Urinary excretion of purine derivatives as an index of microbial protein synthesis under effect of different diets fed to Iranian buffaloes.
To digest the food, ruminant depend on the microbial variety and population in their rumen. That is because these microbes can easily analyze the food with their special enzymes. In addition to their role in analyzing the food, when passing through rumen and reticulum to get to the lower parts of the alimentary system like abomasum and intestines, micro organisms themselves will be dissected and turn into proteins, a nutrition resource for the animals. Therefore, there are two sources of MP available for the ruminants: the true protein in the diet that escapes degradation in the rumen, and the bacterial protein produced by the rumen microbes. The latter can provide the animal with 42% to 93% of the protein . Thus; rumen microbes constitute the major source of protein supply to the ruminant. Rumen microbes are rich in nucleic acid: around 18% of total nitrogen is present on nucleic acids or 11% in purines. The purines from the rumen microbes are metabolized and excreted in the urine as their end products: hypoxanthine, xanthine, uric acid and allantoin. In buffalo and cattle, because of high xanthine oxidase (XO) activity in intestine and blood, hypoxanthine and xanthine convert to uric acid. Therefore, only uric acid and allantoin excreted in urine way . There are various methods to estimate microbial protein synthesis in the rumen, most of which are based on determining microbial indices. The ordinary food of ruminants contains a little of nucleic acids. Under the effect of microorganism in the rumen, these nucleic acids are almost completely analyzed . Therefore, we can say that the nucleic acids, which inter the little intestine, have microbial origins. In the intestine, purine nucleic acids turn into purine nucleosides and free radicals both of which can be absorbed by the intestine. Based on the activity of the existing enzyme in analyzing the purines, these two products can be again used to produce nucleic acid, or else they can turn into their own constituents such as hypoxanthine, xanthine, uric acid and allantoin. These purine derivatives (PD) are often excreting in urine. Therefore, if we know the proportion of purine to microbial nitrogen, as well as digestibility of purine, we can estimate the microbial nitrogen absorbed through the intestines based on observing the amount of absorbed purine in the excreted urine. In 1989, Chen proposed another method based on the above mentioned procedure. This method provides us with an index to estimate the microbial protein available to the animal. One of the advantages of this method is its simplicity, that is to say, the only thing needed is to collect all the urine and analyze it to estimate the purine constituents in it. No surgery is needed in this method. Chen et al.  proposed equations to estimate the amount of absorbed microbial purines in the intestine based on the purine constituents excreted in the urine. Also Chen et al. , Verbic et al.  and Liang et al.  suggested a formula to estimate the amount of synthesized microbial nitrogen (g/day) based on the amount of the absorbed purine derivatives. There have been vast researches by the scientist on the different species in this regard including: sheep [11,4,24], cattle [29,26,28,31], goats [21,6], buffaloes [13,19,28,31], camel [25,17,30] and rabbit . Nonetheless, the data does not seem to be adequate yet. This research was carried out to use excretion of purine derivatives (PD) namely allantoin and uric acid as a parameter to estimate the microbial protein synthesis in the rumen of native swamp buffaloes in north of Iran,
Material and Methods
Buffaloes and experimental conditions:
This study was conducted at 2005-2006 in Animal research center of Islamic Azad University, Ghaemshahr Branch at Iran. Four Iranian male swamp buffaloes with the average live body weight of 140[+ or -]10 kg were used. The experiment duration was 84 days including four periods and each period lasted 21 days (11 days for adaptation and 10 days for urine collection). Animals were housed in four metabolic cages and fitted with urine collection instrument.
Four diets consisting of 0 % (all forage diet), 15%, 30% and 45% concentrate in a Latin square design, in four periods were experimented. Each period lasted 21 days (11 days for adaptation and 10 days for urine collection). Diets were offered twice daily at 8:00 am and 4:00 pm, in two equal meals. Water was available ad libitum. Housing and management conditions were the same for all animals.
Method of Urine collection and urine dilution, as well as keeping the samples until the analysis:
The experiment duration was 84 days including 4 periods and each period lasted 21 days (11 days for adaptation and 10 days for urine collection). After every adaptation period (11day), Urine was collected in a container each day. To prevent microbial degradation of purines, urine was acidified by 10% H2SO4 to a pH of 2-3. Urine was diluted by distilled water to prevent the precipitation of purine derivatives during the storage period. A sub sample of 40 ml was took and stored in -20oc for further experiment.
Analysis method for determining uric acid and allantoin and synthesized microbial protein from excreted purine derivatives in the urine:
In order to measure the uric acid in the urine sample, first samples were diluted to become readable for the spectrophotometer. Then, using a uric acid kit, the amounts of uric acid in the samples were determined. In addition, the Chen et al.  method was implemented in order to measure the allantoin. Obtaining the amounts of uric acid and allantoin in the samples, these amounts are shown based on standard units of milligram/deciliter (mg/dl) and milligram/liter (mg/l). Afterwards, these amount were change into milligram/day (mg/day) and then into millimol/day (mmol/day). The conversions were done so that the units could be used in equations. Then the amounts of uric acid and allantoin in each sample were added to each other to come up with the total of purine derivatives (mmol/day). Then applying the total of excreted purine derivatives in the urine(Y, mmol/day) in the Liang et al.  equation, the amount of absorbed external purine for each buffalo(X, mmol/day) was obtained. Y= 0.12X + 0.20 [W.sup.0.75]
Then applying x in the Chen et al.  formula, the amount of synthesized microbial nitrogen gr/day was measured. Consequently, multiplying the results by 6.25, the amount of microbial protein was measured. X= (Y- 0.20[W.sup.0.75]) / 0.12
It has been experimented four diets in a Latin square design. The model was:
Yij(k) = M+ Ri+ Cj+ T(k)+ Eij(k)
[X.sub.ijk] = Related sample to the [i.sup.th] animal in the [j.sup.th] period under the effect of [k.sup.th] diet.
M = treatment average
Ri = effect of row or animals
Cj = effect of column or periods
T(k) = effect of the treatment or ratios
[E.sub.ijk] = experimental error
Data were statistically analyzed using the General Linear Model procedures of SAS with the Duncan test.
Excretion purine derivatives model:
The results of this research revealed that the excreted purine derivatives in the urine included allantoin and uric acids where allantoin was 88% and uric acid, 12% of the total excreted purine derivatives (mmol/day).
The amount of allantoin, uric acid, total of excreted purine derivatives, absorbed microbial purines, produced nitrogen or microbial protein:
The amount of allantoin, uric acid, total of excreted purine derivatives, absorbed microbial purines, produced nitrogen or microbial protein, provided in Table 2, were increased significantly (p<0.01). By the increase of the level of diet constantrate (0 to 45%). so that significant differences among all diet concentrate (0%, 15%, 30%, 45%) could be observed (p<0.01). The researchers observed the increase of 13.2 to 21.7 (mmol/day) for the allantoin, 14.5 to 24.5(mmol/day) for the total of purine derivatives, 53.3 to 136.7 (mmol/day) for the absorbed microbial purines, 38.7 to 99.4 (gr/day) for the microbial nitrogen, and 242.2 to 621.4(gr/day) for the microbial protein. The amount of uric acid also increased 1.32 to 2.75 (mmol/day) with the increase of the level of concentrate (0% to 45%). However, this increase between the levels of 30% to 45% of the concentrate, also between the levels of 15% and 30% concentrate were not statistically significant(P<0.01), but the differences between the levels of 15% and 45 % of concentrate, and between the level of 30 and an all forage diet were statistically significant(P<0.01). Moreover, the differences among an all forage and the three diets of 15%, 30% and 45% concentrate were also significant (P<0.01).
Daily dry mater intake in relation with excreted purine derivatives and produced nitrogen protein:
The amount of Daily Dry Matter Intake (DDMI, kg [day.sup.-1]) was increased from 3200 to 5100 gr/day significantly (p<0.01) by the increase of the level of the diet concentrate from 0% to 45%. Moreover, there was a high and significant correlation between Daily Dry Matter Intake (DDMI, kg [day.sup.-1]) with excreted purine derivatives and produced nitrogen protein in rumen ([r.sup.2]=0.99).
The results were also analysed by regression where X= Daily Dry Matter Intake (DDMI, kg [day.sup.-1]) and Y=. Total purine derivatives excretion (mmol/day) or microbial protein synthesized in rumen (gr/day).
Excretion purine derivatives model:
In buffalo and cattle, the xanthine oxidase (XO) activity in intestinal mucosa and liver is too high to permit any uptake of salvageable PD, xanthine and hypoxanthine . Therefore, hypoxanthine and xanthine convert to uric acid leaving only uric acid and allantoin excreted in urine way . However, the amount of excreted allantoin as part of total excreted purine derivatives, 88% of the total purine derivatives revealed a little higher than that reported by Chen et al. , 80% to 85% for cow and 60% to 80% for sheep. Also, the amount of excreted uric acid was a little lower than what was reported by them, 15% to 20% for cow and 5% to 10% percentage for sheep. The proportional difference between allantoin and uric acid in the total purine derivatives can be due to the differences between species, since they almost remain alike in the same species [12,13].
Differences in uric acid excretion:
Allantoin was the main excretion product from purine metabolism in agreement with the results of [32,22,11]. The increase of excreted allantoin in the urine connected increase of proportion of diet concentrate can be related to the increase in daily amount of digestible organic matter fermented (DOMR) in agreement with the result of . The increase in concentrate in the diet cause of the increase in Daily Dry Matter Intake (DDMI, kg [day.sup.-1)], and this in turn can increase the excreted allantoin in the urine. The significant difference among the diet of 15%, 30% and 45% concentrate together, as well as with the all forage can be related to the sensitivity of this purine derivative to diet compounds and the level of food intake.
Differences in uric acid excretion:
The increase in the uric acid in the urine can be related to Daily Dry Matter Intake (DDMI, kg [day.sup.-1]), as well as increase in the amount of digestible daily organic matters (DOMR). The lack of significance in the increase in the uric acid between the diets of 30% and 45% concentrate, also between the diets of 15% and 30% concentrate, as well as significance in the amounts of excreted uric acid between diets of 15% and 30% concentrate as well as significance in the amounts of excreted uric acid between diets of 15% and 45%, also significance in the amount of 15%, 30%, 45% with an all forage diet can be explained as the following comparing to allantoin, uric acid has more stability . Thus, in order to make significant changes, it needs more portions in the diet i.e. the increase of the amount of concentrate from 30% to 45% cannot make significant differences in the excreted uric acid; whereas, the increase of concentrate from 15% to 45% causes significant difference in the amount of excretion uric acid in urine.
Differences in total of excreted purine derivatives:
The increase in total of excreted purine derivatives can be justified similar to allantoin with the increase of digestible organic matter fermented (DOMR) in agreement with the result of Chen et al. , Daniels et al.  and Cetinkaya et al, [7-9]. On the other hand, as mentioned before, because allantoin makes the biggest proportion part of the excreted purine derivatives in buffaloes, we can expect that the differences between the amounts of total excreted purine derivatives, like allantoin amounts, be significant in case of all for diets. That is so because the allantoin, being large amount, affects the amount of uric acid and gives significance to the total excreted purine derivates in the urine. This is in line with the results of this research.
Differences in the produced microbial nitrogen and synthesized microbial protein:
Since the produced microbial nitrogen can be measured according to the total amount of the excreted purine derivatives in the urine, it can be expected that the amounts of nitrogen or synthesized microbial protein also increase, and this increase is significant in all levels. This increase can be explained that with the increase of portion of concentrate, Daily Dry Matter Intake (DDMI, kg [day.sup.-1]) also increases. Also with the increase in the port ional concentrate, it can be expected to increase the digestibility of the diet as well as digestible organic matter fermented (DOMR). The increase of the amount of daily digestible organic matter increases the amount of matter fermented in rumen (DOMR), and that ARC[1,2] has used it to explain the produced microbial nitrogen in the rumen.(32 gram for per kg DOMI), ARC  has used 65% coefficient in order to turn DOMI to DOMR. We can also expect that by increasing the concentrate in the diet, total digestible organic matter (TDOM) also increases which in turn can increase the synthesis in microbial protein. Increasing the concentrate portion will also increase the fermentable metabolism energy (FME) in the diet, as well as increasing the synthesized microbial protein in the rumen .
The results of this experiment indicate that increasing the concentrate level in diet of swamp buffaloes would increase the amount of synthesized microbial protein in rumen. The reason can be related to increasing the amount of organic matter fermented in rumen (DOMR) in rumen, as a result of adding concentrate to forage ratio. According to the researchers' observation, there is a high and significant correlation between Daily Dry Matter Intake (DDMI, kg day) with excreted purine derivatives and produced nitrogen protein in rumen ([r.sup.2] =0.99), can be explained by regression equations.
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Kaveh Jafari Khorshidi, Yashar Vakil Faraji
Department of Animal Science, Faculty of Agriculture, Islamic Azad University, Ghaemshahr Branch, Iran.
Kaveh Jafari Khorshidi, Yashar Vakil Faraji; Urinary Excretion of Purine Derivatives as an Index of Microbial Protein Synthesis under Effect of Different Diets Fed to Iranian Buffaloes
Kaveh Jafari Khorshidi, Department of Animal Science, Faculty of Agriculture, Islamic Azad University, Ghaemshahr Branch, Ghaemshahr, Iran.
Table 1: Excretion model of PD in Iranian buffaloes. Diets Allantoin % Uric acid % of total PD of total PD 1 90.8 9.2 2 87.4 12.6 3 88.6 11.4 4 88.7 11.3 average 88.8 11.2 Table 2: Microbial nitrogen synthesis measured in rumen of Iranian buffaloes. Purine derivatives Diets Concentrate 45% Concentrate 30% Allantoin(mmol/d) 21.79[+ or -] 19.15[+ or -] 0.73 (a) 1.44 (b) Uric acid(mmol/d) 2.75[+ or -] 2.44[+ or -] 0.30 (a) 0.12 (ab) Total PD(mmol/d) 24.55[+ or -] 21.59[+ or -] 0.96 (a) 1.50 (b) Microbial nitrogen 99.43[+ or -] 81.48[+ or -] synthesis(g/d) 5.02 (a) 7.36 (b) Purine derivatives Diets Concentrate 15% Concentrate 0% SE Allantoin(mmol/d) 15.84[+ or -] 13.21[+ or -] 0.94 1.26 (c) 0.31 (d) Uric acid(mmol/d) bc 2.28[+ or -] 1.326[+ or -] 0.10 0.22 (bc) 0.16 (d) Total PD(mmol/d) 18.11[+ or -] 14.54[+ or -] 1.06 1.44 (c) 0.36 (c) Microbial nitrogen 60.44[+ or -] 38.76[+ or -] 5.73 synthesis(g/d) 7.99 (c) 2.49 (d) Value in table are mean [+ or -] SD (P<0.01)
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|Title Annotation:||Original Article|
|Author:||Khorshidi, Kaveh Jafari; Faraji, Yashar Vakil|
|Publication:||Advances in Environmental Biology|
|Date:||Sep 1, 2011|
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