Animal (2008), 2:1, pp 4451 & The Animal Consortium 2008 doi: 10.

1017/S1751731107000973

animal

Modelling purine derivative excretion in dairy goats: endogenous excretion and the relationship between duodenal input and urinary output
M. Mota1, J. Balcells1-, N. H. Ozdemir Baber2, S. Boluktepe3 and A. Belenguer1
1 ` Departamento Produccion Animal y Ciencia de los Alimentos, Facultad de Veterinaria, C/Miguel Servet 177, Zaragoza, Spain; 2Turkish Atomic Energy Authoryty Saraykov Nuclear Reserach and Training Center, Ist. Yolu. 30 Km Saray Kazan/Ankara, Turkey; 3Gida Kontrol ve Merkez Arastirma, Enstitusu Mudurlugu, 3 16036 Huririyet/Bursa, Turkey

(Received 22 May 2007; Accepted 17 September 2007)

To determine the endogenous contribution of purine derivatives (PD) to renal excretion and the urinary recovery of duodenal purine bases (PB), ve dairy Granadina goats (initial weight 6 s.e.: 38.6 6 2.78 kg) were each tted 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 conrmed the presence of an endogenous PD fraction (268.5 6 21.98 mmol/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 ndings indicate that lactation in goats did not affect the urinary recovery of duodenal PB but increased the endogenous contribution to urinary excretion of PD.
Keywords: dairy goats, microbial synthesis, milk, purine derivatives, urinary excretion

Introduction The excretion of purine derivatives (PD: allantoin, uric acid, xanthine and hypoxanthine) in urine reects the duodenal ow of their precursors, purine bases (PB: adenine and guanine) (Chen et al., 1990; Balcells et al., 1991). Furthermore, in ruminants, the PB in duodenal digesta are mostly of microbial origin (McAllan and Smith, 1973) and, thus, urinary PD excretion has been used to predict microbial mass owing out of the rumen (Balcells et al., 1993; Perez et al., 1996). However, the relationship between duodenal absorption (exogenous PB) and the urinary excretion of PD is biased by the presence of endogenous PD in urine that are synthesised within the animal. Ruminant species differ in their PB metabolism; unlike sheep and goats, cattle (European and zebu) do not recycle exogenous PB. Cattle also have greater endogenous excretion of PD in the urine (Verbic et al., 1990). In addition, the physiological and nutritional status, e.g. lactation, of animals can affect the metabolism of PB (Balcells et al.,
-

2004). The recovery of duodenal PB and endogenous excretion have been investigated in non-lactating goats (Belenguer et al., 2002) but not in lactating animals. The aim of this study was to dene the response model between the duodenal absorption of PB and their urinary excretion as PD in dairy goats. To that end, the endogenous contribution to urinary PD was determined using the 15N-labelling pro cedure (Perez et al., 1998) and the urinary recovery of duodenal PB was dened using intra-duodenal infusion of RNAPB. The usefulness of milk PD as a predictor of duodenal PB ow was also examined.

Material and methods

E-mail: Balcells@unizar.es

Animals and diets Five multiparous Granadina goats (initial weight 38.6 6 2.78 kg live weight (LW)) tted with a simple T-shaped duodenal catheter (Kehr T-tube, 8 mm external R diameter Silkolatex Willy Rusch AG, Kermen/Germany) were held indoors in metabolism cages. They were fed ad libitum a mixed diet of alfalfa hay and concentrate

44

Purine derivatives in dairy goats

Table 1 Ingredients and chemical composition of the roughage and concentrate supplied to Granadina dairy goats Concentrate Ingredients (%) Alfalfa hay Maize grain Wheat bran Soya-bean meal Vitamin/mineral supplementChemical composition DM (g/kg fresh matter) (g/kg DM) Organic matter Crude protein Neutral-detergent bre Acid-detergent bre Acid-detergent lignin Roughage

100 40 35 23 2 896 933 205 186 60 9 897 916 158 501 400 85

Abbreviation is: DM 5 dry matter. Declared composition: Ca: 155, Mg: 97, P: 97, S: 28, Mn: 3, Fe: 2.7 (g/kg DM); I: 76, Co: 14, Se: 6 (mg/kg DM); vitamin A: 400 000, D3: 80 000, A: 150 (IU/kg DM).

1 mol/l HCl. The solution was infused continuously into the duodenum for 12 h (0800 to 2000 h) at a ow rate of 0.7 ml/min using a peristaltic pump (Minipuls-2 HP 18 Gilson; Villers le Bel, France). The infused doses were calculated to increase two- (phase 1: 0, 6, 12 and 18 g RNA per day) or threefold (phase 2: 0, 12, 24 and 36 g RNA per day) the theoretical basal ow. Baseline duodenal ow of PB was estimated assuming 11 g microbial protein per MJ FME (Agricultural and Food Research Council, 1993) and 1.2 mmol PB per g microbial nitrogen (N) (Balcells et al., 1992). Urine was collected daily during the last 3 days of each infusion period in 50 ml of a H2SO4 solution (10% v/v; nal pH was kept ,3). Daily samples were pooled within each individual, the weight and specic gravity were recorded and two sub-samples (50 ml) were collected and stored at 2208C.

(75 : 25) (Table 1). Each day, food was divided and offered in eight equal fractions and residues were removed and weighed. Animals had free access to drinking water and were hand milked manually at 0800 and 1700 h daily. Animals were handled following the criteria of the European Union for care and use of laboratory animals in research, and the Ethics Committee for Animal Research of Zaragoza University approved the experimental protocol.

Experimental procedure The experiment was divided into two phases. In the rst and second phase, respectively, three (37.4 6 4.34 kg LW) and two (40.4 6 2.23 kg LW) lactating goats were used. The rst phase included 7 days for dietary changeover and 31 days for experimental measurements. The urinary recovery of PD from duodenal PB (day 8 to day 32) and endogenous excretion (day 35 to day 38) was measured. On day 38, goats were sacriced and samples of tissues and digesta (abomasum and duodenum) were collected. In the second phase (32 days : 7 days for dietary adaptation and 25 days for experimental measurements) only the urinary recovery of PD from duodenal PB infusion was measured. Urinary recovery of duodenal PB The recovery of duodenal PB as urinary PD was determined after the peak of lactation, which was assumed to be reached by day 21 after parturition. In order to modify the duodenal ow of PB, four doses, administered at random, of RNA from Torula yeast (Sigma Co. St Louis, MO, USA) were continuously infused into the duodenum during four successive 5-day periods, allowing two days for treatment changeover. The RNA solution (1.1 mmol PB per g RNA) was prepared by diluting yeast doses in 500 ml of alkalinised water (pH 10 to 12 using 1 mol/l NaOH) at 408C. Once the yeast was dissolved, the solution was neutralised with

Endogenous measurements The endogenous fraction was estimated using the enrichment ratio between abomasal PB (obtained by sacrice on day 38) and urinary PD. Abomasal PB were labelled by adding inorganic 15N to the concentrate (eight doses per day). The isotope was supplied as ammonium sulphate (15NH4)2SO4, 10 at% 15N ISOTEC, Inc. USA) to provide 80 mg 15N per day on 3 days (day 35 to day 38). After the start of isotope administration (0 h), urine was collected every 12 h following the protocol described by Perez et al. (1998). Once the collection of urine ended, animals were anaesthetised and the digestive tract was removed, sectioned (rumenreticulum, abomasum and duodenum) and sampled along with several other tissues (liver and muscle). Finally, the goats were slaughtered by bleeding. Analytical procedures To determine the dry matter and organic matter, the samples were dried to constant weight at 1058C, and to ash at 5508C for 8 h. Total N content was determined using the Kjeldahl Method, with Se as the catalyst. PD and creatinine in urine were determined using HPLC (Balcells et al., 1992). PB were analysed in the digesta after perchloric acid hydrolysis following the procedure of Martn Orue et al. (1995). The isotope enrichment of 15N in PB and allantoin was determined using a mass spectrometer (VG PRIM II; IRMS connected in series to a DUMASstyle N analyser EA 1108,Carlo Erba, Milan, Italy). Urinary allantoin-N was isolated after its conversion to allantoic acid by alkali hydrolysis using ion-exchange chromatography following Perez et al. (1998). The PBN for isotope analysis was extracted by specic precipitation with Ag ions following Aharoni and Tagari (1991). Calculations and statistical analysis To estimate the time required for the equilibrium of labelled purine compounds in the urine, the 15N-allantoin abundance
45

Mota, Balcells, Ozdemir Baber, Boluktepe and Belenguer time course was monitored. After the start of isotope ingestion, urine samples were collected over time. A steady state was assumed to be achieved when the 15N abundance in urinary allantoin reached the maximum (plateau) value in a mono-exponential graphical plot (Y 5 a 1 b(1 2 e2kt ), where Y is 15N abundance (at%), a is the basal 15N abundance at time 0, (a 1 b) is the asymptotic or plateau value and t is time after the start of isotope ingestion. The contribution to urinary PD from endogenous sources was calculated at steady-state conditions as follows: Endogenous contribution 1 urinary PD 15 N at% excess= abomasal PB 15 N at% excess: The 15N enrichment (at% excess) of urinary PD was obtained by subtracting the background abundance (at%) of the urinary PD recorded before the administration of 15N. Abomasal PB were obtained by sacrice; therefore, background samples were unavailable, and the natural abundance of abomasal PB was taken from Askar et al. (2005). In the calculation, it was assumed that at short time intervals, the enrichment of endogenous sources would be negligible (Perez et al., 1998). The effect of duodenal infusion on the urinary excretion of PD was assessed using the following model: y m Ai Lj A Lij ij; analysing animal effect (A) and level of PB infusion (L). The model was unbalanced; therefore, to equilibrate the error term, the harmonic (Steel and Torrie, 1960) mean was estimated. Calculations were performed using Statistical Analysis Systems Institute (2000). Results Throughout the experiment, the goats remained in good health and no unusual behaviours were observed in response to the administration of the isotope or the infusion protocol. Even though the experimental procedures were identical in the two phases, the animals used in phase 2 (goats 4 and 5) had higher feed intake and milk production, but lower weight loss, than did the animals in phase 1 (goats 1, 2 and 3) (Table 2).

Excretion of PD in response to the supply of duodenal PB Figure 1 shows the relationship between the duodenal infusion of PB and the urinary excretion of PD in terms of either allantoin or total PD for both phases. Allantoin was the main PD in urine (up to 0.85) and its variation in urine samples mostly explained the response of total PD to the duodenal infusion of PB (P , 0.001; Table 3). In addition, allantoin precursors varied signicantly with PB supply, although more irregularly: from 0.046 to 0.054, 0.048 to 0.031 and 0.042 to 0.013 from the basal to the top level of RNA infusion for uric acid, hypoxanthine and xanthine, respectively) (Table 3). The recovery of duodenal PB in urine was similar in both phases, in terms of allantoin (0.61 6 0.08 and 0.64 6 0.078) and total PD (0.70 6 0.094 and 0.69 6 0.067 in phase 1 and 2, respectively); however, the equation intercepts were lower in phase 1 (8.77 and 17.7 for allantoin and total PD, respectively) than they were in phase 2 (10.6 and 19.9). Some PD were excreted in milk, mostly as allantoin, but also as its precursors (hypoxanthine, xanthine and uric acid). The total PD concentrations in goat milk were almost constant (110.9 6 12.13 mmol/l), with allantoin the most abundant (0.40 to 0.76), followed by uric acid (0.15 to 0.38) and hypoxanthine (0.14 to 0.23). Xanthine was not detected in milk samples. The mean excretion rate of milk PD ranged from 87.7 to 147.4 mmol/day and, on average, it was less than 0.01 of the total excretion (urine plus milk). The proportional contribution decreased from 0.0071 to 0.0031 as the PB infusion increased. Milk PD excretion was not correlated with duodenal PB ow or milk yield. Endogenous excretion of PD After 72 h of isotope administration, the abundances of PBN in abomasal digesta were similar to those found in ruminal bacteria (P , 0.05; Table 4), which conrmed that most of the PB in the abomasum were of microbial origin. The abundances 15N (at%) in the liver and muscle were 0.3893 6 0.044 and 0.3701 6 0.005, respectively (Table 4). After inorganic 15N was added to the diet, isotopic abundance in urinary allantoin increased rapidly during the rst 48 h and slowly, thereafter. A steady state was reached by 66 h. A mono-exponential model was tted to the data and a Students t-test was used to conrm whether the theoretical value at plateau (0.517 6 0.026 at%) differed

Table 2 Feed intake and milk yield in Granadina dairy goats with one kid and fed a mixed diet ad libitum Animal Live weight (kg) Milk yield (ml/day) Dry matter intake (g/day) Roughage Concentrate Total dry matter 1 32.6 1753 921 516 1438 2 33.6 911 921 211 1133 3 41.6 775 892 131 1023 4 51 1682 958 474 1433 5 39.5 1949 1034 972 2006 Mean 6 s.e. 39.7 6 3.31 1414 6 238.2 945 6 24.4 461 6 147.6 1406 6 170.7

46

Purine derivatives in dairy goats

(a)

50

Phase 2 Phase 1

40 Allantoin (mmol /day) y = 0.64x + 17.7 R2 = 0.92 30

20 y = 0.61 + 8.8 R2 = 0.86 10

0 0 (b) 50 Phase 2 Phase 1 40 Total PD (mmol /day) y = 0.69 + 20.0 2 R = 0.95 30 5 10 15 20 25 30 35 40 45

20 y = 0.70 + 10.5 R2 = 0.87 10

0 0 5 10 15 20 25 30 35 40 45 PB infusion (mmol/day)

Figure 1 Mean daily excretion of (a) allantoin and (b) total purine derivatives (PD: allantoin, uric acid, xanthine plus hypoxanthine) and the input of purine bases in two phases: 1 (three goats) and 2 (two goats).

Table 3 Effect of level of RNA infusion (L) into the duodenum and the experimental phase (P) on the excretion of allantoin and total purine derivatives in urine (mmol/day) and milk (mmol/day) in Granadina dairy goats Infused RNA (g/day) Animals (n) Urinary excretion (mmol/day) Allantoin Total PD Mammary excretion (mmol/day) Allantoin Total PD 0 5 12.2 (13.2)14.3 (15.2)64.2 (65.4)102.0 (106.3)6 3 13.0 (17.8) 15.1 (20.0) 67.5 (60.5) 87.8 (95.6) 12 5 21.0 (21.1) 24.2 (24.4) 81.4 (82.6) 116.3 (120.7) 18 3 21.9 (27.0) 25.5 (27.0) 69.1 (75.0) 91.3 (112.7) 24 2 35.1 (30.7) 38.6 (30.7) 75.7 (75.7) 136.1 (114.7) 36 2 42.2 (37.4) 46.9 (37.4) 57.2 (57.2) 147.4 (126.0) r.s.d. s.e. L P

2.34 2.50

1.44 1.54

*** ***

* *

8.21 18.34

8.21 11.19

* NS

NS NS

Abbreviations are: PD 5 purine derivatives; NS 5 not signicant. Estimated harmonic mean for an unequal number of replicates in each treatment. *P , 0.05; ***P , 0.001.

47

Mota, Balcells, Ozdemir Baber, Boluktepe and Belenguer

Table 4 Isotopic abundance (at%) in purine bases isolated from abomasal digesta, rumen bacteria and tissues (liver and muscle), and in allantoin extracted from urine in lactating Granadina dairy goats after 72 h of (15NH)2S04 administration. Estimated contribution of the endogenous fraction to total purine derivative excretion is also presented Animal Goat 1 Goat 2 Goat 3 Mean s.d.

Natural abundance % (Standard I.A.E.A. 0.3663) Induced abundance (%) Purine bases-N Digesta Abomasum Bacteria Tissues Muscle Liver Purine derivative-N Urine Allantoin-N (72 h) Endogenous fraction-

0.5533 0.5458 0.3710 0.3942

0.5755 0.5683 0.3701 0.3933

0.5713 0.5181 0.3691 0.3805

0.5524 0.5441 0.3701 0.3893

0.0128 0.0145 0.0005 0.0006

0.5086 0.251

0.4908 0.426

0.4563 0.437

0.4852 0.358

0.0154 0.063

Endogenous fraction 5 1 2 [urinary PD 15N at% excess/duodenal PB 15N at% excess]. Assuming the natural abundance of allantoin-N (0.3712) determined by Perez et al. (1998) and for purine bases-N the value (0.3670) reported by Askar et al. (2005).

Table 5 Urinary excretion (mmol/kg metabolic weight (LW0.75)) of purine derivatives and creatinine, and the estimated endogenous excretion of allantoin and total purine derivatives in the urine of Granadina dairy goats Animal Urinary excretion (mmol/kg LW0.75) Allantoin Xanthine Hypoxanthine Uric acid Total PD Creatinine Endogenous contribution (mmol/kg LW0.75) Allantoin Total PDGoat 1 Goat 2 Goat 3 Mean 6 s.e.

686 49.8 59.4 99.0 894 335 173 231

630 50.2 63.8 43.0 787 390 253 307

371 48.2 73.9 61.7 540 409 174 267

569 6 62.4 49.4 6 0.60 65.7 6 4.29 67.9 6 16.45 745 6 63.8 382 6 13.1 200 6 26.4 268 6 21.9

Abbreviations are: LW 5 live weight; PD 5 purine derivatives. Endogenous excretion of total PD was calculated assuming that precursor enrichment of the end product (allantoin) should be similar to its precursors (other PD).

signicantly from the abundance at 72 h (0.498 6 0.015 at%). The latter was used to estimate the urinary endogenous contribution to urinary PD, which was 0.386 6 0.070 (Table 4) and the endogenous losses of allantoin was 200.4 6 26.38 mmol/kg LW0.75. Total endogenous losses of PD (268.5 6 21.98 mmol/kg LW0.75) were calculated assuming that the isotopic enrichments in allantoin precursors (i.e. xanthine, hypoxanthine and uric acid) were similar to that in the end product of purine metabolism (allantoin) (Table 5).

Discussion In previous experience in our laboratory Granadina goats showed problems after surgery (Belenguer et al., 2002); therefore, in the present assay, a simple infusion catheter
48

was placed into the proximal duodenum, rather than using cannulated animals. Nevertheless, the dry matter intake recorded in our study was lower and more variable than expected. The animals were identically treated; thus, the high degree of individual variation must be due to uneven adaptation by the goats to the experimental protocol and, therefore, that result must be treated with caution. Scharrer et al. (1981) and Martin Orue (1998) demonstrated in vitro and in vivo, respectively, that the intestinal PB absorption may become saturated. This problem could be particularly important when using lactating animals and increasing doses of yeast RNA. For that reason, in phase 1, the RNA doses were designed to reach about two times the theoretical duodenal ow of PB; however, the initial data (phase 1) showed no irregularities in the PB recovery ratio, and the relationship between duodenal input and urinary output was linear. In the second phase, RNA doses were

Purine derivatives in dairy goats increased to obtain a wider range of responses and closer to the natural PB ow in Granadina goats under normal milk production conditions. considering the high metabolic activity in liver; however, muscular PB indicated a lower level of isotope incorporation (0.016 of digesta PBN enrichment). If muscle constitutes a higher proportion of the total body PB pool, we can assume that the contribution of isotopes from endogenous sources to urinary PD would be similar to the muscle enrichment (1.6 at% excess) and, thus, the error would be negligible. Our study suggests that physiological status had a signicant effect on the contribution of endogenous PD to urinary losses because the value in lactating goats (268 mmol/kg LW0.75) was higher than in goats fed at the maintenance level (202.2 mmol/kg LW0.75; Belenguer et al., 2002). Differences in endogenous PD excretion between physiological status in goats conrmed previous results in cattle (Balcells et al., 2004), which emphasise the need to consider the physiological phase effect in the design of response models of the duodenal input and urinary output of purine compounds.

Endogenous contribution to urinary PD The isotopic (15N) dilution of urinary PD in relation to duodenal PB conrmed an endogenous fraction in the urinary excretion (Table 4), which has been observed in non-isotopic studies of goats (Lindberg, 1989; Belenguer et al., 2002), other ruminants (Balcells et al., 1991; Orellana-Boero et al., 2001; Guerouali et al., 2004) and non-ruminant species (Martn Orue et al., 1995; Balcells et al., 1998). Differences in estimates of the endogenous fraction may partly reect the different experimental techniques adopted. Lindberg (1991) estimated the endogenous fraction in milk-fed goat kids (242 mmol/kg LW0.75), and Belenguer et al. (2002) did so by using fasted goats (202.2 mmol/kg LW0.75). However, those and other procedures, such as intragastric nutrition (rskov et al., 1979) or duodenal digesta draining (Balcells et al., 1991), are too aggressive to use with lactating animals. Initially proposed to be applied in sheep (Perez et al., 1998), the method based on the urinary dilution of labelled exogenous purines allows determination of the endogenous component of the urinary PD without having any adverse physiological effect on the animals. Using that method in sheep, the above authors obtained an endogenous excretion of 115.2 mmol/kg LW0.75, which was in the range (92 to 150 mmol/kg LW0.75) reported by non-isotopic studies based on intragastric or digesta draining methods (rskov et al., 1979; Giesecke et al., 1984; Chen et al., 1990; Balcells et al., 1991). Furthermore, the technique has been used in lactating cows (Gonzalez-Ronquillo et al., 2004), showing that recycling of exogenous PB into the tissues by the salvage mechanism was negligible (Chen et al., 1990). The endogenous contribution to urinary excretion in lactating goats has not been reported. Like sheep, goats exhibited low tissue xanthine-oxidase activity (Belenguer et al., 2002), which suggests a signicant salvage of exogenous PD. That salvage pathway implies isotope enrichment of tissue PD, which might have affected our results, but it should be a time-dependent function. We felt that 48 h were needed to obtain a homogeneous distribution of the isotope in the rumen (Broderick and Merchen, 1992) and that an additional 24 h had to elapse before the 15N enrichment in urinary PD reached a plateau following duodenal absorption (Chen et al., 1990; Balcells et al., 1991). Therefore, 72 h should be sufcient to reach the isotope equilibrium without signicant labelling of endogenous purines (Perez et al., 1998). After 72 h of isotope administration, the enrichment in tissue (liver and muscle) PB conrmed the capacity of goats to recycle exogenous (digestive) purines. In liver tissue, PB became signicantly enriched (0.0011 of the PBN enrichment registered in duodenal digesta) and, thus, liver PB enrichment is an index of the maximum potential error

Duodenal input and urinary output In conventional studies, milk yield and urinary PD excretion were positively correlated. Increases in rumen microbial yield implied an increase in the nutrient supply to the host animal and, consequently, an increase in milk yield (Gonzalez-Ronquillo et al., 2004). The duodenal infusion of RNA allowed us to break down the correlation between milk yield and duodenal PB ow and to have a precise estimation of the variation in the duodenal PB ow. The relationship between duodenal PB input and urinary PD output in goats is similar to that in sheep (Belenguer et al., 2002). Moreover, in our study, the signicant concentrations of salvageable PD (hypoxanthine plus xanthine) in urine (Table 5) and the enrichment in tissue-PB (Table 4) conrmed that in goats, as in sheep, exogenous PB might be recycled and contribute to the biochemical feedback in the de novo synthesis. If that is true, exogenous PD would be recovered in the urine only when intestinal absorption exceeds basal excretion. Once exogenous PB absorption fully replaces the de novo synthesis process, the recovery of duodenal purines as urinary PD would be proportional to the ratio between renal and non-renal losses (e.g. salivary and mammary) (Chen et al., 1990; Balcells et al., 1991). In our study, the urinary excretion of PD (745 mmol/kg LW0.75) in the absence of a supply of duodenal RNA was greater than the endogenous losses (268 mmol/kg LW0.75) and thus urinary recovery of PD should be constant and proportional to the exogenous supply. Effectively the relationship between the daily excretion of PD (y, mmol/day) and the duodenal supply of PB (x, mmol/day) was linear (Figure 1) and the addition of a quadratic component in the calculations did not improve signicantly the amount of variation explained by the model. A linear response between duodenal input and urinary output has been observed in other ruminant, including dry goats (Belenguer et al., 2002), sheep (Balcells et al., 1991),
49

Mota, Balcells, Ozdemir Baber, Boluktepe and Belenguer steers (Orellana-Boero et al., 2001) and milking cows (Gonzalez-Ronquillo et al., 2004). The urinary recovery of PD in lactating goats was in the range observed in cattle 0.75 to 0.8 (Orellana-Boero et al., 2001; Verbic et al., 1990) sheep, (0.7 to 0.8; Balcells et al., 1991; Chen et al., 1990) and camels (0.72; Guerouali et al., 2004), but was higher than the recovery in buffaloes (0.11, Pimpa et al., 2003). The recoveries that we observed in lactating goats were similar to those observed in dry goats (0.74; Belenguer et al., 2002); thus, the metabolic changes during lactation do not affect the metabolism of exogenous PB, and consequently our results suggest that in goats a common coefcient could be used regardless of the individuals physiological state. Unlike in goats, in cattle, lactation affects the recovery of duodenal PB. During peak lactation, the recovery ratio was signicantly lower than it was in late lactation (0.44 v. 0.74, respectively; Gonzalez-Ronquillo et al., 2004). was not detected. Excretion of PD in milk was not a suitable predictor of duodenal ow in Granadina goats. Acknowledgements This work has been supported by the project AGL2001-0941CO2-02 and was part of a coordinated programme of research under the sponsorship of the International Atomic Energy Agency. For isotope analysis thanks are extended to R. Redondo, Faculty of Science, U.A.M.

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Mammary excretion of PD In Granadina goats, the contribution of milk PD to total excretion (0.003 to 0.01) was less than that in ewes (0.022; Martn Orue et al., 1996) and cows (0.016 and 0.034 in early and late lactation, respectively; Gonzalez-Ronquillo et al., 2004), and the PD prole in milk differed from that in urine. Differences in the PD proles of milk and urine were explained previously in dairy ewes (Martn Orue et al., 1996) and cows (Gonzalez-Ronquillo et al., 2004). The absence of uricase and high xanthine oxidase activity in milk and mammary glands (Martn Orue et al., 1996) caused a proportional increase in the uric acid concentration as an end product of PB metabolism in milk. In goats, the duodenal infusion of PB did not affect PD excretion through milk, which is similar to the results observed in lactating cows when a similar experimental protocol was used (Gonzalez-Ronquillo et al., 2004) and lactating ewes (Martn Orue et al., 1996), in which duodenal ow was modied by increasing the dietary supply of rumen by-pass PB. The results of our study, however, contrast with the signicant correlation observed in conventional dairy cows, where increases in organic matter intake and microbial production resulted in parallel increases in milk production and allantoin excretion (Gonda and Lindberg, 1997). In that case, it is not possible to determine whether increases in the mammary excretion of PD are positively correlated with duodenal ow of PB, or rather due to an increase in milk yield. Implications This study dened the positive linear relationship between the duodenal input of PB and urinary output of PD compounds in lactating goats. Lactating animals exhibited a higher level of endogenous losses than did non-lactating goats fed at maintenance, although variation in urinary recovery between mid-lactation and maintenance feeding
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Purine derivatives in dairy goats

McAllan AB and Smith RH 1973. Degradation of nucleic acids in the rumen. The British Journal of Nutrition 29, 331345. Martn Orue SM 1998. Effect of supplementation with degradable protein on rumen fermentation and microbial yield in cattle feed concentrate diets. Thesis, University of Zaragoza, Spain. Martn Orue SM, Balcells J, Guada JA and Castrillo C 1995. Endogenous purine and pyrimidine derivative excretion in pregnant sows. The British Journal of Nutrition 73, 375385. Martn Orue SM, Dapoza C, Balcells J and Castrillo C 1996. Purine derivates excretion in lactating ewes fed straw diets with different levels of shmeal. Animal Feed Science and Technology 63, 341346. Orellana-Boero P, Balcells J, Martn-Orue SM, Liang JB and Guada JA 2001. Modelling purine derivative excretion in cows: endogenous contribution and recovery of exogenous purine bases. Livestock Production Science 68, 243250. rskov ER, Grubb DA, Wenham G and Corrigal E 1979. The sustenance of growing and fattening ruminants by intragastric infusion of volatile fatty acids and protein. The British Journal of Nutrition 41, 553558. Perez JF, Rodrguez CA, Gonzalez J, Balcells J and Guada JA 1996. Contribution of dietary purine bases to duodenal digesta in sheep. In situ studies of purine degradability corrected for microbial contamination. Animal Feed Science and Technology 62, 251262. Perez JF, Balcells J, Cebrian JA and Martn-Orue SM 1998. Excretion of endogenous and exogenous purine derivatives in sheep: effect of increased concentrate intake. The British Journal of Nutrition 79, 237240. Pimpa O, Liang JB, Balcells J, Jelan ZA and Abdullah N 2003. Urinary purine derivative excretion in swamp buffaloes after duodenal purine base infusion. Animal Feed Science and Technology 104, 191199. Scharrer E, Raab W and Amann B 1981. Active absorption of hypoxanthine by lamb jejunum in vitro. Pfugers Archive European Journal Physiology 391, 4143. Statistical Analysis Systems Institute 2000. Statistical analysis systems, released 8.01. Statistical Analysis Systems Institute Inc., Cary, NC. Steel RGG and Torrie JH 1960. Principles and procedures of statistics with special reference to biological sciences. McGraw-Hill Books, New York. Verbic J, Chen XB, MacLeod NA and rkov ER 1990. Excretion of purine derivatives by ruminants. Effect of microbial nucleic acid infusion on purine derivative excretion by steers. Journal of Agricultural Science, Cambridge 114, 243248.

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