The physiological mechanism of low purine derivative excretion in urine of buffaloes
 

Vo Thi Kim Thanh, E R Ørskov^*, Nguyen Thi Thanh and P Susmel^**

Hue University of Agriculture and Forestry, Vietnam
votkthanh@gmail.com
^* Macaulay Institute, UK, **Udine University, Italy

Abstract

Three cattle calves and three buffalo calves were weaned after receiving colostrum and reared by bottle feeding of milk. During the first month the animals did not have access to solid food. Urinary purine derivatives (PD), basal PD excretion and glomerular filtrate rate (GFR) were determined. After one month the animals were given access to solid feed (urea-treated rice straw) to stimulate rumen development.  After three months of age, during which time the solid food was given, urinary PD, basal PD excretion and GFR were again determined.

Urinary PD excretion, both during fasting and milk feeding, was not different between buffaloes and cattle in the milk feeding period, but there were differences between cattle and buffaloes after 3 months of age and two months of access to solid feed. The GFR was lower in buffaloes in both milk fed and solid fed calves, while the excretion of PD in the milk feeding period was similar between the two species.

It is concluded that the lower GFR in buffaloes is a condition allowing PD to stay longer in the blood thus there is more time for recycling to the rumen and to be metabolized by bacteria.

Key words: Buffaloes, cattle, purines, rumen.
 

Introduction

Some recent studies show that purine derivative (PD) excretion in urine could be used as an index to measure microbial protein supply in ruminants, since there is a relationship between microbial nucleic acids reaching the intestine from the rumen and excretion of purine derivative in urine (Chen and Ørskov 2004). While excretion of PD in urine of cattle, sheep goats and camels is closely associated with microbial protein production due to digestion of microbial nucleic acids in the small intestine, there is a problem in using this method for prediction of microbial protein production in buffaloes. This is because excretion of PD in buffaloes is less than 50% of that from other species (Vo Thi Kim Thanh et al 2004; Vo Thi Kim Thanh and Ørskov, 2006; Liang et al 1999 and 2005; Chen and Ørskov 2004). The reasons for this difference are not understood, since measurements of microbial protein production in buffaloes are similar to cattle (Chen and Ørskov, 2004) according to Vo Thi Kim Thanh and Ørskov (2006). Since the differences between cattle and buffaloes occur only after rumen development it is interesting to speculate as to the reason why.  It could possibly be due to differences in the glomerular filtration rate (GFR).  If GFR was lower in buffaloes than cattle the PD would be longer in the blood and exposed to transfer into the rumen for longer or could possibly be also due to differences in permeability between blood and rumen. If these differences in mechanisms are understood, it may be possible to use the PD methodology for measurements of microbial protein production in buffaloes (Chen and Ørskov 2004; Vo Thi Kim Thanh and Ørskov 2006).

The present experiment aimed to test purine excretion from entirely milk-fed animals during feeding and during fasting, and to compare GFR between the milk-fed and solid food periods.

Materials and methods

Three calves of Vietnamese yellow breed (Bos taurus) (17 kg body weight) and three buffalo calves (Bos bubalus) (27 kg body weight) were weaned after receiving colostrum and reared by bottle feeding of milk. The milk was formulated for humans (Vina-milk), and contained (g/liter): lactose 45, fat 32 and protein 32. The feeding level was 0.2 liter of milk/kg W^0.75. The animals were weighed at the start of the experiments.

Experiment 1. Determination of basal purine excretion

During the first month the animals did not have access to solid food so there was no rumen development (Ørskov 1992).

Period 1. Purine excretion from entirely milk-fed calves

After 15 days of milk-feeding, urine was collected for 7 days to determine differences in excretion of PD between buffalo and cattle calves.

Period 2.  Fasting trial

The milk feeding was reduced in steps over two days. No milk was given on the third day. Afterwards, urine was collected for two days to determine differences between the species in fasted purine excretion and between milk-fed and fasted.

Experiment 2. Determination of purine excretion from calves with access to solid feed

The animals had access to solid feed with 80% urea-treated rice straw containing 48.6 % dry matter, 8.5% crude protein and 13.5% ash and 20% molasses, containing 66% dry matter and 7.5 % ash were given in equal amounts for cattle and buffalo calves to stimulate rumen development (Ørskov, 1991). The feeds were offered at 50g/KgW ^0.75.  The  calves were given milk from a bottle at 0.2 liter/kgW^0.75 for a week, then the milk was replaced by water in order to maintain the suckling reflex.

Period 1.  Purine excretion after access to solid feed

After two months (when the animals were 3 months of age), during which time the animals had access to solid feed, urine samples were collected to determine PD excretion.

Period 2. Fasting trial

Milk from bottles, at 0.2 liter/kgW^0.75 was given again to the calves, feed intake was reduced to 10g DM/kgW^0.75 for two days. After that no solid feed (only milk) was given for 9 days, and then the milk was reduced to zero for two days. Afterwards, urine of fasted calves was collected for two days to determine differences between the species in feeding and fasted purine excretion.

Experiment 3. Determination of GFR

Period 1.  GFR from entirely milk-fed calves

For the last 5 days of the entirely milk-fed period blood and urine were collected for creatinine analysis to determine differences in GFR between buffalo and cattle calves.

Period 2. GFR after access to solid feed

In the period when the animals had access to solid feed, after two months urine and blood samples were collected for 5 days to determine GFR.

The GFR was calculated using the equation: (GFR (L/d) = [Urinary creatinine excretion in mmol/d] ÷ [Plasma creatinine concentration in mmol/L]) (Joint FAO/IAEA division, 2003). For comparison of GFR between buffalo and cattle, urinary creatinine was corrected to metabolic weight.

When urine was collected the calves were kept in metabolism cages with urine collected in plastic bottles under the cages that contained 100ml H₂SO₄ 10%. Continuous supervision ensured that there was no contamination of feaces with urine, and that the urine pH was maintained below 3. The urine samples were collected at 18h, mixed and then sampled in 20ml bottles and stored at -20^oC and analysed for allantoin, uric acid and creatinine. The analytical procedure used was according to IAEA (IAEA-TECDOC-945, Joint FAO/IAEA division, 2003).

Blood samples were collected at 0.700h in the morning via a polyvinyl chloride (PVC) catheter fitted into the jugular vein and centrifuged at 4000 rpm. The plasma was collected and stored at -20^oC.

Data processing was done using Excel, Minitab and Pdwork (Joint FAO/IAEA division, 2003).

Results and discussion

Experiment 1. Determination of basal purine excretion

Purine excretion from entirely milk-fed calves

The results from period 1, when the calves had no access to solid food but were fed milk from a bottle are given in Table 1. It can be seen that there were no differences in urinary PD excretion (mmol/kg W^0·75) between the buffalo calves and cattle calves (P >0·05). This is in agreement with previous observations of Vo Thi Kim Thanh and Ørskov (2006).

Purine excretion from fasting milk-fed calves

In order to observe the basal PD excretion, the amount of milk fed was reduced to zero, during which time the animals were fasted for two days. The results of urine analysis for PD excretion after fasting is shown in Table 2 and Figure 1.

There were no differences in PD excretion (mmol/kgW^0.75) between the two species (P>0.05). 

The results above show that the endogenous purine excretion expressed as mmol/kg metabolic weight was not different between the milk fed buffalo and cattle calves. The results of this experiment show also that during fasting of the milk fed animals there were no significant differences between buffalo and cattle calves.

Experiment 2. Determination of purine excretion from calves with access to solid feed

Purine excretion after two months access to solid feed  

After 2 months during which the calves had access to urea treated rice straw to stimulate rumen development, urinary PD excretion expressed as mmol/kg W^0·75 was significantly lower in buffaloes than cattle. The results are given in Table 3. The daily consumption (g dry matter day) was 609 for cattle and 825 for buffalo calves.

Purine excretion from fasting, solid-feed-fed calves

The result of urine PD (mmol/kgW^0.75) excretion during fasting after two months access to solid feed are shown in Table 4.  The PD excretion was significantly different between the two species (P<0.01). It was reported by Liang et al (1999) that endogenous PD excretion in fasting mature buffaloes was lower than in cattle, and this study shows clearly that the differences occur as soon as the rumen has developed.

 Comparison of feeding and fasting PD excretion from cattle and buffaloes in milk fed and solid feed fed periods

The PD excretion rates of calves from cattle and buffaloes (Figures 1 and 2) show clearly the differences between the species for the periods of milk and solid feeding.

Experiment 3. Glomerular filtration rate (GFR)

The cause of low excretion of PD in buffaloes could possibly be due to differences in the glomerular filtration rate (Chen and Ørskov 2004; Vo Thi Kim Thanh and Ørskov  2006). This study was intended to test this supposition. Creatinine is produced from creatine (and creatinine phosphate) in muscle, and is excreted in the urine, and creatinine excretion is correlated with the muscle mass. When expressed as mmol/per kg W^0.75, the daily excretion is relatively constant. Creatinine has been found to be neither reabsorbed from, nor secreted into, the tubule from the primary urine in sheep, cattle, cats and dogs, and may be used as an endogenous/internal marker for estimation of GFR in these animals. Measurements of both the daily creatinine output in urine and creatinine concentration in plasma are required to estimate the GFR (Joint FAO/IAEA division, 2003).

Blood and urine samples were collected in both milk fed and solid feed fed periods, and the samples were analysed for creatinine concentration. The GFR of buffaloes were lower than cattle in both periods of rumen development (Table 4).  This indicates that the lower GFR in buffaloes is a condition allowing PD to stay longer in the blood, thus there is more time for recycling to the rumen and to be metabolized by bacteria.

Conclusions

• The urine PD excretion was similar in calves from cattle and buffaloes before rumen development, but they were one third lower in buffaloes after rumen development.

• GFR in calves from buffaloes were always lower than in calves from cattle, whether the rumen was active or not.

Acknowledgements

The authors would like to thank Sida-SAREC, through the Mekarn project, and the Vietnamese Ministry of Science and Technology, through the Committee of Natural Science for their financial support of the project; thanks also to the students of Hue University of Agriculture and Forestry for participating actively in this study.
 

References

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