D. J. Kyle

The effect of protein infusion on urinary excretion of purine derivatives in ruminants nourished by intragastric nutrition

Two experiments were carried out to determine endogenous excretion of purine derivatives in steers and lambs, and to investigate the relationship between microbial nucleic acid input and urinary excretion of purine nitrogen. The endogenous excretion of allantoin after conversion of hypoxanthine, xanthine and uric acid to allantoin, was calculated to be 72 and 26 mg/kg W 0·75 per day in steers and lambs, respectively, when the dietary protein contained no nucleic acid nitrogen. The excretion of purine derivatives increased linearly with increasing microbial nucleic acid input in lambs. The excretion of purine derivatives in excess of endogenous contribution was closely related to the theoretically expected values. The average recovery was calculated as 0·96 for one sheep and 1·0 for the other.

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.

The voluntary intake of hay by sheep in relation to its degradability in the rumen as measured in nylon bags

The voluntary intake and digestibility of four hays were measured with eight sheep using two 4 × 4 Latin squares. Measurements were made during the last 8 days of each 3-week period. The degradation characteristics of the hays were measured by incubating samples (in nylon bags) for 12, 24, 48 or 72 h in the rumen of four sheep fitted with rumen cannulae and given a good hay. The exponential p = a + b(1 − e −α ) where p = degradation loss, t = time, and a, b and c are constants, was fitted. The potential degradability (defined as a + b) of the dry matter (DM) of the four hays was 0·76, 0·66, 0·54 and 0·46 with corresponding voluntary intakes of 71, 62, 52 and 45 ± 2·9 g DM per kg M075 per day. The in vivo digestibilities were 0·61, 0·59, 0·46 and 0·45 (s.e. 0·013) respectively and corresponded to 23-, 25-, 31- and 67-h degradation. Voluntary intake was better related to potential degradability (and degradability at 12, 24, 48 and 72 h) than to in vivo digestibility. It is concluded that the degradation characteristics of forages may have useful application in predicting voluntary intakes, and that potential degradability could be used to define the rumen (jegradable nitrogen content necessary with any particular forage.

Flow of nitrogen from the rumen and abomasum in cattle and sheep given protein-free nutrients by intragastric infusion

1. Three experiments were conducted to determine the flow of nitrogen through the rumen and abomasum when cows, steers and lambs were totally nourished on volatile fatty acids infused into the rumen. 2. In two dairy cows (650-700 kg) and two large steers (370-405 kg) the daily flow of non-ammonia-N (NAN) from the rumen was 50.7 and 58 mg/kg live weight (W)0.75 respectively. 3. The flows of NAN through the rumen and abomasum in four young steers (240-315 kg) were 85.0 (SE 21.0) and 195 (SE 7.0) mg/kg W 0.75 respectively. 4. In the third experiment the effects of altering rumen pH and osmotic pressure on flow of NAN through the rumen and abomasum were investigated in lambs. While rumen pH and osmotic pressure influenced rumen volume and outflow they had no significant effect on NAN flow. The mean values for NAN outflow from the rumen and abomasum were 76 and 181 mg N/kg W 0.75 respectively. 5. Abomasal NAN flow increased with increasing abomasal pH. When osmotic pressure was greater than about 330 mosmol/l in the rumen there was a net inflow of water, while below this value there was net loss of water. 6. For all experiments the flow of N both from the rumen and abomasum was highly variable; this has to be considered if a constant value is used for endogenous N in estimating dietary N in the abomasum. 7. With N-free infusion the rumen NH3 concentration varied from 50 to 120 mg NH3-N/l. 8. The amino acid composition of rumen and abomasal N was also determined. Relative to tissue N it contained a higher proportion of cysteine.

Undernutrition in sheep. The effect of supplementation with protein on protein accretion.

In a comparative-slaughter experiment, individually rationed wether lambs initially of 42 kg were given 235, 362 or 456 kJ metabolizable energy (ME)/kg live weight (LW)0.75 per d as sodium hydroxide-treated barley straw with urea (six lambs per treatment), or NaOH-treated barley straw with urea plus 125 g/d white-fish meal to give 307 or 488 kJ ME/kg LW0.75 per d (seven lambs per treatment) for 92 d. All unsupplemented lambs lost both fat and body protein. The changes in fat were -3.53, -2.75 and -1.40 (SE 0.59) kg (initial value 8.6 kg), and the changes in body protein were -0.47, -0.09 and -0.14 (SE 0.13) kg (initial value 4.9 kg) for the three unsupplemented groups respectively. When supplemented with fish meal, fat was again lost as -1.53 and -0.93 (SE 0.55) kg, but wool-free body protein was increased, and gains were 0.48 and 0.89 (SE 0.12) kg for the two supplemented groups respectively. All animals lost wool-free body energy, total changes being -150, -111, -59 and -49 and -16 MJ respectively. When corrected to an equal ME intake the supplemented lambs, when compared with the unsupplemented lambs, gained (instead of losing) body protein (P less than 0.001) and lost less fat (P less than 0.05). Wool growth did not respond to supplemental protein, but was related to ME intake with an increase of 0.78 g wool fibre for each additional MJ ME. The maintenance requirements of the unsupplemented and supplemented groups respectively were estimated by regression analysis to be 554 and 496 kJ ME/kg LW0.75 per d. The apparent utilization of ME below energy equilibrium (km) was 0.31 (SE 0.08) for the unsupplemented animals, and 0.12 (SE 0.10) for the supplemented animals, well below a km of 0.70 which current UK standards (Agricultural Research Council, 1980) would predict. Most of these differences could be reconciled if basal metabolism was assumed not to be constant. It is concluded that lambs in negative energy balance can continue lean body growth at the expense of body fat, provided sufficient dietary protein is available. It is also concluded that since the animals at the lowest ME intakes required less ME than predicted by current feeding standards, the effect was that it would have been difficult to distinguish between the apparent utilization of ME for maintenance (km) and for fattening (kf).

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.

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.

Undernutrition in sheep. Nitrogen repletion by N-depleted sheep

Wether lambs of 29-44 kg live-weight, totally nourished by the infusion of volatile fatty acids (VFA) into the rumen and casein into the abomasum, were given five treatments in consecutive periods. The treatments were (daily amounts per kg live weight (W)0.75): (a) high-protein for 7 d (2500 mg nitrogen, 650 kJ VFA); (b) low-protein for 7-15 d (525 mg N, 650 kJ VFA); (c) N-free for 7 d (no N, 450 kJ VFA); (d) very-low-protein for 24-28 d (300 mg N, 400 kJ VFA); (e) high-protein for 40 d (2500 mg N, 650 kJ VFA). Nine lambs were subjected to treatments (a), (b) and (c) (Expt 1) and four of the lambs additionally received treatments (d) and (e) (Expt 2). In Expt 1 all nine lambs had a positive N retention on treatment (a) but abrupt change to treatment (b) resulted in substantial negative N balances initially, and a period of approximately 5 d adaptation was required before N equilibrium was re-established. Animals again exhibited negative N balances when the N-free infusion (treatment c) was introduced and during that period there was no evidence of adaptation. Basal urinary N excretion was estimated to be 356 (SE 12) mg N/kg W 0.75. In Expt 2 all four lambs were depleted of N when receiving the very-low-protein treatment (d). The progressively decreasing N losses recorded during days 1 to 12 of the treatment period were slightly greater than those recorded during days 13 to 28 but the difference between the means was not significant (P greater than 0.05). There was no evidence of an adaptation in N retention between days 13 and 28 of the treatment. As assessed during days 13 to 28 of the treatment the efficiency of utilization of infused casein N was 1.0; this compared with a value of 0.66 recorded during treatment (b) in Expt 1. Live weight loss during the period of N depletion was 101 (SE 27) g/d. When lambs were given treatment (e) during the last period of Expt 2, N repletion was rapid and complete within a few days. Ten days after the introduction of the treatment the rate of N retention was estimated to be 1019 (SE 38) mg/kg W 0.75 per d and this value declined at a rate of 9.5 (SE 1.9) mg N/kg W 0.75 per d for the following 30 d.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of glucose supply on fasting nitrogen excretion and effect of level and type of volatile fatty acid infusion on response to protein infusion in cattle

Two experiments were carried out on cattle nourished entirely by intragastric infusion, to determine the extent to which glucose or a glucose precursor determines the response to protein infusion in energy-undernourished animals. In order to determine the requirement for glucose in 1-year-old fasting cattle, glucose was infused at increments to supply 0, 1.5, 2.5, 3.5, 4.5, 5.5 and 6.5 g/kg metabolic body weight (W0.75) and the effects on plasma beta-hydroxybutyrate and N excretion were measured. At 5.5 g glucose/kg W0.75 plasma beta-hydroxybutyrate was reduced to a basal level of 1.65 mmol/l and fasting N excretion reduced from 529 to 280 mg N/kg W0.75. No further reduction was observed with the higher level of 6.5 g glucose/kg W0.75. In the second trial, three steers were used in a 3 x 3 Latin square design and infused with a volatile fatty acid mixture of 65, 27 and 8 mol acetic, propionic and butyric acids respectively/100 mol, either at an estimated maintenance energy level of 450 kJ/kg W0.75 and supplying a calculated glucose equivalent level of 13.0 g/kg W0.75 (M1A), or at 1.5 x maintenance supplying a glucose equivalent of 20 g/kg W0.75 (M1.5A). Another mixture infused at the maintenance energy level contained 49, 43 and 8 mol acetic, propionic and butyric acids respectively/100 mol but with a glucose equivalent of 20 g/kg W0.75 (M1P). Casein was infused at each of these energy treatments to supply 0, 200, 400, 800, 1600 and 2500 mg N/kg W0.75 daily, and N balance and blood metabolites were measured. N retention increased linearly (r 0.98) with casein infusion. The coefficients for N retention were 0.55, 0.57 and 0.64 for M1A, M1.5A and M1P respectively. The mean efficiency of N utilization was 0.58. The results suggest that provided the glucose need is met there is no relationship between energy supply and efficiency and level of protein retention. However, the results also indicate that glucose requirement in cattle may be higher than that previously observed in sheep.