Technical note: Evaluation of urinary purine derivatives in comparison with duodenal purines for estimating rumen microbial protein supply in sheep1 G. V. Kozloski,*2 C. M. Stefanello,* L. Oliveira,* H. M. N. Ribeiro Filho,† and T. J. Klopfenstein‡ *Departamento de Zootecnia, Universidade Federal de Santa Maria, Santa Maria, RS, 97105-900, Brazil; †Departamento de Zootecnia, Centro de Ciências Agroveterinárias, Universidade do Estado de Santa Catarina, Lages, SC, 88520-000, Brazil; and ‡Animal Science Department, University of Nebraska, Lincoln 68588
the endogenous excretion of PD, and the slope was lower than 1 (P < 0.05), indicating that only[AU1: Throughout the paper, should values for PB be converted to percentages rather than set as decimal values, e.g., 43% instead of 0.43 in “that only 0.43 of the PB”? Please check and indicate any changes that should be made throughout.] 0.43 of the PB in the duodenum was excreted as PD in urine. The Nm supply estimated by either approach was linearly related (P < 0.05) to the digestible OM intake. However, the Nm supply estimated through either of 3 published PD-based equations probably underestimated the Nm supply in sheep.
ABSTRACT: A data set of individual observations was compiled from digestibility trials to examine the relationship between the duodenal purine bases (PB) flow and urinary purine derivatives (PD) excretion and the validity of different equations for estimating rumen microbial N (Nm) supply based on urinary PD in comparison with estimates based on duodenal PB. Trials (8 trials, n = 185) were conducted with male sheep fitted with a duodenal T-type cannula, housed in metabolic cages, and fed forage alone or with supplements. The amount of PD excreted in urine was linearly related to the amount of PB flowing to the duodenum (P < 0.05). The intercept of the linear regression was 0.180 mmol/(d·kg0.75), representing
Key words: purine bases, purine derivatives, rumen microbial protein synthesis, sheep
© 2017 American Society of Animal Science. All rights reserved. INTRODUCTION
J. Anim. Sci. 2017.95:1–8 doi:10.2527/jas2016-0840
lantoin, uric acid, xanthine, and hypoxanthine) has been used as an alternative approach for estimating duodenal flow of PB and therefore of rumen Nm supply. The major advantage of the PD method is to avoid the need for both fistulated animals and measurements of duodenal digesta flow. However, not all PD in urine originates from absorbed PB (i.e., urinary PD also comes from endogenous origins), and not all PB flowing to the small intestine are excreted as PD in urine. Chen et al. (1990b) observed in sheep totally nourished by intragastric infusions of VFA and casein and given abomasal infusion of levels of microbial nucleic acids that the endogenous PD excretion decreased quadratically at increased levels of infused PB. Moreover, assuming a constant contribution of endogenous excretion, the recovery rate of the infused PB as exogenous urinary PD varied from 0.36 to 0.69. Variable recovery rates of intestinal PB as urinary PD were also obtained in trials conducted with
Proteins from rumen bacteria cells are the major source of AA for ruminants. The microbial N (Nm) supply to the small intestine cannot be directly measured, and despite uncertainties regarding their origin (Vicente et al., 2004; Askar et al., 2005), purine bases (PB) flowing to the duodenum are among the most used markers for estimating Nm yield. However, under increased concern about animal welfare, urinary excretion of purine derivatives (PD; the sum of al1This work was partially supported by the Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq, DF, Brazil) and by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, DF, Brazil). 2Corresponding author: [email protected]
Received July 26, 2016. Accepted November 23, 2016.
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Table 1. Descriptive variables of the digestibility trials carried out with sheep fitted with duodenal T-type cannula1 n
BW, kg Urinary excretion, mmol/(d·kg0.75) Allantoin Uric acid
Total purine derivatives Duodenal purine bases, mmol/(d·kg0.75) Digestible OM intake, g/(d·kg0.75)
185 185 185
0.51 0.72 28.4
0.11 0.11 9.4
1.55 2.14 56.5
0.256 0.439 10.68
(2009), Hentz et al. (2012), Kozloski et al. (2012), Reiter (2012), Schnaider et al. (2014), and Zeni et al. (2016).
RS; 29°4¢S, 53°4¢W) and at the Universidade do Estado de Santa Catarina (Lages, SC; 27°5¢S, 50°2¢W) using male sheep (8 trials, n = 185) fitted with a duodenal T-type cannula, housed in metabolism cages, and fed forage alone or with supplements. The forages offered in both trials included Pennisetum purpureum, Arachis pintoi, Lolium multiflorum, Cynodon sp., and Sorghum sudanense, and the supplements were corn cracked grain, canola meal, and crude glycerin. The data source and general description of the relevant variables are shown in Table 1. The Latin square design was used for all trials with experimental periods varying from 15 to 21 d, with 10 to 14 d for adaptation and 5 to 7 d for sample collection. In all trials the feed offered, refusals, and feces were weighed, recorded, and sampled daily during the collection periods, and duodenal digesta samples (i.e., approximately 100 mL) were obtained at 2 h or 3 h intervals over a 24-h period. All samples were dried at 55°C in a forced-air oven, ground through a 1-mm screen, and pooled by animal within each experimental period for analysis. Urine was collected daily during the collection period in buckets containing 100 mL of 3.6 N H2SO4. The total volume of urine was measured, and a sample of 10 mL was taken, diluted to 50 mL with distilled water, and stored frozen (−20°C). For analysis, urine samples were pooled by animal and period using proportional aliquots of the volume excreted daily in all trials.
sheep fed normal diets, such as 0.80 by Balcells et al. (1991), 0.57 by Pérez et al. (1996), and 0.83 by Ma et al. (2013). Moreover, the urinary recovery of allantoin administered directly into the blood by jugular infusion has also been incomplete and variable, ranging from 62% to 105% (Chen et al., 1991; Surra et al., 1997; Kahn and Nolan, 2000; Prasitkusol et al., 2004[AU2: Prasitkusal changed to Prasitkusol to match reference; please check.]). Despite these discrepancies, some equations have been proposed for use as tools for estimating the Nm supply in sheep on the basis of allantoin or total PD excretion ([AU3: Year of Chen and Gomes changed to 1992 to match reference; please check.]Chen and Gomes, 1992; Puchala and Kulasek, 1992; Ma et al., 2013). However, these equations were established using data from single trials conducted with animals fed specific diets, and their validity to estimate Nm supply under different dietary conditions has not been evaluated. The present study compiled data from digestibility trials with sheep under diverse dietary conditions to examine the relationship between the duodenal PB flow and urinary PD excretion and the validity of different equations on estimating Nm supply on the basis of urinary PD in comparison with estimates based on duodenal PB.
MATERIALS AND METHODS All procedures were conducted in accordance with the guidelines set out by the Brazilian College of Animal Experimentation in the Code of Practice for the Care and Use of Animal for Experimental Purposes and were reviewed and approved by the Ethics Committee on Use of Animal for Research of the Universidade Federal de Santa Maria and Universidade do Estado de Santa Catarina. Data Collection A data set of individual observations was compiled from digestibility trials conducted in southern Brazil at the Universidade Federal de Santa Maria (Santa Maria,
Chemical Analysis The DM content of feed, refusals, and duodenum and fecal samples was determined by drying samples at 110°C overnight (Ahn et al., 2014), and ash was determined by combusting at 600°C for 3 h (AOAC, 1997). The ADF concentration excluded ash and was analyzed according to method 973.18 of AOAC (1997), except samples were weighed into polyester filter bags (25 [AU4: Is m correct as the unit in “filter bags (25 m porosity)”? Or should this be µm? Please check.] m porosity) and treated with acid detergent solution in an autoclave at 110°C for 40 min (Senger et al., 2008).
Markers of rumen microbial protein synthesis
Statistical Analysis Data analysis was performed using PROC MIXED of SAS (SAS Inst. Inc., Cary, NC). The statistical model was Yij = β0i + β1jXij +Ej + εij, where Yij is the value of the dependent variable for the ith animal of the jth study; b0 is the intercept and b1 is the slope of the regressions; Ej is the random effect of the jth study, ~N(0, ss2); and eij is the residual error, ~N(0, ss2). Quadratic and cubic effects were previously tested and were not significant. Thus, using the model above, the following linear relationships were obtained: 1) duodenal flow of PB vs. urinary excretion of PD, 2) Nm supply estimated through PB vs. those estimated through either PD-based equation, and 3) Nm supply estimated through PB or through either PD-based equation vs. DOMI. The confidence interval (95%) of the equation parameters was calculated on the basis of SE values (i.e., ±2 SE) and was used to evaluate the deviation of either the slope from 1 or intercept from 0. Significance was declared at P ≤ 0.05.
The duodenal flow (g/d) of DM was estimated on the basis of ADF concentration in duodenal digesta and feces as follows: [fecal DM (g/d) ´ fecal ADF (g/ kg of DM)]/duodenal ADF (g/kg of DM). The option to use this marker was based on results from previous studies with sheep fitted with reentrant duodenal cannula (Porter and Singleton, 1971; Charmley and Veira, 1990), where most of the cellulose entering the duodenum was excreted in feces, and from a study with cattle in which the mobile nylon bag technique was used (Kozloski et al., 2014). In this last study, no significant disappearance of ADF in the lower gastrointestinal tract was observed, and ADF was a more precise and accurate marker for estimating duodenal digesta flow than n-alkanes or sulfuric acid lignin. The apparent digestible OM intake (DOMI) was calculated as OM intake (kg/d) minus fecal OM (kg/d). The Nm flow (g/d) to the small intestine was estimated by 1 of the following methods: 1) on the basis of duodenal flow of PB (g/d), assuming that the ratio of N:PB in mixed microbial mass is 4.3 ([AU5: Year for Chen and Gomes changed to 1992 to match reference; please check.]Chen and Gomes, 1992), 2) using the equation described by[AU6: Year for Chen and Gomes changed to 1992 to match reference; please check.] Chen and Gomes (1992) in which the amount of PB absorbed from the small intestine (x, mmol/d) was estimated on the basis of the amount of PD excreted (y, mmol/d, considering 158 mg/mmol of allantoin and 168 mg/mmol of uric acid) as y = 0.84x + (0.150 BW0.75 e−0.25X), where BW is body weight (kg), and the duodenal flow of PB was then calculated assuming that only 0.83 of PB entering the duodenum was absorbed, 3) using the equation described by Ma et al. (2013) in which Nm (y, g/d) was directly estimated from the PD excretion (x, g/d) as y = 0.030 + (0.741x),
or 4) using the equation described by Puchala and Kulasek (1992) in which Nm (y, g/d) was estimated from the urinary excretion of allantoin N (x, g/d) as y = exp (0.830 + 2.089x). For this last calculation it was assumed that the N content in allantoin was 354 mg/g.
Dried duodenal samples were analyzed for PB concentration after extraction with 2 M perchloric acid using the spectrophotometric method of Makkar and Becker (1999). In urine samples allantoin and uric acid concentrations were determined colorimetrically according to Chen and Gomes (1992). Uric acid was determined using a commercial kit (LABTEST, Lagoa Santa, MG, Brazil) after xanthine and hypoxanthine were converted to uric acid with xanthine oxidase. Thus, the uric acid values were calculated as the sum of uric acid, xanthine, and hypoxanthine, and the total PD was calculated as the sum of uric acid and allantoin. Duplicates or triplicates were used for all analysis, and values were accepted only when the difference between replicates, relative to the average value, was 5% or less.
The amount of PD excreted in urine was linearly (P < 0.05) related to the amount of PB flowing to the duodenum (Fig. 1). The intercept of the linear regression was positive and different from 0 (P < 0.05), and the slope was lower than 1 (P < 0.05). The Nm supply estimated through either PD-based equation was linearly related (P < 0.05) to that estimated through PB (Fig. 2), but the slope of the linear relationships was less than 1 (P < 0.05) for all PD-based equations. The Nm supply (g/d) estimated through PB or through either PD-based equation was linearly related (P < 0.05) to DOMI (kg/d; Fig. 3), and the slope of the linear relationships was 20.64, 7.67, 14.36, and 11.34 for the PB, Puchala and Kulasek (1992), [AU7: Year for Chen and Gomes changed to 1992 to match reference; please check.]Chen and Gomes (1992), and Ma et al. (2013) approaches, respectively. DISCUSSION
[AU8: Year for Chen and Gomes changed to 1992 to match reference; please check.]Chen and Gomes’s (1992) equation assumes that 0.83 of PB entering the duodenum is absorbed and 0.84 of absorbed
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Figure 1. Relationship between the amount of purine bases flowing to the duodenum and urinary excretion of purine derivatives in sheep (n = 185). The intercept was different from zero (P < 0.05), and the slope was different from 1 (P < 0.05). Different symbols denote different trials.
The intercept of the linear relationship between these variables is 0.180 mmol/(d·kg0.75), which indicates the endogenous excretion of PD when PB absorption is null. This value is higher than that obtained by Chen et al. (1990) [AU11: Please indicate if 1990a, 1990b, or both is meant in “obtained by Chen et al. (1990).”] using female lambs nourished by continuous infusion of volatile fatty acids into the rumen and casein into the abomasum (i.e., 0.150 mmol/[d·kg0.75]). The Nm supply estimated through PD was linearly related to that estimated through PB for all equations. However, as a consequence of the low recovery of duodenal PB as PD in urine, the Nm supply estimated through PD represented, on average, only 0.32, 0.55, and 0.44 of that estimated through PB using the equations of Puchala and Kulasek (1992), [AU12: Year for Chen and Gomes changed to 1992 to match reference; please check.]Chen and Gomes (1992), and Ma et al. (2013), respectively. Independent of the approach used to estimate Nm, the intercept of the relationship between Nm supply (g/d) and DOMI (kg/d) was not different from zero, which is expected since microbial protein synthesis should be null at zero DOMI. However, for all approaches, the precision of the relationship between these variables was not high, showing relatively high root-mean-square error values. Particularly, the PDbased equation of Puchala and Kulasek (1992) used allantoin N instead of total PD as the Nm predictor, and the allantoin excretion as a proportion of total PD showed high variability, from a minimum of 0.53 to a maximum of 0.95, with an average value of 0.82 ± 0.079 (data not shown). The slope of the linear re-
PB is excreted as PD in urine, representing a constant recovery rate of the duodenal PB as PD in urine of 0.70. However, the amount of PD excreted in urine represented, on average, only 0.43 of the duodenal PB in the present study and only 0.57 in the study of Pérez et al. (1996). In fact, as pointed out by Kahn and Nolan (2000), the recovery rate of either duodenal PB or blood allantoin as PD in urine has been broadly variable both within and between studies, which raises uncertainties on using this approach to estimate Nm supply. The incomplete recovery of absorbed PB as urinary PD may be due to both nonrenal losses of PD and/or PB retention in splanchnic tissues. In this way, Chen et al. (1990a) suggested that the salivary excretion of allantoin and uric acid would be equivalent to about 0.10 of the urinary excretion, a proportion that could be increased in animals fed roughages. However, Surra et al. (1997) and Kahn and Nolan (2000) observed that the salivary loss of PD was negligible, lower than 0.01 of the urinary PD loss, which suggests direct secretion of PD to the postruminal gut is likely the main nonrenal way of PD excretion. [AU9: As meant to change “In other way” to “In other words” in “In other way, since the sheep”? If not, please clarify.]In other words, since the sheep intestinal mucosa has only trace xanthine oxidase activity (Chen et al., 1990b), absorbed PB could be retained in splanchnic tissues instead of converted to PD. [AU10: Should “any” be “no” in “However, any study has examined”? Please clarify.]However, any study has examined what proportion of absorbed PB really enters the hepatic blood as PD.
Markers of rumen microbial protein synthesis
Figure 2. Relationship between microbial N supply estimated through duodenal flow of purine bases (Nm-PB) and estimates based on urinary excretion of purine derivatives using the equation of Puchala and Kulasek (1992; Nm-PK), ([AU18: Year of Chen and Gomes changed to 1992 to match reference; please check.]Chen and Gomes (1992; Nm-CG), or Ma et al. (2013; Nm-Ma). The slope was different from 1 (P < 0.05) in all linear relationships. Different symbols denote different trials
gressions between Nm and DOMI represents the efficiency of rumen microbial protein synthesis (EMPS). The EMPS were, on average, 48, 90, 71, and 129 g of microbial CP (MCP, calculated as Nm ´ 6.25) per kilogram of DOMI using the equations of Puchala and Kulasek (1992), [AU13: Year for Chen and Gomes
changed to 1992 to match reference; please check.] Chen and Gomes (1992), and Ma et al. (2013) and the PB approach, respectively. The first value is below and the other 3 values are within the range reported in the literature. The equation of Ma et al. (2013) was generated from data of a trial with sheep fed a diet with
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Figure 3. Relationship between digestible OM intake and microbial N supply estimated through either purine bases flowing to the duodenum (NmPB) or through the purine derivative–based equation of Puchala and Kulasek (1992; Nm-PK), [AU19: Year of Chen and Gomes changed to 1992 to match reference; please check.]Chen and Gomes (1992; Nm-CG), or Ma et al. (2013; Nm-Ma). The intercept was not different from zero in all linear relationships. Different symbols denote different trials.
Markers of rumen microbial protein synthesis
ria changes throughout microbial pools (i.e., liquid- vs. particle-associated microorganisms) and diet type, the same ratio of total N:PB in mixed microbial mass was used for calculations in the present study. Although there is not a standard procedure for obtaining representative rumen microbial samples, it is probable that the use of a more appropriate and specific total N:PB ratio for the different dietary conditions of trials included in the data set would improve the precision of the relationship between Nm and DOMI. Nevertheless, because the same ratio was used for all markers within individuals, this discrepancy does not invalidate marker comparison in the present study. The factors that could potentially contribute to the discrepancy in results obtained using PD include 1) analytical underestimation of PD concentration in urine, 2) overestimation of either the recovery rate of duodenal PB as urinary PD or the endogenous contribution to total PD excretion components in the Chen and Gomes equation, and 3) losses of PD during urine processing and storage. The Rimini-Schryver colorimetric reaction was used for allantoin analysis, which is reported to be nonspecific with uric acid among the contaminant chromogens (Young and Conway, 1942). However, uric acid concentration in sheep urine is usually low, the potential of uric acid in producing color is only 1/8 of that of allantoin (Young and Conway, 1942), and the PD-based Nm supply was apparently underestimated instead of overestimated. The PD excretion at zero duodenal PB flow in the present study was higher than the value used in the Chen and Gomes equation (i.e., 0.180 vs. 0.150 mmol/[d·kg0.75]), and Kahn and Nolan (2000) reported that 0.94 of the intravenously infused allantoin was recovered in urine of sheep, indicating negligible nonrenal excretion of PD. These results suggest, consequently, that the PB digestibility in the small intestine of sheep is lower than 0.83 or some of the absorbed PB does not enter the hepatic blood as PD. However, even using the urine collection and dilution procedures recommended by[AU14: Year for Chen and Gomes changed to 1992 to match reference; please check.] Chen and Gomes (1992), the losses of PD during urine processing and storage should not be disregarded. In conclusion, a high relationship between PB flowing to the duodenum and urinary excretion of PD was observed, confirming the potential of using urinary PD as an index of rumen microbial protein synthesis. However, all of the equations proposed for use as a tool for estimating Nm from urinary PD in sheep have generated unreliable estimates of the Nm supply. Most modeling uncertainties are related to the incomplete recovery of absorbed PB as urinary PD.
a roughage:concentrate ratio of 55:45, where the Nm supply at the duodenum was estimated using the 15N marker. The reported values of total DOMI and Nm supply in their study were, on average, 0.8 kg/d and 10.26 g/d, respectively, which represent an EMPS of 80 g MCP/kg DOMI. In turn, most current nutritional systems assume that the EMPS in animals fed diets containing medium to high levels of fiber is, on average, 130 g MCP/kg of total digestible nutrients intake, with a downward adjustment for diets containing less than 200 g/kg of effective NDF (Cannas et al., 2004). However, because most of the sheep in the present study were fed only forage, results indicated the Nm supply was, on average, accurately estimated through PB but underestimated by using the PD approach. When the duodenal PB approach is used for estimating the Nm supply, the estimation of duodenal digesta flow is also of concern. The present study used ADF measured in the feces and duodenum as a single internal marker for calculating duodenal digesta flow and, consequently, PB flow into the duodenum, assuming that the ADF was indigestible throughout the lower gastrointestinal tract. Although hindgut microorganisms might use cellulose as energy source, the cellulose in ADF residue reaching the hindgut is present in a cell wall matrix that was previously available to and fermented by rumen microorganisms. The mean retention time of digesta in the rumen is much longer than the retention time in the hindgut (Mertens, 1993), and thus, it is likely only a negligible proportion of the cellulose not fermented in the rumen was fermented in the hindgut. Moreover, eventual ADF disappearance in the hindgut would result in underestimated duodenal PB flow and, consequently, in an overestimated urinary PD recovery rate, which is unlikely since relatively low PD recovery values were obtained. In fact, there is not a standard method for measuring duodenal digesta flow, as inaccurate estimates might be obtained even when more complex double- or triple-marker approaches are used (Ortigues et al., 1990; Titgemeyer, 1997). In turn, the PD excretion was measured through total urine collection in all trials, and thus, a more precise relationship between DOMI and PD-based Nm supply would be expected. However, no improvement was obtained by using this approach. In most trials sheep were fed ad libitum and probably showed an irregular pattern of both nutrient ingestion and PD excretion throughout collection days. This result raises a concern about how long the collection period should be in digestibility trials with sheep fed ad libitum to obtain more precise and accurate data of urinary PD excretion. Moreover, although previous in vitro (Carro and Miller, 2002) and in vivo (Rodríguez et al., 2000) studies have reported that the chemical composition of rumen bacte-
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