others but differences between treatments were not significant. However, if we accept this difference as an index of the contribution of rumen microbial protein to total purine turnover, the calculations outlined above (p. 80) yield a value of 1-71 g day-' for total microbial N production in the rumen when glucose was given as a supplement. Urea metabolism The concentrations of urea and glucose in plasma and the kinetics of urea metabolism are given in Table 4. As in previous work (Oncuer et al. 1990; Whitelaw et al. 1990), plasma urea concentrations remained remarkably stable throughout each 24 h blood-sampling period. The imposed treatments had no significant effect on the concentrations of either urea or glucose in plasma but when the one anomalous value for plasma urea concentration was omitted (see p. 81), the mean urea concentration for the glucose group was significantly lower than the values for the other three treatments (P < 0-05). Urea ILR did not differ significantly between treatments, although there was a tendency for values to be higher on the two treatments in which additional energy as VFA was infused. Urinary urea excretion was significantly lower (P < 0-05) when the glucose supplement was given but otherwise did not differ between treatments. Urea degradation in the GI tract was lowest on the control treatment but the values recorded, whether expressed as g urea day-1 or as a proportion of

7 UREA DEGRADATION IN SHEEP 83 Table 3. Mean liveweights, energy inputs and nitrogen (N) inputs, excretions and retentions in sheep nourished by intragastric infusion and given additional energy into the rumen in the form of mixed volatile fatty acids (VFA), butyric acid or glucose (mean values for four animals) Treatments 'Control' 'Mixed VFA' 'Butyric' 'Glucose' S.E.D. P Live weight (kg) n.s. Energy input (kj (kg W075)-l day-') Nitrogen input (g day-1) Faecal nitrogen (g day-') n.s. Urinary nitrogen (g day-') ** Urinary urea-nitrogen (g day-') ** Urea N: total N in urine * Nitrogen retention (g day-') n.s. Urinary purine excretion n.s. (mg (kg W075)-1 day-1) S.E.D. = standard error of differences; P = statistical significance: n.s. = non-significant; * P < 0 05; ** P<001. ILR, did not differ significantly between the four treatments. A simple comparison of the control treatment with the means of all three supplemented groups indicated, however, that urea degradation was significantly higher (P < 0 05) when the higher energy inputs were given (6 18 vs. 8&62 g urea day-1; S.E.D., 0 9). Urea pool size and volume of distribution showed no significant changes in response to the imposed treatments but omission of the value relating to the very high plasma urea concentration gave a revised urea pool size for the glucose treatment which was significantly lower than the values recorded for the others (P < 0 05). The calculated endogenous urea clearance, which represents the volume of plasma cleared of urea by transfer to the GI tract (Engelhardt et al. 1978), was lowest on the control treatment and highest in animals receiving the glucose supplement. Considerable variability was evident, however, and differences between treatments were not significant, whether or not the means were adjusted for the high plasma urea value noted above. As in previous work (Milne, Whitelaw, Price & Shand, 1990; Oncuer et al. 1990; Whitelaw et al. 1990), plasma urea concentration was highly correlated with other indices of urea metabolism. The relationships between these variables were examined by regression analysis and the more important of these are summarized in Table 5. For each relationship in which plasma urea concentration was a variable, the pair of observations containing the anomalous plasma urea value (p. 80) was omitted from the analysis. Significant linear relationships existed between plasma urea concentration on the one hand and urea pool size (P < 0-001) and ILR (P < 0-001) on the other. Urea degradation in the GI tract was significantly related to ILR (P < 0-001) and to plasma urea concentration (P < 0 05) but was not related to rumen NH3 concentration, rumen urease activity, the molar proportions of acetic, propionic or butyric acids in rumen fluid or to endogenous urea clearance values. A further examination of the urea degradation-ilr relationship by covariance analysis showed that the intercept for the glucose-treatment set differed significantly from those of the other three treatments (P < 0-01). The other intercepts did not differ one from the other and the slopes of the regression relationships also did not differ between the four treatment

8 84 F. G. WHITELAW AND J. S. MILNE 00 rli I'D r- tn CP w CA C. w' w w coi C> AS b tn C> c> c, r- tn 400 C-1 r-ol ON WI).O C,4 00 C-4 'IO W) C) C) m C) w tq CO) X r2 v en C) en tn lz CZ4 C', + + ZS 'IO all tn 4 'IO C> r,5 "O 00 m C> CO C> 60 C;-) 1.r Ei V.14 (U X C44 c5n CZ4.b t-.- Ob d> 6 V (U > Ci Wz).j q6) '--b t3 oo C) r- en tn 00 tn tn tn (71, W) WI lqt WI C. Ob C.) t;-) %b Ob 6 6 6N 0 8 'Ti w.0.0 tn > I::) +1 +-O 11 - CP t. -7' qn -7' lr 'T C? N 0 00 en C) CD C.) 00 - w -, -0 1:$ > tz, - u o 04 S. w ea) 00 El Z5 Q 00 t 0 'a (u.- t3 m m m m I-Ou I r. EE t= qj (Z. (D 0 o CZ (U 8 > o r4 (U >,. to ed C '.J '.J C's -0 0 t) ;.. -Z t. > 0 0 W too 0 to - Ea.2 q6) L. CIS Po CO cd - Cid qj (U :3 0 0 ra cd :3 8 cd o 0 10 to (U e4o > 1=4 cu > (U cts0 $. $-4 la 0 "46) M.0 C13 C-i Cis 2 C-d CZ ra c-d zo. (U > 00 (U cqs

9 UREA DEGRADATION IN SHEEP 85 a 15 I., A0 b l 10 -A O l Fig. 1. Urea ILR (g day-1) Relationship between urea degradation (g day-') and urea irreversible loss rate (ILR; g day-') for animals maintained by intragastric nutrition, either at a maintenance level of energy (O) or when given supplements of mixed VFA (A), butyric acid (A) or glucose (A) by infusion into the rumen. Twhe regression lines represent animals given supplementary glucose (a), and animals on all other treatments (b). groups. When constrained to a common slope, the relationships between these variables were, for the glucose treatment: Y= 0-976X-4329 and for the other three treatments combined: Y = 0 796X- 6A65, where X and Y are ILR and urea degradation respectively. These relationships were highly significant (P < 0-001; R.S.D., 01902)and are illustrated in Fig. 1. DISCUSSION A unique feature of the rumen microbial populations which developed in these sheep in response to infusions of glucose is that they did so in the total absence of an exogenous supply of N. Microbial growth was thus dependent on the transfer of urea or other endogenous N into the rumen and, ultimately therefore, on the reutilization of waste products arising from the digestion of casein absorbed at the duodenum. The bacterial species present were not identified in this experiment, but in preliminary work with other animals given glucose or lactose by infusion the predominant organisms in both aerobic and anaerobic culture were found to be Gram-positive cocci or rods and were provisionally identified as Lactobacillus species (F. G. Whitelaw, J. S. Milne & S. Duncan, unpublished observations). Lactic acid, however, was not detected in rumen fluid. Microbial protein synthesis in this experiment was assessed by reference to the daily excretion of purine derivatives in urine (Chen et al. l1990a). The mean daily purine excretion of the three 'non-glucose' treatments was taken as the endogenous excretion value and calculations based on this gave a microbial N production of 1d71 g day-' in animals given glucose. The mean daily glucose input to the sheep was 97i 8 g DM and this was presumed

10 86 F. G. WHITELAW AND J. S. MILNE to be wholly fermented since plasma glucose values showed no change relative to the other treatments (Table 4). The microbial N yield corresponding to this intake can be calculated from stoichiometric principles using the factor of 17-5 g microbial N kg-1 organic matter fermented, as proposed by Czerkawski (1986) for the efficiency of synthesis when all microbial matter is formed from glucose and NH3. This calculation yields a value of 1 71 g day-' microbial N, identical to that derived above. Thus efficiency of microbial synthesis in these induced fermentations is of the same order as that observed on normal diets (Czerkawski, 1986) and there is no indication that microbial growth was limited by N supply. This synthesis, however, relates to a glucose input equivalent to only 23 % of the total energy normally supplied to the rumen and problems of N supply from endogenous sources might arise at glucose inputs higher than this. Rumen fermentation and urea metabolism It is clear from Tables 2 and 4 that glucose was the only form of supplementary energy to effect a change in the process of urea metabolism in this experiment. In all animals, urinary urea excretion decreased when glucose was given and in three of the four animals there were significant decreases also in plasma urea concentration, urea pool size and rumen NH3 concentration. Similar findings have been reported in other studies in which supplements of fermentable energy have been given to animals exhibiting normal rumen function (Engelhardt & Hinderer, 1976; Potthast et al. 1977; Kennedy, 1980; Kennedy et al. 1981; Norton et al a; Whitelaw et al. 1990) and there is clearly a strong suggestion that the changes seen were a direct consequence of the microbial population present. The interpretation of these findings in terms of microbial activity invokes a sequence in which the addition of fermentable energy causes an initial change in the protein: energy ratio of the rumen substrate. This results in an increase in the scavenging of NH3 within the rumen pool by the rumen microbes and eventually in a reduction in rumen NH3 concentration. This in turn creates a change in the NH3 concentration gradient between epithelial tissue and rumen contents and leads to an increased rate of hydrolysis of urea to NH3 in the keratinized layer of the epithelium under the influence of bacterial urease (Houpt & Houpt, 1968). An increased rate of removal of urea from blood then causes a lowering of plasma urea concentration and this is reflected in a reduction in the clearance of urea by the kidney and a decrease in urinary urea excretion. Many of the steps in this sequence have been identified in other studies (see Kennedy & Milligan, 1980; Egan et al. 1986) but what has not been clearly demonstrated before is that the initial step requires the presence of a microbial population subsisting on an energy-adequate, N-deficient substrate. Urea production and degradation Despite the control achieved in regulating the input of energy and total N (Table 3) and the very satisfactory 'steady-state' conditions which can be maintained in plasma by the use of the intragastric infusion system, considerable variability was encountered in the measured values for urea production (ILR) and urea degradation (Table 4). Kennedy (1980) and Egan et al. (1986) have also made reference to a high variability in measurements of urea kinetics in ruminants and Hibbert & Jackson (1990) have reported similar problems in studies on human subjects. In the present experiment, this variability was such that a significant difference in urea degradation could be detected only between the control treatment on the one hand and the three energy-supplemented treatments on the other. This difference was about 2 4 g urea day-' and represented an increase of about 40 % over the degradation values recorded on the control treatment. Thus despite some

11 UREA DEGRADATION IN SHEEP variability in the response, additions of energy either as VFA or as a fermentable substrate clearly had the effect of increasing urea degradation in the GI tract. The linear relationship between urea degradation and ILR in this experiment (Table 5) was almost identical to that reported in our previous work (Oncuer et al. 1990). It was noted however that this relationship must be treated with caution, since [14C]urea on hydrolysis gives rise to unlabelled NH3 and this NH3 will contribute anew to measured ILR unless it is incorporated into microbial or tissue protein or other large molecules (Oncuer et al. 1990). Thus the measured ILR will be higher the greater the proportion of hydrolysed urea which is simply reabsorbed as NH3 from the GI tract. Conversely, if a dietary or physiological change results in a greater proportion of recycled urea N being incorporated into larger molecules, this will be reflected in a lower ILR for a given level of urea degradation. In theory therefore different relationships will exist between degradation and ILR depending on the fate of the NH3 produced in the hydrolysis of urea. Analysis of the relationships between urea degradation and ILR for the four dietary treatments showed that the slopes of the regression lines did not differ but that the regression for the glucose treatment had a significantly higher intercept than those for the other treatments (Fig. 1). This difference is consistent with the hypothesis developed above and indicates that a portion of the NH3 arising from urea degradation when glucose was given had a different metabolic fate from that produced when no fermentable substrate was available. Incorporation into microbial protein is clearly the most likely route of capture of this NH3 and the difference in intercept between the two regressions (p. 85) suggests that 2-33 g urea (1 09 g N) was removed from, and did not return to, the urea/nh3-n pool in animals given glucose. It is evident also from Fig. 1 that increments in ILR above a baseline value (given by Y = 0) are due entirely to NH3 arising from endogenous recycling. It would appear, however, from the variability seen in Fig. 1, that this process is not well regulated and the extent of NH3 recycling might vary even on a day-to-day basis. This could clearly account for a large part of the variability in ILR and degradation noted when '4C dilution methods are employed. The estimate derived above for NH3-N removed from circulation (1-09 g) is not, of course, equivalent to microbial N synthesized since it does not include urea produced in the subsequent catabolism of the microbial protein. Further inevitable losses from this moiety will occur in faeces and as nucleic acids in urine and it is perhaps not surprising that the increase in N supply as microbial protein was not reflected in an improved N retention in animals given glucose (Table 3). Indeed, it is interesting to note that there are remarkably few reports in the literature in which a measured increase in urea degradation has been shown unequivocally to result in a net gain of N to the host animals (Norton et al b; Whitelaw et al. 1990). Rumen VFA and urea recycling As indicated above, additions of energy as a mixed VFA solution or as butyric acid alone appeared to stimulate the transfer of urea to the GI tract. The precise site of transfer cannot be established from the present work but the rumen epithelium would appear to be the most likely location for this exchange (Dinsdale, Cheng, Wallace & Goodlad, 1980; Oncuer et al. 1990). The rumen is also the major site of VFA absorption (Stevens, 1970) and the three nutritionally important VFAs, particularly butyric acid, are known to undergo metabolism within the rumen epithelium (Masson & Phillipson, 1951). In vivo absorption studies have shown that these VFAs, and again particularly butyric acid, also have the effect of increasing blood flow to the rumen epithelium (Sellers, Stevens, Dobson & McLeod, 1964; 4 EPH 76 87

12 88 F. G. WHITELAW AND J. S. MILNE Dobson, Sellers & Thorlacius, 1971; Engelhardt & Hinderer, 1976). It is tempting therefore to assign urea transfer to this organ and to suggest that it results from an increase in metabolic activity in, and an increased blood flow to, the rumen epithelium. Dobson et al. (1971), however, were unable to demonstrate an effect of rumen blood flow on urea clearance in a single experiment on a cow. Although some indirect support for this observation has been presented by Engelhardt et al. (1978), it has been questioned by others (Kennedy, 1980; Kennedy & Milligan, 1980; Egan et al. 1986). The limited information available is at best equivocal, and the whole question of rumen blood flow and urea clearance would seem to warrant further study. Norton et al. (1982a) infused butyric acid to the rumen of sheep and reported a significant increase in both the amount and proportion of total urea synthesis that was degraded in the rumen. They proposed that butyric acid might stimulate microbial activity in the keratinized layers of the rumen epithelium, leading to increased disruption of this layer and an increase in its permeability to urea (Norton et al. 1982a). An alternative suggestion was that metabolism of butyric acid in the epithelium might stimulate urea transfer through an effect on the rate of cell division and shedding (Norton et al. 1982a). As yet neither of these proposals has been confirmed experimentally. Also, the effects attributed to butyric acid by Norton et al. (1982a) could equally well have been due to a change in energy supply, since this was some 15 % greater in their butyric acid treatment than on the others. In the present work there was no indication that the supplement of butyric acid was any more effective than the mixed VFA solution in promoting urea degradation. Butyric acid additions caused a reduction in rumen NH3 concentration and an increase in urinary N excretion but at present we can offer no satisfactory explanation fo'r these effects. Similarly, the reason for the increase in rumen outflow rate on the two treatments involving additions of VFA to the rumen must remain a matter for conjecture. Conclusions We conclude from these findings that additions of energy to the rumen of sheep nourished by infusion can result in increases of up to 2-4 g day-1 in urea degradation in the GI tract. The response is similar whether the additional energy is given as VFA or as a fermentable substrate and butyric acid does not appear to have any special properties with respect to urea recycling. The evidence suggests that degraded urea will contribute to the N economy of the animal only when the energy supply results in an increase in microbial fermentation and the incorporation of urea N into microbial protein. In the absence of these features, or when the supply of N to the rumen microbial population is already adequate, it seems likely that degraded urea will simply revolve around a continuous hydrolysis-regeneration cycle. We are indebted to Mr G. Wenham for surgery on the experimental animals and to Mrs Hazel Vint and Dr M. F. Franklin for assistance with statistical analysis. We are grateful also to Miss Maureen Annand and Mr R. S. Smart and their respective colleagues for their help in the analysis of plasma and digesta samples. REFERENCES CHEN, X. B. (1989). Excretion of purine derivatives by sheep and cattle and its use in the estimation of absorbed microbial protein. Ph.D. Thesis, University of Aberdeen. CHEN, X. B., HOVELL, F. D. DEB, 0RSKOV, E. R. & BROWN, D. A. (1990a). Excretion of purine derivatives by ruminants: effect of exogenous nucleic acid supply on purine derivative excretion by sheep. British Journal of Nutrition 63,

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