Duodenal Purine Bases Research Articles

Technical note: Evaluation of urinary purine derivatives in comparison with duodenal purines for estimating rumen microbial protein supply in sheep.

A data set of individual observations was compiled from digestibility trials to examine the relationship between the duodenal purine bases (PB) flow and urinary purine derivatives (PD) excretion and the validity of different equations for estimating rumen microbial N (Nm) supply based on urinary PD in comparison with estimates based on duodenal PB. Trials (8 trials, = 185) were conducted with male sheep fitted with a duodenal T-type cannula, housed in metabolic cages, and fed forage alone or with supplements. The amount of PD excreted in urine was linearly related to the amount of PB flowing to the duodenum ( < 0.05). The intercept of the linear regression was 0.180 mmol/(d·kg), representing the endogenous excretion of PD, and the slope was lower than 1 ( < 0.05), indicating that only 0.43% of the PB in the duodenum was excreted as PD in urine. The Nm supply estimated by either approach was linearly related ( < 0.05) to the digestible OM intake. However, the Nm supply estimated through either of 3 published PD-based equations probably underestimated the Nm supply in sheep.

The relationship between microbial N synthesis and urinary excretion of purine derivatives in Dorper × thin-tailed Han crossbred sheep

This study aimed to investigate the relationship between microbial N (MN) yield and urinary excretion of purine derivatives (PD) from the rumen in Dorper×thin-tailed Han crossbred sheep. Twelve Dorper×thin-tailed Han crossbred, non-castrated male lambs (41.3±2.8kg body weight) fitted with ruminal and duodenal cannulae were randomly assigned to one of three levels of DM intake: ad libitum intake, 70%, or 50% of the ad libitum intake, with four lambs at each level. The MN yield from the rumen was measured using 15N or purine bases (PB) as a microbial marker, and urinary PD were determined by colorimetric methods. Microbial components (OM, N, PB and ratio of PB to N) were not affected (P>0.05) by dietary treatment. The MN yield measured using PB was slightly lesser and more variable than using 15N. No difference in MN yield was found between markers. The efficiency of MN synthesis (g N/kg organic matter apparently digested in the rumen) was not affected (P>0.05) by feed intake regardless of the marker used. Urinary PD decreased significantly (P<0.05) with decreasing level of feed intake and the molar recovery of duodenal PB measured with digesta markers as urinary PD was 0.83 (r2=0.89, P<0.001). Urinary PD excretion was linearly correlated with DOMI (r2=0.94, P<0.001), and MN yield estimated by using either 15N or PB (r2=0.91 and 0.80, respectively, P<0.001). These results suggest that urinary PD excretion is an accurate indicator of MN synthesized in the rumen, and an equation, MN (g/d)=0.030+0.741×PD (mmol/d), is proposed to predict the MN yield from the rumen of Chinese sheep.

Modelling purine derivative excretion in dairy goats: endogenous excretion and the relationship between duodenal input and urinary output

To determine the endogenous contribution of purine derivatives (PD) to renal excretion and the urinary recovery of duodenal purine bases (PB), five dairy Granadina goats (initial weight ± s.e.: 38.6 ± 2.78 kg) were each fitted with a duodenal infusion catheter. Animals were offered ad libitum a mixed diet (75 : 25; alfalfa hay : concentrate), which was supplied in equal portions every 3 h. To label microbial PB, (15NH4)2SO4 was added to the concentrate. The lower enrichment of urinary PD (15N-allantoin) compared with duodenal PB enrichment confirmed the presence of an endogenous PD fraction (268.5 ± 21.98 μmol/kg weight0.75 or 0.386 of the total PD excretion). The recovery of PD in urine and milk increased linearly in response to increasing amounts of duodenally infused RNA (starting on day 21 after parturition). On average, 0.74 of infused PB from RNA was recovered in urine. Milk PD constituted a minor (<0.01) fraction of the total PD excretion and this fraction decreased as the amount of infused PB increased. Our findings indicate that lactation in goats did not affect the urinary recovery of duodenal PB but increased the endogenous contribution to urinary excretion of PD.

Microbial contribution to duodenal purine flow in fattening cattle given concentrate diets, estimated by purine N labelling (15N) of different microbial fractions

AbstractThe origin of duodenal purine bases (PB) was studied in a digestion experiment with four heifers, cannulated in the rumen and duodenum, which received a basal concentrate (152 g crude protein (CP) per kg dry matter (DM)) together with barley straw (85: 15 fresh weight basis) or the same concentrate supplemented with soya-bean meal, carbohydrate-treated soya-bean meal, maize gluten meal or fish meal to increase its protein content to 192 g/kg DM. Tr eatments were assigned to the four animals in five experimental periods according to an incomplete Latin-square design. Each 30-day period included 20 days of change-over adaptation and 10 days of experimental measurements. The flow of digesta entering the duodenum was estimated using Yb and acid-detergent insoluble ash as indigestible markers according to a double-marker system and microbial nitrogen (N) and PB were labelled with15N infused into the rumen. The proportion of duodenal PB of microbial origin estimated from15N enrichment of PB-N averaged 0·66 (s.e. 0·029) and did not differ between treatments nor when protozoa or bacteria associated with liquid (LAB) and solid (SAB) fractions were used as a reference sample. On average microbial contribution to duodenal non-ammonia N was higher when estimated from the PB/N ratio than from15N (0·67 v. 0·55 (s.e. 0·015)) although differences were small and not significant when LAB was the reference sample (0·58 v. 0·52 (s.e. 0·018)) reflecting the higher PB/N ratio of this fraction compared with SAB and protozoa (2·04 v. 1·65 and 1·60 (s.e. 0·04) mmol/g). Considering only the duodenal PB of microbial origin resulted in estimates of microbial N synthesis from the PB/N ratio of SAB similar to those derived from15N enrichment of both bacterial fractions (12·9 v. 13·5 and 13·3 (s.e. 0·83) g/kg of organic matter apparently digested in the rumen OMADR)) but underestimated the values derived from LAB (9·9 g/kg OMADR). Regardless of the estimation method, neither the duodenal flow of microbial N nor the efficiency of microbial synthesis differed between treatments. These results suggest that a significant proportion of duodenal PB have a non-microbial origin which may lead to overestimation of microbial yield when PB are used as a marker. Differences in PB/N ratio between microbial fractions is another important factor to be considered.

Urinary purine derivative excretion in swamp buffaloes after duodenal purine base infusion

Four male swamp buffaloes ( Bubalus bubalis) each fitted with a duodenal catheter, with an initial body weight of 244±19.8 kg were used to study the recovery rate of urinary purine derivatives (PD) after duodenally infused with incremental amounts of purine bases (PB). During the experiment, the buffaloes were fed, at a maintenance energy level, a diet containing 40% oil palm frond and 60% concentrates (DM basis). Purine bases in the form of adenosine (50%) and guanosine (50%) were infused into the duodenum in five incremental rates equivalent to 0, 24, 47, 71 and 95 mmol purine per day. Urinary allantoin, the principal PD and uric acid were linearly correlated with PB input, while the contributions of other PD were not effected by treatments. The relationship between daily urinary PD (i.e. allantoin, uric acid) excretion (mmol) and duodenal PB infused (mmol per day) was Y=0.12 X+12.78 ( r 2=0.45, P<0.001), suggesting that only 12% of supplied exogenous PB were excreted in the urine. The intercept suggested that PD excreted at maintenance energy level was 12.8 mmol per day. Urinary PD excretion rate of swamp buffaloes was much lower than those reported for cattle. We postulate that the discrepancy between the two species of ruminants could be due to a lower rate of PD absorption in the small intestine and/or recycling of plasma PD in buffaloes.

Urinary excretion of duodenal purine derivatives in Kedah-Kelantan cattle

Three male Kedah-Kelantan (KK) cattle each fitted with a ruminal and a T-shaped duodenal cannulae, with an initial body weight of 178.3±5.78 kg were used to study the recovery rate of urinary purine derivatives (PD) after duodenal infusion of incremental amounts of purine bases (PB). During the experiment, the cattle were fed at a maintenance energy level with a diet containing 40% oil palm frond and 60% concentrates. Basal purine flows into the duodenum from the maintenance diet were estimated. Purine bases in the form of adenosine (46%) and guanosine (54%) were infused into the duodenum in four incremental rates equivalent to 10, 15, 30 and 45 mmol purine per day. Urinary allantoin, the principal PD was linearly correlated with PB input, while the contributions of other PD were not affected by treatments. The relationship between daily urinary PD (allantoin, uric acid, hypoxanthine and xanthine) excretion (mmol per day) and duodenal PB flow (mmol per day) was Y=0.847 X+7.146 ( r 2=0.50, P<0.001), suggesting that 0.85 of the supplied exogenous PB were excreted in urine, with an endogenous excretion of 7.15 mmol per day. Urinary PD excretion rates of zebu cattle are similar to those of European cattle.

Excretion of purine derivatives in cows: endogenous contribution and recovery of exogenous purine bases

Two dry cows fitted with simple cannula in the rumen and duodenum, and fed with a mixed diet (straw:barley, 50:50), were used to determine endogenous losses and response of urinary purine derivatives (PDs) to duodenally infused yeast-RNA. Duodenal flow of purine bases (PBs) was determined by a dual marker system, and 15N was infused continuously into the rumen to label microbial PBs. The isotope dilution of urinary PDs in relation to duodenal PBs confirmed the presence of an endogenous fraction (236 μmol/kg LW 0.75) bigger than in sheep and lower than values estimated in cows with impaired rumen fermentation. Excretion of PDs increased linearly in response to incremental supply of PBs with an equimolar recovery of 0.84 mol/mol. However, net recovery of duodenal PBs was 0.67 for the basal diet and 0.65, 0.90, 0.79 and 0.82 for the four levels of PB infusion. It is suggested that differences in digestibility between yeast-RNA and duodenal PBs might explain differences in recovery estimations.

Microbial nitrogen production in growing heifers: direct measurement of duodenal flow of purine bases versus urinary excretion of purine derivatives as estimation procedures

The present study compares estimates of rumen microbial nitrogen (MN) production derived from duodenal flow measurements with those obtained from urinary purine derivatives (PD) excretion. Four crossbred Holstein–Friesian heifers (initial weight 306±6.0 kg) fitted with rumen and duodenal cannulae were randomly allocated in a double 2×2-crossover design to one of two experimental diets. Both diets were composed of (g/kg as fed) 250 barley straw and 750 concentrate, given together twice daily at a fixed rate close to ad libitum intake. The concentrate consisted of (g/kg as fed) either 655 corn and 225 barley (diet C) and 225 corn and 655 barley (diet B), respectively. Heifers receiving every experimental diet were infused with four levels of effective rumen degradable protein (ERDP) as urea:casein (1:1 on N basis) equivalent to 0, 25, 50 and 75 g/kg of concentrate. Duodenal flow of purine bases (PB) was determined either by using urinary PD excretion or by the dual-phase marker technique during continuous intra-ruminal infusion of Cr-EDTA and Yb-acetate. Microbial contribution to duodenal non-ammonia N (NAN) flow was estimated by the PB/N ratio in bacterial fractions extracted from the liquid (LAB) and the solid (SAB) fraction of rumen digesta. Digestible organic matter intake was higher in animals fed barley than corn diets (4.86 versus 4.25 kg/day) and increased with the ERDP supplementation (4.36–4.70; P<0.05) although most of the response was observed at the first level (25 g/kg) of supplementation. The microbial PB/N ratio was different between LAB (1.16±0.196) and SAB (0.62±0.226) leading to different estimates of MN yield from direct measurements of duodenal PB (93.3±2.68 versus 175.3±3.67 g/day) or urinary excretion of PD (62.9±1.44 versus 118.3±1.98 g/day). Urinary PD closely reflected relative differences among treatments obtained in digesta flows measurements, although urinary PD rendered consistently lower absolute values.

Microbial contribution to the duodenal flow of purine bases in cattle

It is widely accepted that purine bases (PB) could overestimate microbial production when used as microbial markers as result of rumen by-pass of dietary PB. Free nucleic acids are rapidly degraded when incubated in rumen content either in vitro or in vivo (McAllan and Smith, 1973) and nylon bag disappearance of food PB seems to confirm their low contribution to duodenal flow (Pérez et al. 1996). However isotopic labelling of microbial PB (Pérez et al. 1997) have resulted in much higher estimates of non microbial PB arriving to duodenum, which were attributed to differential uptake of dietary PB by bacterial and protozoal populations leading to lack of representativeness of the reference bacterial sample. The present experiment aimed to know if estimation of microbial contribution to duodenal PB could be biased by differences between rumen bacterial and protozoal populations in 15N enrichment of PB.

Urinary excretion of purine derivatives as an index of microbial-nitrogen intake in growing rabbits.

Three experiments were carried out to establish a response model between intake and urinary excretion of purine compounds. In Expt 1 the relationship between the intake of purine bases (PB) and the excretion of total purine derivatives (PD) was determined in seven growing rabbits with a mean initial live weight (LW) of 2.03 (SE 0.185) kg, aged 70 d, each fitted with a wooden neck collar to prevent caecotrophagy. They were fed on five experimental diets formulated with different levels of nucleic acids (0.00, 3.75, 7.50, 11.25, 15.00 g yeast-RNA/kg diet). The relationship between intake of purine (x, mumol/kg W0.75) and total urinary PD excretion (y, mumol/kg W0.75), y= 0.56 + 0.67x (r2 O.86; RSD 0.338), indicated that about 70% of duodenal PB were recovered as urinary PD and that the endogenous contribution was constant and independent of dietary PB supply. Endogenous excretion of PD (allantoin and uric acid) was measured in a second experiment using six rabbits fed on a purine-free diet and fitted with neck collars to avoid caecotrophagy. Basal daily urinary excretion values for allantoin and uric acid were 532 (SE 33.9) and 55 (SE 7.3) mumol/kg W0.75 respectively; xanthine and hypoxanthine were not found in urine samples and therefore the sum of allantoin and uric acid should comprise the total excretion of PD (588 (SE 40.1) mumol/kg W0.75). The xanthine oxidase (EC 1.2.3.2) activity in plasma, liver, duodenum, jejunum and kidney was measured in a third experiment. The activities of xanthine oxidase in duodenal and jejunal mucosa, liver and kidney were: 0.61 (SE 0.095), 0.37 (SE 0.045), 0.035 (SE 0.001) and 0 units/g fresh tissue respectively and in plasma 2.96 (SE 0.094) units/1. The results show that urinary excretion of PD may be a useful tool to estimate duodenal PB input and microbial protein intake once the relationship between PB and N has been established in caecal micro-organisms.

Excretion of endogenous and exogenous purine derivatives in sheep: effect of increased concentrate intake.

The present study examined the endogenous urinary excretion of purine derivatives (PD; allantoin, uric acid and xanthine plus hypoxanthine) in fed animals. Four Rasa Aragonesa ewes fitted with simple cannulas in the rumen and proximal duodenum were used. Animals were given a lucerne (Medicago sativa) hay diet, as sole feed (A) or supplemented, respectively, with 220 (B), 400 (C), and 550 (D) g rolled barley grain/d following a 4 x 4 random factorial design. Duodenal flow of purine bases (PB) was determined by the dual-phase marker system. 15N was infused continuously into the rumen to label exogenous or microbial PB. Duodenal PB flow and urinary excretion of PD increased with digestible organic matter intake showing a constant recovery of duodenal PB. The isotope dilution of PD in urine samples confirmed the presence of an endogenous fraction, originating from tissues, that increased from 115.2 (SE 5.84) mumol/kg W0.75 for the basal diet to 304.2 (SE 7.6) mumol/kg W0.75 at the highest level of duodenal PB.

Rumen microbial production estimated either from urinary purine derivative excretion or from direct measurements of 15N and purine bases as microbial markers: effect of protein source and rumen bacteria isolates

AbstractFour ewes fitted with ruminal and duodenal T-piece cannulae were each given six diets in a 6 × 4 factorial design. Diets or experimental treatments consisted of two ratios of forage: concentrate (700:150 (LC) and 400: 600 (HO). Forage was ammonia-treated straw and the concentrate was formulated with barley supplemented with one of three protein sources: sunflower meal, soya-bean meal or fish meal. Duodenal flows ofdigesta were estimated by the dual-phase technique using Co-EDTA and Yb acetate as liquid and solid markers. Microbial nitrogen (N) was estimated from the digesta flow of purine bases and 15N enrichment using as reference samples, bacterial isolates from the liquid (LAB) or solid (SAB) phase of rumen digesta.Duodenal flow of purine bases (mmol/day) was lower on LC (12·9) than HC (17·7) diets but in both treatments it was depressed by fish meal (12·3) compared with either soya-bean (17·3) or sunflower meal (16·3) as supplements (s.e. 1·13). Urinary excretion of purine derivatives showed a similar trend, 8·6 v. III mmol/day in LC and HC respectively and 8·8 v. 10·4 and 10·5 mmol/day in fish meal, soya-bean and sunflower meal diets (s.e. 0·56), respectively. Variation in excretion of urinary purine derivatives was mainly associated with digestible organic matter intake with an average ratio of 1·7 (s.e. 0·11) mmol per 100 g digestible organic matter intake. Irrespective of the microbial marker used, microbial yield was higher in animals offered HC than in those offered LC and with soya-bean or sunflower meal compared with fish meal supplemented diets. The microbial purine bases/N (mmol/g) ratio varied between LAB (1·99, s.e. 0·092) and SAB (1·69, s.e. 0·071) isolates leading to different estimates of microbial-N yield (g) from duodenal purine bases (7·76 (s.e. 2·84) v. 9·13 (s.e. 3·24)), urinary excretion of allantoin (5·57 (s.e. 2·0) v. 6·57 (s.e. 2·03)) or total purine derivatives (6·43 (s.e. 2·39) v. 7·56 (s.e. 2·77)). Urinary excretion of allantoin or total purine derivatives provided consistently lower estimates of duodenal microbial-N than duodenal purine bases or 15N, although it closely reflected the pattern observed in direct measurements.

Contribution of dietary nitrogen and purine bases to the duodenal digesta: comparison of duodenal and polyester-bag measurements

AbstractFour ewes fitted with ruminal and duodenalT-piece cannulas were given fourdietsin a 4 × 4 factorial design. Diets consisted of 700 (HF) or 400 (LF) g/day of ammonia-treated barley straw supplemented respectively with 150 or 600 g/day of concentrate made up with barley plus either soya-bean meal (SBM) or fishmeal (FM) as the protein source, offered at 2-h intervals. Duodenal flowsof digestawere estimated by the dual-phasetechniqueusing CoEDTAand Yb-acetate as markers and (15NH4)2SO4 was infusedinto the rumento label microbial N. Bacteria were isolated from the liquid (LAB) or solid (SAB) rumendigesta. Purinebases (PB) were isolated by precipitationin an acid solution of AgN03, and microbial contribution either to the duodenalnitrogen(N) or PB were determinedby 15N measurements induodenaldigesta and bacteria.Simultaneously, therumen degradation of Nand PB contained in SBM and FM was studiedby incubating supplements in polyesterbags in the rumen.PBcontent (mmol/g dry matter)and guanine: adenine(G/A) ratio of barley strawwas 2·89 and 5·23; barley grain,7·91 and 111;SBM, 18·8 and 1·26; and FM, 58·9 and 6·96, respectively. Duodenal flow ofPB(mmol/day)was significantly higher than PB intake on all diets and G/A ratio showed a meanvalue of 0·97, similarto the ratios determined in SAB(0·80) and LAB (1·04) and muchlower than diets(1·31 to 4·32). Microbial contribution to duodenal Nflow ranged from43·3% to 61·0%, beinghigherin SBM(59·0%)thanin FM(46·7%)diets. However, microbial contribution to duodenal PB was not affected by the experimentaltreatment, accounting for proportionately 0·77 of total PB at the duodenum. Rumen degradability of PB was much higher than that of total N and in both cases degradability was higher in SBM than FM. Direct measurements of non-microbialN were significantly higher than values determined by the polyester-bagmeasurements. However, once corrected forthe endogenousN (52 mgN per kg live weight)contribution, results show edan acceptable agreement. Duodenal flow of PB non-attributable to microbes (unlabelled PB) showed a mean value of 3·25 mmol/daywithouta significanteffect of dietary treatment. However, undegradablePBsupply determinedfor0·02, 0·05 and 0·08 per h fractional out flow rates were proportionately lower than 0·025 with SBM and 0·100 with FM diets of the estimated duodenalPB flow. Despite the magnitudeof the unlabelledduodenalPB, the close agreement between G/A ratios in duodenaldigesta and bacteria suggests thatthe contribution of dietary PB to the duodenalflow was low and seemsto confirm the reliability of values obtained from polyester-bag measurements.