J. Balcells

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.

Rumen digestion and urinary excretion of purine derivatives in response to urea supplementation of sodium-treated straw fed to sheep.

The present study examined the effect of urea-N supplementation of a N-deficient diet on digestion and metabolism in the rumen. Five Rasa Aragonesa ewes, each fitted with a rumen cannula, were offered alkali-treated barley straw ad lib. alone or supplemented continuously via the cannula with four levels of urea-N (3, 6, 9 and 12 g/d). Rumen NH3 concentrations increased in response to urea infusion (6-128 mg/l; P < 0.001). At the highest level of rumen NH3 concentration there was a significant increase, compared with the unsupplemented treatment, in dry matter (DM) intake (846-1206 g/d; P < 0.001) and apparent digestibility of DM (0.38-0.43), organic matter (0.38-0.45) and neutral-detergent fibre (0.41-0.49; P < 0.01). Rumen outflow rates of particulate matter and potential DM disappearances, assessed using nylon bags, were not affected by the experimental treatments, although fractional rate of DM disappearance increased significantly with increasing levels of urea infusion (2.4-4.6 per h). Urinary excretion of total purine derivatives increased with N supplementation, although the response was exclusively due to an increase in allantoin excretion (26.9-66.4 mg/kg live weight (W)0.75 per d; P < 0.001). Xanthine, hypoxanthine and uric acid excretion rates were constant, averaging 1.8 (SE 0.17); 5.4 (SE 0.21) and 7.2 (SE 0.36) mg/kg W0.75 per d respectively. The maintenance of a minimum rumen NH3 concentration (approximately 50 mg/l) was necessary to avoid significant reductions in DM intake and fermentation rate. Higher levels, however, may further increase microbial N flow at the duodenum, as suggested by the response in urinary allantoin excretion over the range of rumen NH3 concentrations.

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

a ´ Abstract 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 0.75 level. The endogenous contribution of purine derivatives was determined at fasting (11.34 mg N / W or 202.2 0.75 mmol / W ) 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.  2002 Elsevier Science B.V. All rights reserved.

Quantification and chemical composition of mixed bacteria harvested from solid fractions of rumen digesta: effect of detachment procedure

Four ewes were given two diets made up with two ratios of rolled barley grain and ammonia treated straw (80/20, diet C and 20/80 diet R) twice a day. Two microbial markers, purine bases (PB) and 15N, were used as internal and external markers, respectively. Ruminal bacteria from the liquid (LAB) and solid (SAB) fractions of digesta were harvested from samples obtained at 1 and 6 h after feeding. Bacteria were separated from the particulate material by cooling plus homogenisation (B) and also by applying tertiary butanol (TB), methylcellulose (M) or changes of temperature (CHT). The most effective procedures to remove bacteria from the solid phase were M and CHT although CHT showed the highest level of losses and the lowest total recovery of the bacterial pellet. There were no differences between B and TB in both detaching efficiency and total recovery ratio of adherent bacteria. Ratio of recovery of detached material as a bacterial pellet was 32.0, 32.2, 33.3 and 27.8% for B, TB, M and CHT, respectively. Diet did not interact with detaching efficiency of the experimental treatment although concentration of total N (g/100 g OM) and PB (μmol/g OM) content concentration were higher in pellets obtained with diet C (9.11 and 125) than with R (8.20 and 107, respectively, P<0.05). Postprandial differences were not significant. Bacterial samples extracted from the liquid phase contained significantly (P<0.001) more total N (9.21 vs. 8.51) PB (160.5 vs. 104.3) and PB/N (1.73 vs. 1.23 μmol/mg) than those samples extracted from the solid phase. There were no differences in the chemical composition of the microbial sample after detachment by B, BT and M but bacterial extract obtained after CHT treatment showed significant changes in PB, N content and PB/N ratio (P<0.01).

Contribution of dietary purine bases to duodenal digesta in sheep. In situ studies of purine degradability corrected for microbial contamination

The dietary contribution of purine bases (PB) to duodenal flow was evaluated by the in situ method after correcting for microbial contamination using 15N as microbial marker and rumen solid associated bacteria as reference sample. Four ruminally fistulated sheep were offered at 4 h intervals a mixed diet 2:1 vetch-oat hay:concentrate, and 179 mg of ((15NH4)2SO4) were continuously infused in the rumen during a minimum of 5 days prior to incubation and rumen sampling. The following feeds were tested: meat meal (MM), fish meal (FM), gluten feed (GF), barley grain (BG), corn grain (CG), sunflower meal (SFM), soya bean meal (SBM), dry brewery distilled grain (DBDG), alfalfa hay (AH) and barley straw (BS). The largest residual proportion of microbial purine bases was found in BS followed by AH, CG, BG, DBDG, GF, SFM, MM, SBM and FM, in that order. For all feeds, the extent of degradation of dietary purine bases at the studied fractional outflow rates increased after the correction for microbial contamination. Apparent and true degradability were significantly different (P < 0.05) in DBDG, CG, AH, GF and MM, ranging the feeds in that descending order; whereas they did not differ in BG, SBM, SFM and FM. Correction for contamination of feeds by microbial purine bases did not substantially affect the proportion of dietary purines escaping ruminal degradation ranging from 5 to 17% for AH, BG, GF, SFM, CG, from 11 to 23% for MM, SBM and from 20 to 40% for FM and DBDG when fractional rates of outflow from the rumen of 0.02–0.08 were assumed.

Influence of dietary rumen-degradable protein supply on rumen characteristics and carbohydrate fermentation in beef cattle offered high-grain diets.

Four crossbred Holstein‐Friesian heifers (initial live weight 306 6:1 kg) fitted with rumen and duodenal cannulae were randomly allocated to one of two dietary treatments in a double 2 2 crossover design. Both diets were composed of (g/kg as fed) 250 barley straw and 750 concentrate. The concentrate consisted of (g/kg as fed) 655 corn and 225 barley (Diet C) and 225 corn and 655 barley (Diet B), respectively. During Period 1, two heifers were given Diet C and the other two heifers were given Diet B and all four heifers were infused intraruminally, during four sequential 16-day intervals, with four levels of effective rumen degradable protein (ERDP). ERDP was given as an iso-nitrogenous mixture of urea and casein at 0, 25, 50 or 75 g/kg of concentrate intake. Animals offered Diet B ate more DM, OM and NDF than those offered Diet C (97.6, 89.9 and 37.6 g/kg versus 94.4, 87.3 and 31.9 g/kg metabolic live weight (W 0.75 ), respectively (P < 0:05). Starch digestion did not differ significantly between diets, but fibre was better digested in Diet C than in Diet B, i.e. 56.5% versus 47.5%, 51.3% versus 36.4% and 50.5% versus 40.2% for arabinose, xylose and cellulose‐glucose digestibilities, respectively (P < 0:05). Mean rumen ammonia concentrations increased linearly from 29.1 mg/l when no ERDP was infused to 184.5 mg/l when ERDP was infused at the highest level. Ruminal pH was lower (P < 0:05) in animals offered Diet B than those offered Diet C (6.29 versus 6.46) and in ERDP-supplemented rather than unsupplemented diets (6.73 versus 6.28). However, pH never fell below 5.5. There were no differences in effective rumen degradability between Diets B and C, and increasing the ERDP supply promoted an increase in straw (P < 0:05) and corn (P < 0:1) DM disappearance from polyester bags. The diets without ERDP infusion were apparently deficient in degradable N because rumen microbial yield increased from 76.0 to 102.5 g N/d (P < 0:05) when ERDP infusion rate was increased from 0 to 25 g/kg of concentrate, irrespective of which type of grain concentrate was used. With further increases in ERDP, microbial yield maintained constant and there was no further

Effect of ammonia treatment and carbohydrate supplementation of barley straw on rumen liquid characteristics and substrate degradation by sheep

Abstract The effect of ammonia treatment and supplementation of barley straw with increasing levels (0, 200, 400 and 600 g day −1 ) of meadow grass hay (H), rolled barley (B) or pelleted sugar-beet pulp (P) on ruminal characteristics and straw degradation was studied using eight adult ewes, divided in two groups, receiving either untreated (US) or ammonia treated (TS) straw, supplemented with H, B or P consecutively. Urea (30 g kg −1 straw) was added to US 24 h before feeding. Sheep fed US showed higher ammonia-N (NH 3 N) concentration than those fed TS, either as the only feed (17.5 and 12.3 mg per 100 ml) or supplemented (15.0 and 12.7 mg per 100 ml). There was no effect of ammoniation over rumen pH or total volatile fatty acid (VFA) concentration. Supplementation with B or P depressed pH and enhanced VFA concentration, whereas H did not have any great effect. Extent of straw rumen degradation was enhanced by ammoniation (maximum degradation of 593 and 741 g kg −1 for US and TS), but rate of degradation and lag time remained unaffected. Both rate and extent of straw degradation were decreased to a similar extent when supplemented with B or P, while with H this effect was only noticeable when it was included in the diet at 600 g day −1 .

Validation of use of purine bases as a microbial marker by 15 N labelling in growing lambs given high-concentrate diets: effects of grain processing, animal age and digesta sampling site

The origin of post-ruminal purine bases ( PB) was studied in 24 growing lambs that were given a pelleted concentrate plus barley straw (C) or whole barley grain plus protein supplement ( WB). Six lambs from each treatment were slaughtered at 10 and 30 days post weaning after 15 N labelling of microbial nitrogen (N) and PB. Microbial contribution to digesta nonammonia N (NAN) and PB was lower ( P < 0·01) when estimated from duodenal rather than abomasal samples (0·36 v. 0·52 (s.e.d. 0·021) for NAN and 0·47 v. 0·77 (s.e.d. 0·029) for PB) as a result of endogenous contamination. In comparison with 15 N, total PB/N led to higher estimates ( P < 0·01) of microbial contribution to abomasal NAN in WB treatment (0·62 v. 0·46 s.e.d. 0·049). The difference was removed after correcting for microbial PB, while this effect was not observed with < the C diet, resulting in a marker by diet interaction ( P < 0·05). Abomasal PB flow increased ( P < 0·1) from 10 to 30 days after weaning mainly due to the higher proportion of microbial PB (0·70 v. 0·81 (s.e.d. 0·047)). Rumen apparent PB degradation did not differ between diets in older lambs, but it was proportionally 0·39 lower for WB treatment ( P < 0·05) in younger lambs. When the microbial PB flow was estimated indirectly from labelled microbial N and the PB/N ratio of bacterial extracts the estimates were in agreement with those derived from PB- 15 N in the WB treatment but resulted in unrealistic values in lambs on diet C. Results suggest that significant proportions of dietary PB can escape rumen degradation which may lead to overestimation of microbial contribution to abomasal NAN when the PB/N ratio is used as marker. The extent of the overestimation is affected by the lamb age and grain processing.

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.