Livestock Research for Rural Development 23 (6) 2011 Notes to Authors LRRD Newsletter

Citation of this paper

Effect of feeding regime on the nutritional status and purine derivative excretion of milking cows

T Seresinhe

Department of Animal Science, Faculty of Agriculture, University of Ruhuna, Mapalana, Kamburupitiya, Sri Lanka   ;


Daily excretion of purine derivatives (PD), milk yield and composition by lactating cows under two different nutritional regimes were examined to determine the validity of their use as indicators of nitrogen (N) and nutritional status.  Ten crossbred (Sindhi x Sahiwal) milking cows between 2nd – 4th lactation (230 ▒11 kg of live weight, 119 ▒ 10 days in lactation) were fed on a traditional ‘on farm’ diet with 115g of crude protein (CP)/kg of feed on a dry matter (DM) basis for 20 days followed by an experimental diet (137.5 g of CP/kg of feed) for another 20 days.


Intake of organic matter, N, milk yields, fat and total solids were higher (P<0.05) with the experimental diet due to higher (P<0.05) dry matter intake, however, the “Nitrogen Use Efficiency” (NUE) of milk was reduced in this period. The estimated microbial nitrogen yields were 28 and 33 g/day for the ‘on farm’ and ‘experimental’ diets, respectively. PDC Index or allantoin/PD ratios were not affected (P>0.05) by dietary treatments. However, the PDC Index and PD output showed a positive correlation (r2=0.74).   It can be concluded that allantoin alone or PD could be used as indicators of microbial nitrogen production and the nutritional status of Sindhi x Sahiwal milking cows.  

Keywords: Allantoin, Creatinine, Dry matter intake, Microbial nitrogen, Nitrogen use efficiency


Dairying is exclusively a small holder activity in Sri Lanka but produces only marginal profits for farmers. Poor breeding, scarcity and low quality of feed, improper management practices etc. result in low productivity levels of dairying mainly based on indigenous cross-bred cattle. Therefore, one strategy for improving production has been to maximize the efficiency of utilization of available feed resources by providing optimum conditions for ruminal microbial growth. Ruminants meet 50 to 100% of their total crude protein requirements from rumen microbes (Johnson et al 1996). Microbes are assimilated primarily as amino acids and nucleic acids, both of which undergo a series of metabolic processes. The catabolism of purine bases usually yields purine derivatives (PD), which are principally allantoin, uric acid, xanthine and hypoxathine. Allantoin is quantitatively the most abundant in the urine of ruminants, while the other three vary from one species to another (Balcells et al 1991). It is therefore, not surprising that a close relationship exists between urinary excretions of PD and duodenal supply of purines (Balcells et al 1993, Giesecke et al 1984, Fujihara et al 1987).  Likewise Soejono et al (2004) demonstrated that PD excretion concentration in the urine was increased significantly with intake and higher nutrient status in Ongole cattle. However, due to the need for total urine collection in with this technique, the potential application under farm conditions is limited. Several authors (Nsahlai et al 2000, Cetinkaya et al 2000) observed that the concentration of PD in urinary spot samples could be used as an index of nitrogen (N) intake or status.


In the present study, the daily excretion of PD and creatinine (CR) by lactating cows under two nutritional regimes was in order to test as to whether PD could be used as indicators of N and nutritional status under local feeding conditions.


Materials and Methods


Animals and diets


The experiment was conducted at Mapalana farm, Kamburupitiya, Sri Lanka. This farm is situated in the low country-wet zone of Sri Lanka at an elevation of 58 m above sea level (latitude 6.20 N; longitude 80.460E ), with a mean temperature of 280C and a relative humidity of 78% (Seresinhe and Pathirana 2000). The experimental design was a switch over design with two treatments (“on farm” and “experimental”) with 10 replicates (cows).


Experimental procedure


Ten crossbred (Sindhi X Sahiwal) milking cows of 230 ▒11 kg of live weight, 119 ▒ 10 days in lactation and 3rd – 4th lactation were housed in concrete floor- tie stalls with concrete feeding troughs. They were fed for 20 days an “on farm diet” composed of: 80% of total diet dry matter of natural grasses (Brachiaria brizantha- Panicum maximum- mid bloom stage 260 g of DM and 105 g of CP/kg of grass) and 20% a cattle  concentrate (270 g of DM and 178 g of CP/kg of concentrate). The whole diet had 270 g of DM and 115g of CP/kg of total feed. Fresh grasses were harvested in the morning, chopped to 10-20 cm length, thoroughly hand mixed and fed ad libitum (10% refusal rate) at 10:30h and 15:30h on the same day. Dairy mash was fed separately daily before milking. Thereafter an experimental diet with 310 g of DM, 137.5 g of CP/kg of feed made of 70% pre bloom stage grass and 30% concentrates (dairy mash + sesame oil meal) was fed for another 20 days.. Milk yields, samples of milk, feeds and spot urine samples were collected as mentioned above. Feed composition and intake are presented in Table 1.


Milk yields were recorded daily while milk and feed samples were collected during last 3 days of the adaptation period. Spot urine samples (at least 4 samples from each animal) were collected during the last two days of each period between 08-12, 12-15, 16-20 and 20 - 8 h (Chen and Gomez 1995). Water was available ad libitum. A mineral mixture was given daily with concentrates at a rate of 50 g per animal.


Forty ml of 10% H2SO was added into containers before urinary collection. After each collection period, 25 ml of urine were transferred using a syringe to storage bottles and diluted four-folds with distilled water. Four aliquots of 10 ml were taken from each diluted urine sample and stored in glass bottles at -200C for subsequent analyzes.


Body weights were recorded using a weigh bridge at the beginning of the experiment, at 20 days and at the end.


Milk samples were analyzed for fat and total solids while feeds and faeces were analyzed for DM, N, crude fiber (CF) and crude fat (CF) according to A.O.A.C (1985).


Urine samples were analyzed for allantoin and, uric acid representing the purine derivatives (PD) and for creatinine (CR) following the procedures of IAEA TECDOC 945 (1997).




PD: Creatinine ratio (PDC INDEX) was calculated according to the following formulae (Seresinhe et al 2004).



Where W is the body weight in kg and PD and Creatinine are their concentrations are in mmol/l.


PD excretion (mmol/d) was calculated using the following equation.


PD excretion (mmol/d) = (PDC index)*C


Where C is the daily creatinine excretion (mmol/kg W0.75) for these crossbred animals typical for Sri Lanka


Creatinine excretion averaged 0.96 mmol/kg W0.75 /d (Seresinhe et al 2004).


Using the above data, absorbed exogenous purine concentration (mmol/d) was determined as follows.


Y= (0.385kg W0.75) + 0.85X


Where  Y = Urinary PD excretion (mmol/d)

X= absorbed exogenous purine as mmol/d

W = live weight (kg)


Microbial N yield was calculated following Chen et al 1995b using the above sata as follows.



Statistical analysis


Treatment effects were analyzed using the SAS procedure ANOVA and differences among mean values were determined by the least significant difference test at t. The level of significance is 0.05. Regression equation and correlation coefficient between PDC index and PD output were calculated using the Excel system.


Results and Discussion


Overall nutritional status of lactating cows


The experimental diet with higher contents of  DM, organic matter (OM) and  N resulted in significantly higher (P<0.05) intake of DM, OM and N compared with the ‘on farm’ diet. Improved quality of the experimental diet (higher contents of N, fat, nitrogen free extracts (NFE) and a lower content of crude fibre) was due to better quality of the offered grass (pre-bloom heading stage) together with inclusion of Sesame oil meal in the dairy mash.  Therefore, the results  reflect  the  higher nutritional status of   cows on the experimental diets compared to the “on farm” diet, the experimental diet with a superior nutritional quality resulted in a higher intake of feed and therefore, N and other nutrients as well.


Table 1: Composition and intake of ‘on farm’ and experimental diets


On farm diet

Experimental diet

Ingredient composition1



Brachiaria brizantha and Panicum maximum



Mixture (mid-bloom stage)2



B. brizantha and P. maximum



(pre-bloom stage-heading) 3



Concentrates mixture



Proximate composition (g/kg) - Total Ration



Dry matter

270 ▒ 2.1

310 ▒ 2.4

Organic matter

188 ▒ 1.8

207 ▒ 1.9


18.4 ▒ 2.4

22 ▒ 2.7

Crude protein

115 ▒ 15

137.5 ▒ 16.9

Crude fiber

359 ▒ 5.6

318 ▒ 4.4

Crude fat

18 ▒ 1.1

29 ▒ 1.3

Nitrogen free extract

436 ▒ 5.6

444 ▒ 4.7

Intake  (Total ration)6



Dry matter (kg/head/day)

6.50b ▒ 0.14

7.98a ▒ 0.18

Organic matter (kg/head/day)

6.0b ▒ 0.21

7.15a ▒ 0.19

Nitrogen (g/head/day)

140 b 0.18

179 a ▒ 0.17

Crude protein (g/head/day)

872 b ▒ 1.12

1116a  ▒ 1.06

1On DM basis

2  Grass –mid bloom (DM 270 g/kg; CP 105 g/kg)

3 Grass –Pre bloom (DM  260 g/kg; CP 115 g/kg)

4 On farm’ conc. – (Dairy mash DM 950 g/kg; CP 178.1g/kg )

5 Experimental ’ conc. Dairy mash and Sesami oil meal  (Dm 950 g/kg ;CP 250 g/kg ) 1:1 ratio

6Values are means of four animals

a, bValues within rows followed by different superscripts differ P<0.05


Milk yield and composition are presented in Table 2. The experimental diet significantly increased (P<0.05) the average milk yield to 4.03 kg/head/day compared 3.64 kg/head/day.  All other milk parameters, except protein and NUE were also significantly higher. Hence the experimental diet resulted in a higher plane of nutrition useful to test the effectiveness of urinary excretions of PD as indicators of N and nutritional status in general. On farm and experimental diets had 18.4 and 22 g N/kg respectively. Therefore, both diets had N contents in excess of the 14.4 g N/kg (90 g CP) which is perceived as the minimum to meet microbial N requirement (Nsahlai et al 2000). However, an increased intake of 3.6 g nitrogen/head/day in the experimental diet compared with the “on farm” diet by itself could not account for the increased milk yield and higher fat content. Therefore, the experimental diet seems to have resulted in an increased supply of N through microbial synthesis compared to the on farm diet. In fact the estimated microbial N contents in this study following Chen et al (1995b) were 28  and 33 g of N/day for “on farm” and “experimental diets” respectively. On the other hand, a decreasing tendency was observed in the NUE of cows fed with the experimental diet (10.73%) as compared with “on farm” diet (12%). Available evidence suggested that the NUE is largely controlled by rumen fermentation process. By feeding the experimental ration, N intake increased up to 3.6g /head/day but energy intake may have not increased proportionately. As reported by Kauffman and St. Pierre (2005) the higher amount of protein could be rapidly broken down when entering the rumen and in the absence of sufficient energy for rumen microbes the liberated nitrogenous compounds absorbed from the rumen and excreted in the urine. Indeed excess dietary protein   results in higher urinary urea excretion the largest source of NH3  released into the environment from dairy cattle. Wright (2003) with similar findings stressed that in order to ensure efficient rumen fermentation it is necessary to match ruminally available protein with the necessary readily fermentable carbohydrates to maximize microbial protein production.


Table 2: Milk yield and composition in lactating cows fed the ‘on farm’ and experimental diets1


On farm diet

Experimental diet

Milk yield (kg/d)



Milk fat (g/kg)


50.7a▒ 8.0

Milk fat (kg/d)

0.142b ▒0.04


Total solids (g/kg)



Total solids (kg/d)


0.614a ▒0.12

Milk N (g/kg)



Milk proteins (g/kg)



Milk proteins (kg/d)



N use efficiency (%)


10.73a   ▒1.71

1Values are means of four animals

a, b Values within  rows  followed by different superscripts differ P<0.05


Further to the findings of Kauffman and St. Pierre (2005) in mature, non-growing lactating cows the N retentions should be near zero because N intake and output (Milk N + faecal N + Urine N) are almost equal. Therefore, an increase in NUE could be expected with an improved diet. The present study was conducted also with non-growing lactating cows but the energy content of the experimental ration relative to the CP content  probably, unexpectedly a decreased  the NUE  with  the experimental ration.


The results of this study confirm that the urine output of PD, creatinine (CR) and the PDC index with the two diets could be considered as indicators of protein status from microbial synthesis but it is evident that better response could have been obtained in the experimental diet with a higher supply of readily fermentable energy.


Response of diet on PD excretion rate


Daily excretion patterns of allantoin, uric acid, CR, total PD, and the PDC indexes are presented in Table 3.  Times of the day had no effect (P>0.05) on excretion patterns of individual or the total PD, CR and the index. However, there was a clear allantoin, CR, PD and allantoin/PD excretions were higher at night with the ‘on farm’ diet and lower with the experimental diet while an inverse effect was observed with uric acid and PDC index. Although the time of sampling had no effect (P>0.05) on any of the parameters, dietary treatments, however, had a strong influence on the concentrations of allantoin, creatinine and PD with the experimental diet resulting in higher (P<0.05) excretions. Although uric acid, PDC index and allantoin/PD ratio tended to be higher with the experimental diet, values were not significantly different (P>0.05).


Table 3: Ranges and daily patterns of PD and creatinine urine excretion  of cross-bred milking cows from spot samples


Sampling time (hrs)

Allantoin (mmol/l)

Uric acid (mmol/l)

Creatinine (mmol/l)

PD1 (mmol/l)

PDC2 Index

Allan/PD ratio

Farm Diet










2.76 0.12






























Experimental Diet















88 ▒0.37



















4.57▒ 0.11






















*Significant (P<0.05)

1Purine derivatives (allantoin plus uric acid)

2PD/creatinine W 0.75


PDC Index and total PD (mmol/l) excretion in spot urine samples were closely and positively related (r2 = 0.75) as illustrated in Figure 1.


Figure 1 Relationship between PDC Index and PD (mmol/l) in the urine of milking cows


Not only did allantoin alone increased significantly in response to the experimental diet, but also the PD increased significantly, although the amount of uric acid was non-significant different between both diets. Allantoin and uric acid results therefore clearly indicated that allantoin is not only the major component in PD, but also the most sensitive indicator of protein and nutritional status of cattle. In addition to allantoin, creatinine also responded significantly in the same direction, while the PDC Index and allanatoin/PD ratio were not significant as indicators. These findings are consistent with those reported by numerous  studies the efficiency of microbial protein synthesis as measured by urinary PD reflected dry matter and CP and energy intake (Soejono et al 2004,Fujihara et al 2005, Puchala and Kulasek 1992, Dapoza et al 1999, Shem et al 1999). Further, Antoniewicz et al (1980) reported that the endogenous PD excretion can change as a result of alternations in the protein supply. Other workers (Nsahlai et al 2000, Long et al 1999) also confirmed the same trend, allantoin excretion in close agreement with the present findings.


Present  results are further supported by the findings of Chen et al 1995b who observed that the sampling period had no influence on the PD excretion in milking cows  neither on the f PD or creatinine concentration nor on the PD: CR ratio in urine (Nsahlai et al 2000, Fujihara et al 2005). Numerous workers (Seresinhe et al 2004, Odeja et al 2005, Wang et al 2009, Chen et al 1995a) confirmed the linear relationship between urinary PD and digestible organic matter intake (DOMI) and thus suggested that PD could be used as an indicator of microbial protein supply. Similarly, PDC index in spot urine samples was positively correlated (r2=0.74) with PD output (Figure 1). Therefore, present results indicate the potential use of PDC as index for microbial protein supply in crossbred milking cows. Similar to the above findings, plasma PD concentration, CR and Microbial N content of steers positively responded to the feed intake (George et al 2007).


It can be concluded that these results confirm previous reports that allantoin is the major PD and by itself could be used as indicator of microbial synthesis and of the general nutritional status of milking cows, irrespective of the sampling time.




The author gratefully acknowledges the dedicated work of Miss. Indika Udulanayanie during data collection.   




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Received 3 January 2011; Accepted 1 March 2011; Published 19 June 2011

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