Duodenal Flow Of Purine Bases Research Articles

Integration of Ruminal Metabolism in Dairy Cattle1,2

An important objective is to identify nutrients or dietary factors that are most critical for advancing our knowledge of, and improving our ability to predict, milk protein production. The Dairy NRC (2001) model is sensitive to prediction of microbial protein synthesis, which is among the most important component of models integrating requirement and corresponding supply of metabolizable protein or amino acids. There are a variety of important considerations when assessing appropriate use of microbial marker methodology. Statistical formulas and examples are included to document and explain limitations in using a calibration equation from a source publication to predict duodenal flow of purine bases from measured urinary purine derivatives in a future study, and an improved approach was derived. Sources of specific carbohydrate rumen-degraded protein components probably explain microbial interactions and differences among studies. Changes in microbial populations might explain the variation in ruminal outflow of biohydrogenation intermediates that modify milk fat secretion. Finally, microbial protein synthesis can be better integrated with the production of volatile fatty acids, which do not necessarily reflect volatile fatty acid molar proportions in the rumen. The gut and splanchnic tissues metabolize varying amounts of volatile fatty acids, and propionate has important hormonal responses influencing milk protein percentage. Integration of ruminal metabolism with that in the mammary and peripheral tissues can be improved to increase the efficiency of conversion of dietary nutrients into milk components for more efficient milk production with decreased environmental impact.

Comparison of purines and nitrogen-15 as microbial flow markers in beef heifers fed barley- or corn-based diets

The objective of this study was to estimate the contribution of microbial purine bases to duodenal purines and to purine derivatives [allantoin and uric acid (PD)] excreted in the urine. Additionally, microbial protein (MCP) flow estimated using duodenal flow of purine bases was compared to estimates using 15N as a microbial marker. Four beef heifers were fed two diets, barley silage/barley grain/soybean meal (diet B) or corn silage/corn grain/corn gluten meal (diet C), in a cross-over design study. (15NH4)2SO4 was infused in the rumen for 8 d to label ruminal microorganisms and their purine bases. Rumen contents, duodenal digesta, urine, and feces were sampled during the last 2 d of tracer infusion and for 48 h after the infusion ceased. The animals consumed more (P < 0.01) dry matter (DM), organic matter (OM), N, and neutral detergent fiber (NDF) with diet B than with diet C. Total tract digestibilities of DM, OM, and NDF were also higher (P < 0.01) with diet B. Ruminal ammonia (P < 0.01), volatile fatty acids (P < 0.05), and acetate (P < 0.01) concentrations and xylanase activity (P < 0.05) were higher with diet B compared with diet C. Flow of MCP to the duodenum was estimated from duodenal samples using purines or 15N as microbial markers, or from urinary PD excretion. The effects of diet or method of measurement on MCP flow were not significant. However, when the urinary PD method was excluded from the analysis, MCP flow was greater (by 26%; P = 0.01) when estimated using 15N vs. the purine-based method. The difference was mainly due to underestimation of the proportion of microbial N in the liquid duodenal digesta with the purine method. Feed purines contributed from 3.5 (liquid digesta phase) to 19.7% (solid digesta phase) of the total purine flow at the duodenum. 15N enrichment of urinary PD was 1.08 of the enrichment of duodenal purines, suggesting that feed purines contributed little N to urinary allantoin and uric acid in cattle. Key words: Allantoin, cattle, microbial protein synthesis, nitrogen-15, purine derivative

Purine Derivative Excretion in Dairy Cows: Endogenous Excretion and the Effect of Exogenous Nucleic Acid Supply

An experiment was conducted with dairy cows to study the partitioning of excreted purine derivatives between urine and milk and to quantify the endogenous contribution following the isotopic labeling of microbial purine bases. Three lactating cows in their second lactation that had been cannulated in the rumen and the duodenum were fed a mixed diet (48:52, roughage/concentrate ratio) distributed in equal fractions every 2h, and duodenal flow of purine bases was determined by the dual-phase marker system. Nitrogen-15 was infused continuously into the rumen to label microbial purine bases, and the endogenous fraction was determined from the isotopic dilution in urinary purine derivatives. Urinary and milk recovery of duodenal purine bases were estimated at early (wk 10) and late (wk 33) lactation by the duodenal infusion of incremental doses (75 and 150mmol purine bases/d) of RNA from Torula yeast. Each period was 6 d, with RNA being infused during the last 4 d, followed by measurement of the flow of purine bases to the duodenum. The isotope dilution of purine derivatives in urine samples confirmed the presence of an endogenous fraction (512±36.43μmol/W0.75 or 56.86mmol/d) amounting to 26±3.8% of total renal excretion. Total excretion of purine derivatives in urine plus milk was linearly related to the duodenal input of purine bases, but the slopes differed (P<0.005) between lactation stages resulting in a lower equimolar recovery in early (y=58.86 (±3.89)+0.56(±0.0164) x; r=0.90) than late lactation (y=58.86 (±3.89)+0.70 (±0.046) x; r=0.80). Excretion of purine derivatives through milk represented a minimum fraction of total excretion but responded significantly to the duodenal input of purine bases. No differences between lactation stages were detected, and variations in milk yield did modify significantly the amount of purine derivatives excreted through the milk.

Urinary excretion of purine derivatives and prediction of rumen microbial outflow in goats

The present study examined the relationship between duodenal flow of purine bases and urinary excretion of their derivatives (i.e., Allantoin, uric acid, xanthine and hypoxanthine) in selected milk goats. Three adult Granadina goats fitted with a T-shaped cannula in the abomasum were used to determine the endogenous contribution to renal excretion of purine derivatives and urinary recovery of abomasaly infused purine bases as yeast-RNA. Animals were fed alfalfa at maintenance level. The endogenous contribution of purine derivatives was determined at fasting (11.34 mg N/W 0.75 or 202.2 μmol/W 0.75) and it was similar to that obtained in sheep but lower than that reported in cattle. Urinary PD excretion responded linearly to incremental supply of purine bases throughout the abomasal cannula, these recovery (%) averaged 76. Xanthine oxidase activities in goat tissues were, in plasma 0.001 (S.E. 0.0001) i.u./ml, in liver 0.12 (S.E. 0.021) i.u./g and in duodenum 0.0009 (S.E. 0.00026) i.u./g. Again, activities were lower than those detected in cows but close to values determined in sheep. A similar response model between both species (sheep and goat) is suggested.

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.

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.

Composition of liquid-and particle-associated bacteria and their contribution to the rumen outflow

A method is described to estimate the composition and rumen outflow of microbes associated with liquid (LAB) and solid (SAB) digesta. Four rumen-cannulated Rasa Aragonesa ewes were given, in random order, the following 4 diets: (1) NaOH-treated barley straw, as a sole diet (700 g/day, TS); (2) NaOH-treated barley straw mixed (50 : 50) with 400 g/day of rolled barley grain (BS); (3) Diet 1 with addition of 8 g/day of urea; and (4) Diet 2 with addition of 16 g/day of urea. Co-EDTA was used as a marker for the liquid phase to estimate rumen outflow of liquid-associated purine bases (PB), and urinary purine derivatives were used as an indirect marker of total duodenal flow of PB. Solid-associated PB were calculated by the difference between both estimates. Urea infusion increased ammonia-N concentration in the rumen fluid from 4·8 to 15·9 mg/100 mL (P &lt; 0· 05) and enhanced dry matter intake of TS diets (from 343±63· 5 to 556±41·2 g/day, P &lt; 0·001). Significant differences were observed in the PB/N ratio of bacteria harvested from the liquid phase compared with that isolated from the solid phase (1·89±0·25 v. 1·66±0·32 mol/mg in LAB and SAB, respectively). Because of the differences observed between the liquid- and solid-associated bacteria, estimated values of bacterial N supply varied depending on which bacterial extract was used as reference. The fractional contribution of LAB and SAB to the postruminal bacteria was significantly influenced by the experimental diets, mainly through variations in the amount of LAB flowing out of the rumen.

Purine derivatives excretion in lactating ewes fed straw diets with different levels of fish meal

Purine derivatives (allantoin, uric acid, xanthine and hypoxanthine) concentration in blood, urine and milk samples, from a group of 20 lactating ewes, were analysed by high performance liquid chromatography (HPLC). The mammary gland contributed, together with the kidney and salivary glands, to purine derivative (PD) excretion. Daily excretion of PD and allantoin in milk were 280 ± 22.9 and 178 ± 10.7 μmol day −1, respectively, representing 2.2 ± 0.16 and 1.6 ± 0.10% respectively of the total (mammary plus renal) excretion. Problems with the endogenous uric acid contribution of the mammary gland to milk PD excretion are discussed. Measurements of PD losses in milk would improve the efficiency of response models relating duodenal flow of purine bases with urinary excretion of PD, although milk PD excretion in ewes does not seem to be a reliable alternative to such methodologies.

Determination of rumen microbial-nitrogen production in sheep: a comparison of urinary purine excretion with methods using 15N and purine bases as markers of microbial-nitrogen entering the duodenum

The present study compares estimates of rumen microbial-N production derived from duodenal flow measurements (15N and purine bases) with those from measurements of the urinary excretion of purine derivatives. Four Rasa Aragonesa ewes fitted with simple cannulas in the rumen and proximal duodenum were used. Four diets consisting of 550 g lucerne (Medicago sativa) hay/d as sole feed or supplemented with 220, 400 and 550 g rolled barley grain/d were given in a 4 x 4 random factorial arrangement. Duodenal digesta flows were determined by the dual-phase marker technique during continuous intraruminal infusions of Co-EDTA and Yb-acetate. Microbial contribution to the non-NH3 N (NAN) flow was estimated from 15N enrichment and purines: N ratio in duodenal digesta and bacterial fractions isolated from the rumen content. Whole tract organic matter (OM) digestibility and duodenal flow of OM and NAN increased (P < 0.001) with the level of barley supplementation. Digestible OM intake ranged from 19.0 to 42.7 g/kg metabolic weight (W0.75) and the duodenal flow of purine bases and the urinary excretion of allantoin increased linearly (P < 0.001) from minimum values of 7.47 (SD 1.524) and 4.65 (SD 0.705) mmol/d respectively on the basal diet to 18.20 (SD 1.751) and 11.62 (SD 0.214) mmol/d on the 400 g barley diet; a further increase in barley supplementation decreased both variables (13.50 (SD 2.334) and 8.77 (SD 0.617) mmol/d respectively). Urinary excretion of uric acid and hypoxanthine showed a slight but significant increase (P < 0.05) over all levels of barley. Molar recoveries of duodenal purine bases as purine derivatives or allantoin in the urine were 0.78 (SD 0.156) and 0.65 (SD 0.130) respectively. The increase on barley supplementation significantly augmented microbial-N, but large differences between microbial markers employed were observed. Mean values of microbial-N estimated from the duodenal purine bases or urinary allantoin excretion were on average 18 and 29% lower than those measured by 15N.

Relationship between the excretion of allantoin in urine and milk of dairy cows

The overall efficiency of nitrogen utilisation by ruminants is variable, but often very low. The efficiency of capture of degraded nitrogen by rumen microbes is an important determinant of the overall efficiency of utilisation of nitrogen by dairy cows (Dewhurst et al, 1996). Existing marker-cannula techniques for assessing rumen microbial efficiency, and their derived models, do not provide a useful description of the situation on individual farms. There is considerable scope for and improvement in the efficiency of utilisation of nitrogen within dairy systems if a simple diagnostic test of rumen function could be applied. It is envisaged that such a test would contribute to more refined use of protein supplements. A considerable amount of work (see review by Stangassinger et al, 1995) has shown that urinary allantoin excretion is a good index of microbial yield which closely correlates with duodenal flow of purine bases (which originate largely from rumen microbes). However, urine samples are not easy to obtain in the practical situation and so this work investigated the possibility of using allantoin in milk (which is easily sampled) as a microbial index by comparing it with urinary allantoin (the existing technique) and milk protein yield (a product of microbial protein).

Relationship between the excretion of allantoin in urine and milk of dairy cows

The overall efficiency of nitrogen utilisation by ruminants is variable, but often very low. The efficiency of capture of degraded nitrogen by rumen microbes is an important determinant of the overall efficiency of utilisation of nitrogen by dairy cows (Dewhurst et al, 1996). Existing marker-cannula techniques for assessing rumen microbial efficiency, and their derived models, do not provide a useful description of the situation on individual farms. There is considerable scope for and improvement in the efficiency of utilisation of nitrogen within dairy systems if a simple diagnostic test of rumen function could be applied. It is envisaged that such a test would contribute to more refined use of protein supplements. A considerable amount of work (see review by Stangassinger et al, 1995) has shown that urinary allantoin excretion is a good index of microbial yield which closely correlates with duodenal flow of purine bases (which originate largely from rumen microbes). However, urine samples are not easy to obtain in the practical situation and so this work investigated the possibility of using allantoin in milk (which is easily sampled) as a microbial index by comparing it with urinary allantoin (the existing technique) and milk protein yield (a product of microbial protein).