X. B. Chen

Excretion of purine derivatives by ruminants: endogenous excretion, differences between cattle and sheep.

The endogenous urinary excretion of the purine derivatives allantoin, uric acid and xanthine plus hypoxanthine were measured in twenty-nine lambs, ten cattle (six steers, one cow and three preruminant calves) and four pigs. The sheep and mature cattle were nourished by intragastric infusion and the calves were given a milk-substitute. The pigs were fed on a purine-free diet. The excretion of total purine derivatives was substantially greater by the cattle, being 514 (SE 20.6) mumol/kg live weight (W)0.75 per d compared with 168 (SE 5.0) mumol/kg W0.75 per d by the sheep and 166 (SE 2.6) mumol/kg W0.75 per d by the pigs. Plasma from normally fed sheep, cows and pigs was incubated with either xanthine or uric acid. Sheep and pig plasma had no xanthine oxidase (EC 1.2.3.2) activity whereas plasma from cattle did. Uricase (EC 1.7.3.3) was not present in plasma of cattle and pigs and appeared to be present in trace amounts only in sheep plasma. It is suggested that the species differences in endogenous purine derivative excretion were probably due to the different profiles of xanthine oxidase activity in tissues and particularly in the blood. This is because a high xanthine oxidase activity would reduce the chance to recycle purines, by increasing the probability of degradation to compounds which could not be salvaged.

Evaluation of the use of the purine derivative: creatinine ratio in spot urine and plasma samples as an index of microbial protein supply in ruminants: studies in sheep

In ruminants, the urinary excretion of purine derivatives (PD) reflects the absorption of microbial purines and can be used as an index of microbial protein supply. The objective of this study, carried out in Aberdeen, 1992, was to examine whether PD concentrations in spot urine or plasma samples vary diurnally during a given feeding regime and if they reflect differences in daily PD excretion induced by varying feed intake. Sixteen sheep were offered ad libitum one of four diets (fresh weight basis, the rest of each diet being minerals and vitamins): (1) 99.9 % lucerne (pelleted); (2) 50 % hay, 30% barley, 9% fishmeal and 10% molasses; (3) 72% straw, 7% molasses and 20% molassed sugarbeet pulp; and (4) 97 % barley. Measurements were made for 1 week after a 2-week adaptation period. Urine was collected daily on days 1-4 and hourly on days 5-7. Hourly urine collection was achieved using a fraction collector. Plasma samples were collected hourly from 09.00 to 17.00 h on day 4. Feed intake varied considerably (347-1718 g DM/day) between diets and between animals. Daily excretion of PD (7.1-22.6 mmol/day) was linearly related to DM intake (r = 0.85, n = 16), and so was the microbial N supply (3.9-19.5 g N/day) estimated from daily PD excretion (r = 0.87). In hourly urine samples, the ratio of PD:creatinine concentrations showed no significant difference between sampling times, and was linearly correlated with the daily PD excretion (r = 0.92). Similarly, plasma PD concentration also showed little diurnal fluctuation. Glomerular filtration rate (GFR) increased with feed intake. Plasma PD was not well correlated with daily PD excretion in urine (r = 0.57). The tubular load of PD (plasma PD x GFR) was better correlated with the daily excretion (r = 0.80). It appears that when sheep are fed ad libitum, PD in spot urine may provide a practical indicator of microbial protein supply status.

Measurement of allantoin in urine and plasma by high-performance liquid chromatography with pre-column derivatization

A method is reported for determination of allantoin in urine and plasma based on high-performance liquid chromatography (HPLC) and pre-column derivatization. In the derivatization procedure, allantoin is converted to glyoxylic acid which forms a hydrazone with 2,4-dinitrophenylhydrazine. The hydrazone appears as syn and anti isomers at a constant ratio. These derivatives are separated by HPLC using a reversed-phase C18 column from hydrazones of other keto acids possibly present in urine and plasma and then monitored at 360 nm. All components were completely resolved in 15 min. Both the reagents and derivatization products are stable. Recovery of allantoin added to urine and plasma was 95 +/- 3.7% (n = 45) and 100 +/- 7.5% (n = 64), respectively. The lowest allantoin concentration that gave a reproducible integration was 5 mumol/l. The between-assay and within-day coefficients of variation were 2.8 and 0.6%, respectively.

Influence of dietary rumen-degradable protein on bacterial growth in the rumen of sheep receiving different energy sources

An experiment with ruminally cannulated sheep was undertaken to determine how the dietary energy source affected the ability of rumen fermentation to respond to pre-formed amino acids compared to non-protein nitrogen in the diet. Four sheep received diets of grass hay (H) or molassed sugar beet pulp (BP) supplemented with 9.3 g N kg−1 DM added either as urea (U) or casein (C) in a 4 × 4 latin square with a factorial design. Measurements of rumen fermentation, microbial numbers and purine excretion were made in the third week of each 21-day period. Rumen total volatile fatty acids concentrations were higher with the BP diets (130 vs. 91 mM; P 0.05). A microbial response to rumen-degradable protein therefore occurred only with the more rapidly degraded BP diet, consistent with the idea that rumen microbial growth responds to pre-formed amino acids only when the energy source is fermented rapidly.

Urinary excretion of purine derivatives and tissue xanthine oxidase ( EC 1.2.3.2) activity in buffaloes ( Bubalis bubalis ) with special reference to differences between buffaloes and Bos taurus cattle

The urinary excretion of purine derivatives (PD) was measured in six buffaloes (Bubalis bubalis) during fasting and in fourteen buffaloes given four restricted levels of roughage (2.5-4.8 kg DM/d). Only allantoin and uric acid, not xanthine and hypoxanthine, were present in the urine, the pattern of excretion being similar to that in cattle. The fasting PD excretion amounted to 0.20 (SD 0.06) mmol/kg metabolic weight (W)0.75 per d, and the rate of PD excretion as a linear function of feed intake was 5.2 mmol/kg digestible organic matter intake. Both values were considerably lower than the values for cattle reported in the literature. Creatinine excretion values were 0.33 (SD 0.06) and 0.44 (SD 0.09) mmol/kg (W)0.75 per d determined in fasting and feeding periods respectively. Fasting N excretion was 257 (SD 49) mg N/kg (W)0.75 per d. Both creatinine and fasting N excretions were also lower than in cattle. The activities of xanthine oxidase (EC 1.2.3.2) in plasma, liver and intestinal mucosa were determined in buffaloes, cattle and sheep. Xanthine oxidase activities in buffaloes were 24.5 (SD 2.7) unit/l plasma and 0.44 (SD 0.02) and 0.31 (SD 0.10) unit/g fresh tissue in liver and intestinal mucosa respectively. These activities were higher than those in cattle and sheep. Xanthine oxidase was practically absent from plasma and intestine of sheep. It is suggested that the differences in PD excretion between buffaloes and cattle were probably due to the smaller proportion of plasma PD that was disposed of in the urine of buffaloes.

Excretion of purine derivatives by ruminants: recycling of allantoin into the rumen via saliva and its fate in the gut.

The saliva of sheep was shown to contain significant concentrations of uric acid (16(SD 4.5) mumol/l) and allantoin (120(SD 16.4) mumol/l), sufficient to recycle purine derivatives equivalent to about 0.10 of the normal urinary excretion. When allantoin was incubated in vitro in rumen fluid, it was degraded at a rate sufficient to ensure complete destruction of recycled allantoin. In a series of experiments in which allantoin was infused into the rumen of sheep fed normally, or into the rumen or abomasum of sheep and the rumen of cattle completely nourished by intragastric infusion of volatile fatty acids and casein, no additional allantoin was recovered in the urine. These losses were probably due to the degradation of allantoin by micro-organisms associated with the digestive tract. It is concluded that all allantion and uric acid recycled to the rumen via saliva will be similarly degraded. Therefore, the use of urinary excretion of purine derivatives as an estimator of the rumen microbial biomass available to ruminants will need to be corrected for such losses.

Effects of long-term protein excess or deficiency on whole-body protein turnover in sheep nourished by intragastric infusion of nutrients.

The effect of long-term dietary protein excess and deficit on whole-body protein-N turnover (WBPNT) was examined in lambs nourished by intragastric infusions of nutrients. Ten sheep were given 500 mg N/kg metabolic weight (W0.75) per d from casein for 2 weeks and then either 50 (L), 500 (M) or 1500 (H) mg N/kgW0.75 per d for 6 weeks. Volatile fatty acids were infused at 500 kJ/kgW0.75 per d. Daily WBPNT was measured by continuous intravenous infusion of [1-13C]leucine 3 d before, and on days 2, 21 and 42 after the alteration in protein intake. Whole-body protein-N synthesis (WBPNS) was calculated as the difference between WBPNT and the protein-N losses as urinary NH3 and urea. Whole-body protein-N degradation (WBPND) was then estimated from WBPNS minus protein gain determined from N balance. Fractional rates of WBPNS and WBPND were calculated against fleece-free body N content. WBPNS rates at the L, M and H intakes were respectively 35.1, 41.5 and 63.7 g/d (P < 0.001) on average over the 6 weeks and WBPND rates were 39.5, 41.1 and 56.8 g/d (P < 0.001). The fractional rates of WBPNS were 5.01, 6.37 and 7.73% per d (P < 0.001) while those of WBPND were 5.64, 6.29 and 6.81% per d (P < 0.005) respectively. On days 2, 21 and 42, WBPNS rates at intake H were 54.0, 61.8 and 75.4 g/d (P = 0.03) respectively, and WBPND rates were 43.2, 56.4 and 70.9 g/d (P = 0.03); at intake L the amounts were 38.2, 34.2 and 32.8 g/d for WBPNS (P = 0.003) and for WBPND were 43.4, 38.0 and 36.9 g/d (P = 0.016) respectively. There were no significant (P > 0.05) differences in fractional rates of WBPNS and WBPND with time at either the L or H intake. We concluded that absolute protein turnover was affected both by dietary protein intake and body condition while the fractional rate of turnover was predominantly influenced by intake.

The effect of starch supplementation of straw on microbial protein supply in sheep

The effect on microbial N (MN) supply of supplementation of straw with a mixture of maize starch and ground barley was examined. Urinary excretion of purine derivatives (PD) was used as an index of microbial protein supply. Six wether sheep of 42–48 kg with rumen cannulae were offered ad libitum either barley straw with 5% molasses, or barley straw with 15.5 or 31.0% of a mixture of maize starch and ground barley (1.2:1.0), and 9 and 13% molasses respectively. All sheep received all three diets for periods of 21 days, excreta being collected during the last 7 days. Digestible organic matter intake (DOMI) increased with starch inclusion as 435, 750 and 1043 (SED 47) g day−1 for the three diets respectively. Total PD excretion was 7.0, 12.3 and 21.1 (SED 0.92) mmol day−1, and the estimated MN supply was 5.7, 10.5 and 18.2 (SED 0.84) g day−1. Expressed as per unit DOMI, MN supply was 12.8, 14.1 and 17.5 (SED 1.3) g N kg−1 DOMI. Rumen liquid and solid outflow rates (fraction of rumen volume h−1) were 0.047, 0.054 and 0.073 (SED 0.0074) and 0.078, 0.105 and 0.130 (SED 0.0083) respectively. MN supply per unit DOMI increased probably due to a greater rumen outflow rate. The practical implication is that with low quality straw, supplementation of starch concentrate up to 31% of the whole diet increased both voluntary food intake and the supply of microbial protein per unit of DOMI.

The modification of an in vitro gas production test to detect roughage related differences in in vivo microbial protein synthesis as estimated by the excretion of purine derivatives

The relationship between variations in in vitro microbial biomass production and microbial protein supply to the host animal was examined in 13 Malawian goats fed on stover leaves from two varieties of maize at Bunda College, Malawi, in 1995. The in vitro parameters were analysed based on the concept of the partitioning factor (PF) which is obtained by the combination of gas volume measurements with determinations of the amount of substrate truly degraded. The PF reflects substrate-dependent variation in the in vitro partitioning of degraded substrate between short chain fatty acids (SCFA), gases and microbial biomass. The in vivo microbial protein synthesis was estimated by the urinary excretion of the purine derivatives (PD) allantoin, uric acid and xanthine + hypoxanthine. For the two types of stover leaves examined, the higher microbial efficiency of one variety in vitro was reflected by different PD excretions on practically identical digestible dry matter intakes in vivo. It is concluded that substrate-dependent variations in microbial efficiencies as detected by the PF are also evident and relevant for in vivo microbial protein synthesis.

Intake, digestion and small intestinal protein availability in sheep in relation to ammoniation of wheat straw with or without protein supplementation

The effects of ammoniation of wheat straw with or without supplementation of protein sources of either high (casein) or relatively low (potato protein) rumen degradability on intake and digestion were studied with four sheep in a 4 x 4 Latin square design. Rations offered were: (1) untreated wheat straw (UWS), (2) ammoniated wheat straw (AWS), (3) AWS supplemented with 3.2 g casein/kg live weight (W)0.75 per d (AWSC) and (4) AWS supplemented with 3.9 g potato protein/kg W0.75 per d (AWSP). Straw was offered ad lib. and all rations were supplemented with sugarbeet pulp and a mineral mixture. NH3 treatment increased intake and digestion. Supplementation of AWS with potato protein increased total digestible organic matter intake (DOMI) compared with AWS whereas supplementation with casein did not affect total DOMI. Protein supplementation of AWS significantly reduced rumen digestion of cellulose, and when the supplementation was with casein it reduced rumen digestion of neutral-detergent fibre and hemicellulose also. This lower rumen digestion was compensated by a higher proportion of digestion occurring in the hindgut for hemicellulose (P < 0.05 for AWSC, P > 0.05 for AWSP), but not for cellulose. Across all rations, rumen fluid volume increased with increasing cell-wall intake. The efficiencies of microbial protein synthesis were (average of three different methods of estimation) 23.3, 26.2, 34.8 and 31.7 g N/kg apparently-rumen-degraded organic matter for UWS, AWS, AWSC and AWSP respectively. The difference between UWS and AWS was not significant, but values for AWSC and AWSP were significantly higher than that for AWS. The rumen digestion of feed amino acid-N (AA-N) was significantly higher for AWSC than for the other rations. The apparent small-intestinal digestion of AA-N and N was significantly higher for AWSP than for the other rations. The true small-intestinal digestion values were 0.86, 0.84 and 0.68 for AA-N, N and non-protein-N respectively. Ileal endogenous losses of AA-N were approximately 6 mg/g duodenal non-protein dry-matter flow. Linear relationships were observed between DOMI and N balance and truly absorbed AA-N, indicating that DOMI could have been limited by small-intestinal amino acid availability. Regression of N balance v. truly absorbed AA-N resulted in an estimate of net efficiency of utilization of truly absorbed AA-N or 0.54.