Preliminary studies on urinary excretion of purine derivatives and ...

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Previous measurements of the excretion of purine derivatives (PD) (i.e. allantoin, uric acid, hypo- xanthine and xanthine) in ruminants have indicated that there ...
Journal of Agricultural Science, Cambridge (1999), 133, 427–431. Printed in the United Kingdom # 1999 Cambridge University Press

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Preliminary studies on urinary excretion of purine derivatives and creatinine in yaks R. J. L O N G*, S. K. D O N G, X. B. C H E N" , E. R. Ø R S K O V"    Z. Z. H U Department of Grassland Science, Gansu Agricultural University, 730060 Lanzhou, China " Rowett Research Institute, Bucksburn, Aberdeen AB21 9SB, UK (Revised MS received 3 August 1999)

SUMMARY Field experiments were conducted at the farm of Qinghai Academy of Animal and Veterinary Science, Xining, China during 1996\97 to determine the effects of level of food intake on the urinary excretion of purine derivatives (PD), creatinine and nitrogen in yaks (Bos grunniens). Two experiments were carried out with three female yaks (initial body weight 173–187 kg, age 5 years). For Expt 1 a 3i3 Latin square experimental design was used with three levels of oat hay (nitrogen 13n5 g\ kg dry matter (DM)) intake treatments, i.e. 0n3, 0n6 and 0n9 of voluntary intake (1n3–3n5 kg DM\d). Each treatment lasted for 17 days and the samples were collected during the last 7 days of each period. For Expt 2 the animals were fed the same oat hay as in Expt 1 for 3 weeks at a level equivalent to the estimated energy maintenance requirement (M) (1n5–2n2 kg DM\d). The intake was then reduced to 0n6 M on day 1, 0n3 M on day 2 and zero from day 3 until day 10. The animals were re-fed in the reverse order for 3 days. Of the PD, only allantoin and uric acid were present in the urine. The proportions of allantoin and uric acid were 0n86 and 0n14 respectively for both experiments. There was no response of creatinine and nitrogen excretions to feed intake. The rates of PD excretion per kg digestible organic matter (DOM) or digestible dry matter (DDM) were 13n5 and 13n6 mmol respectively. As expected, urinary PD excretion increased significantly (P 0n001) with increasing intake of DDM and DOM. The daily fasting PD, creatinine and nitrogen excretions amounted to 0n22p0n02 (..), 0n25p0n01 mmol\kg W!n(& and 314p24n2 mg\kg W!n(& respectively. The results suggest that it is possible to establish a method for estimating intestinal microbial protein flow based on PD excretion in yaks. INTRODUCTION Previous measurements of the excretion of purine derivatives (PD) (i.e. allantoin, uric acid, hypoxanthine and xanthine) in ruminants have indicated that there are marked differences in the amount and composition of the endogenous PD between ruminant species. The average daily endogenous PD excretions were about 500, 200 and 210 µmol\kg W!n(& in cattle, sheep (Chen et al. 1990 a ; Lindberg & Jacobsson 1990 ; Balcells et al. 1991) and buffaloes (Samaraweera et al. 1994 ; Chen et al. 1996), respectively. Cattle and buffaloes seem to excrete a higher proportion of PD in form of allantoin (0n86–0n92) (Verbic et al. 1990 ; Chen et al. 1992 ; Chen et al. 1996) than sheep (0n37–0n86) (Chen et al. 1990 a). Hypoxanthine plus

* To whom all correspondence should be addressed. Present address : Rowett Research Institute, Bucksburn, Aberdeen, AB21 9SB, UK. Email : rjl!rri.sari.ac.uk.

xanthine are important PD components in sheep urine (0n17 and 0n05 of total PD, respectively) but hardly detectable in urine of cattle and buffaloes. Thus, different equations based on the measurement of the excretion of total PD in urine had to be developed to estimate the supply of microbial protein to the host animal in sheep and cattle respectively (Chen et al. 1990 a ; Verbic et al. 1990). Yak (Bos grunniens), a member of the bovine genus, is the most important ruminant species in Qinghai–Tibetan Plateau (Long 1994 ; Long & Ma 1997 ; Long et al. 1999 a, b). There is no information in the literature on the excretion of PD by yaks. It would be useful to establish whether the approach of PD excretion to estimate microbial protein supply was applicable to yaks. The objective of the present work was to examine the responses of PD excretion to different levels of feeding and to fasting, and the variances between yak and other ruminants (cattle and buffaloes) in PD, N and creatinine excretions were also discussed.

. .    

428

MATERIALS AND METHODS The field experiments were carried out from November 1996 to July 1997 in Qinghai Academy of Animal and Veterinary Sciences, Xining, China, altitude 3100 m, latitude 36h8m N and longitude 101m 37h E. Three yaks were used and two experiments were conducted. Experiment 1 Three adult female yaks (initial body weight 173– 187 kg, age 5 years) were given oat hay ad libitum for 10 d to measure their voluntary intake (VI). They were then given three levels of intake at 0n3, 0n6 and 0n9 of VI in an experiment with a 3i3 Latin square design using three consecutive 17 d periods. The oat hay contained (g\kg DM), respectively : organic matter 921 ; nitrogen 13n5 ; calcium 3n9 ; phosphorus 1n8 ; neutral detergent fibre 674 and metabolizable energy (in vivo) 8n3 MJ. Water was always freely available. The animals were housed individually in metabolism crates to facilitate faeces and urine collection. Urine was collected through a mesh by a plastic funnel into a plastic tray placed under the metabolism crate. Trays were provided with a fine plastic mesh (2 mm) attached to the top edge to avoid contamination with faeces. Faeces were collected daily from the mesh every 2 h from 06.00 to 24.00 h in the last 7 d of each period. Total daily faeces were weighed and subsampled. The daily faecal subsamples from each animal were bulked for the whole period and oven dried (65 mC for 24 h). Daily excretion of urine was collected from animals into buckets during the last 7 d of each period. The buckets contained 10 % (V\V) sulphuric acid to ensure a final pH between 2 and 3 to avoid microbial growth. The urine was weighed daily, filtered through four layers of gauze, mixed thoroughly and subsamples were placed in 25-ml glass vials for storage at k25 mC for analysis of PD, creatinine and nitrogen. Experiment 2 At the end of Expt 1, the same three yaks were fed the same oat hay as in Expt 1 for 3 weeks at a level equivalent to the energy maintenance requirement (M) as estimated by the Agricultural Research Council (1984) i.e. an oat hay intake of 1n5–2n2 kg DM\d. The intake was then reduced to 0n6 M on day 1, 0n3 M on day 2 and zero from day 3 until day 10. The animals were re-fed in the reverse order for 3 days. Water was always freely available. Urine and faeces were collected from the second week of the maintenancefeeding period until the end of the experiment, using the procedure described for Expt 1. Chemical and statistical analysis Urine samples were analysed for allantoin by high-

E T A L.

performance liquid chromatography (HPLC) with pre-column derivatization according to Chen et al. (1993) and for uric acid and hypoxanthine plus xanthine according to Chen et al. (1990 b). In the latter method, hypoxanthine and xanthine were determined collectively as uric acid after treatment with xanthine oxidase. Urine samples were diluted with distilled water before the assays, by 40 times for allantoin and 10 times for uric acid and hypoxanthine plus xanthine. Creatinine in urine was determined using the method of Larsen (1972). N content of urine samples was measured by the method of Davidson et al. (1970). All daily urine samples were analysed individually. DM and ash contents of feed and faeces were determined by the method of the Association of Official Analytical Chemists (1965). Analysis of variance was carried out with the aid of Genstat 4 (Lawes Agricultural Trust 1983) to examine the effects of fasting and intake level on urine PD, creatinine and N concentrations. RESULTS In both experiments, xanthine and hypoxanthine were hardly detectable in the urine, therefore the sum of allantoin and uric acid comprise virtually all the purine derivative compounds excreted in yak urine. The ratio of allantoin and uric acid was about 0n86 : 0n14, and this proportion was affected neither by feed levels nor by fasting. Expt 1. Urinary excretion of purine derivatives, creatinine and nitrogen at different levels of feed intake The differences in the daily PD excretion between the three levels of intake were statistically significant (P 0n01) (Table 1) probably due to the considerable differences (P 0n01) in the digestible organic matter intake (DOMI). The response of total PD excretion in yak urine expressed in mmol\kg of metabolic weight (mmol\kg W!n(&) to digestible dry matter intake (DDMI) and DOMI expressed in g\kg of metabolic weight (g\kg W!n(&) were significant. The relationships between daily PD excretion (Y, mmol\kgW!n(&) and DDMI (X, g\kg W!n(&) and DOMI (X, g\kg W!n(&) were : Y l 0n148 (.. 0n055)j0n0135 (.. 0n0018) X, DDMI (r 0n943, n 9, P

0n001)

(1)

Y l 0n156 (.. 0n051)j0n0136 (.. 0n0017) X, DOMI (r 0n949, n 9, P

0n001)

(2)

The fitted value for the daily endogenous excretion from either Eqn 1 or 2 (0n15 or 0n16 mmol\kg W!n(&) was lower than the estimated endogenous excretion (0n22 mmol\kg W!n(& per d) (see next

429

Pattern of purine derivatives in urine of yaks It is the first time that measurements of PD and creatinine excretion in yaks have been reported. The pattern of PD excreted in the urine of yaks was similar to that of cattle and buffaloes, i.e. xanthine

15n8 12n6 22n8 275 309 283 0n07 0n02 0n02 0n67 0n60 0n52 0n02 0n01 0n02

.. Mean ..

Nitrogen (mg\kg W!n(&) Creatinine (mmol\kg W!n(&)

Mean .. Mean

0n39 0n54 0n70 0n00 0n00 0n00 0n07 0n08 0n09 0n02 0n01 0n02 0n32 0n46 0n61 0n84 0n39 1n07 16n4 31n0 37n7 1n01 0n52 0n08 17n2 32n1 38n1 0n682 0n656 0n598 0n658 0n628 0n563

* Voluntary intake ; † Body weight (meanp..).

DISCUSSION

175p10n9 180p10n8 186p12n4

PD excretion decreased rapidly when the feed intake was reduced from a level equivalent to estimated energy maintenance requirement to zero within 3 d, and reached a low and relatively constant level during fasting. The lowest PD value for the three yaks all appeared on day 2 followed by a relatively high level on day 3 compared with the remaining days of fasting (Fig. 1). The daily PD excretion during the feeding period was 0n38 (.. 0n04) mmol\kg W!n(& which was significantly higher (P 0n05) than that during the fasting period. If the data from the last 4 d during the fasting period were taken as the endogenous excretion, the mean of the endogenous excretion of PD in the urine was 10n5 (.. 1n0, .. 10) mmol\d or 0n22 (.. 0n02, .. 10) mmol\kg W!n(& per day. There was no significant difference (P  0n05) for daily creatinine excretion between fasting (0n25p 0n01 (..) mmol\kg W!n(&) and feeding (0n32p 0n10 mmol\kg W!n(&) periods. However, the average N excretion between the two periods (fasting : 314p24n2 and feeding : 119p6n6 mg\kg W!n(&) was significantly different (P 0n01).

0n3 VI* 0n6 VI 0n9 VI

Expt 2. Urinary excretion of purine derivatives, creatinine and nitrogen during feeding and fasting

..

paragraph). Creatinine excretion was not significantly affected by level of feed intake. There was no clear trend of changes in N excretion between intake levels.

Mean

Fig. 1. Total urinary purine derivative excretion during feeding (from day 1 to 9 and day 19) and fasting periods (from day 11 to 17) in three yaks (Expt 2).

..

19

Mean

17

..

15

Mean

13

..

9 11 Day

Mean

7

OMD

5

DMD

3

BW† (kg)

1

Intake level

0·1

Total PD (mmol\kg W!n(&)

0·2

Uric acid (mmol\kg W!n(&)

0·3

Allantoin (mmol\kg W!n(&)

0·4

DOMI (g\kg W!n(&)

0·5

DDMI (g\kg W!n(&)

PD excretion (mmol/kg W 0·75 )

0·6

Table 1. Feed intake, dry matter digestibility (DMD), organic matter digestibility (OMD), daily intake of digestible dry matter (DDMI ), and of digestible organic matter (DOMI ) and daily urinary excretion of purine derivatives (PD), creatinine, and nitrogen in three yaks (Expt 1 ; Mean and S.E. ; error D.F. 19)

Purine derivative excretion in yaks

430

. .    

and hypoxanthine were not detectable ; and the proportion of allantoin : total PD (0n86, .. 0n03) was within the range previously observed in cattle (0n86– 0n91 ; Verbic et al. 1990 ; Chen et al. 1992) and in buffaloes (0n86–0n92 ; Chen et al. 1996). This proportion of total PD excreted as allantoin was affected neither by feed levels nor by fasting. Chen et al. (1996) reported that buffaloes had a similar profile of urinary PD to that of cattle, which was expected in the light of high xanthine oxidase activities in plasma and the tissues. Whether this is also the case in the plasma and tissues of yak, or whether other mechanisms are involved, is not known. Excretion of purine derivatives, creatinine and N during fasting and in response to levels of feed intake In Expt 2, if the value for the fasting period (0n22 mmol\kg W!n(&per d) is taken as an estimate of the endogenous PD excretion, the endogenous excretion in yak is only about 40 % of that previously noted in cattle (0n530 mmol\kg W!n(& ; Chen et al. 1990 a ; 0n485 mmol\kg W!n(& ; Verbic et al. 1990) but is similar to that found in buffaloes (0n20 mmol\kg W!n(& ; Chen et al. 1996). It is interesting also to note that the daily fasting N excretion of 316 mg\kg W!n(& was similar to that found for buffaloes (275 mg\kg W!n(&) (Chen et al. 1996). This suggests that yaks could have evolved a mechanism to recycle more N to the rumen than cattle. Unlike endogenous N excretion, the daily fasting creatinine excretion of yaks (0n25 mmol\kg W!n(& was lower than the values reported for buffaloes (0n33–0n44 mmol\kg W!n(& ; Chen et al. 1996) and cattle (0n90–1n00 mmol\kg W!n(& ; Chen et al. 1992 ; Daniels 1993). This may be attributed in part to the lower body weight of the yaks used in the present study (173–187 kg) compared to that of the buffaloes (219 kg, Chen et al. 1996) and of the cattle (508 kg, Daniels 1993) used in previous work. Fiems et al. (1997) reported that heavier animals excreted more creatinine in urine than lighter ones, even if expressed per unit of metabolic weight.

E T A L.

In Expt 1, the excretion of PD was linearly related to the level of intake. Equations 1 and 2 describe the response of PD excretion to levels of DDMI and DOMI. The daily endogenous PD excretion, 0n148 and 0n156 mmol\kg W!n(& given by Equation 1 and 2 as extrapolated values, was lower than the determined value of fasting PD excretion (0n22 mmol\kg W!n(&). This suggests that yaks might have a low concentration of intestinal xanthine oxidase and exogenous purines are utilized by other tissues as well as by intestinal mucosa. The slope of the regression line (13n6 mmol PD\kg DOMI, Equation 2), which indicates the rate of PD excretion per kg DOMI, was lower than the values obtained from cattle (18n5 mmol PD\kg DOMI reported by Giesecke et al. 1993 ; and 18n4 mmol PD\kg DOMI by Daniels 1993). However, it was 2n5 times higher in yaks than in the buffaloes which excreted 5n2 mmol PD\kg DOMI (Chen et al. 1996), and this seems not to be fully accounted for by the dietary differences. The values for excretion of creatinine in yak urine were in agreement with those of Han et al. (1992) and Chen et al. (1996) in that creatinine excretion was not related to feed intake. In conclusion the results suggest that it may be possible to establish a method for estimating intestinal microbial protein flow based on PD excretion in yaks. Future work will need to focus on determining purine derivatives in plasma and tissue xanthine oxidase activity in yaks in order to establish the appropriate relationship. This work was supported by grants from Gansu Nurture Foundation for Science (GNFS No. 9660483), International Atomic Energy Agency (IAEA : RAS\5\030) and International Foundation for Science (IFS : RB\2093–1). The authors thank X. T. Han, Y. S. Mao, J. J. Shi and Q. M. Dong for assistance with samples collection and D. J. Kyle for his skilled analytical help, and R. N. B. Kay for criticism of the manuscript. R. J. Long is grateful to the Royal Society of London, UK for providing the Royal Fellowship.

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