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Department of Animal Science, Cornell University, Ithaca, NY 14853
3 Correspondence:
272 Morrison Hall (phone: 607-254-4910; fax: 607- 255-1335; E-mail:
jcm41{at}cornell.edu).
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Key Words: Biochemical Transporters • Heifers • Kinetics • Nitrogen Metabolism • Urea Recycling
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Physiological changes, such as reduced plasma filtration by the kidney (Leng et al., 1985; Cirio and Boivin, 1990) and an increase in urea reabsorption from the initial inner medullary collecting ducts of the kidney (Isozaki et al., 1994) have been reported in animals consuming low-N diets, salvaging urea from excretion. An increase in the GI tract clearance rate of urea, in animals fed low-N diets, has been previously reported (Ford and Milligan, 1970; Kennedy and Milligan, 1980), but the specific mechanism of action has not yet been described. The discovery of urea transporters (UT) in the gastrointestinal tract of ruminants (Ritzhaupt et al., 1997; 1998) might be the mechanism responsible for this increase in the transfer of urea into the GI tract.
The objective of the present research was to quantify the transfer of urea to the GI tract and the use of this recycled N by growing heifers fed highly fermentable, isocaloric diets with varying N contents. An additional objective was to confirm the presence of UT in the ruminal wall of cattle and investigate the effect of N intake on UT expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
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Heifers were individually fed 90 g of DM/kg BW0.75 daily of a diet composed of 30% brome hay and 70% of a pelleted feed every 2 h using automatic feeders. There were two pellets, one for the 1.45% N diet (Low pellet, Table 1
) and the other for the 3.40% N diet (High pellet, Table 1). By mixing these two pellets in the proportions of 3:1, 1:1, and 1:3, respectively, the 1.89, 2.50, and 2.97% N diets were obtained. The actual N content of the diets is reported in Table 2. Diets were formulated to be isocaloric on a ME basis and to contain similar levels of NDF, which was accomplished by substituting soybean meal with citrus pulp (Table 1). Each experimental period was 21 d long and included a 15-d adaptation to the diet, a 5-d total collection of fecal and urinary excretions, and a final day when blood, saliva, and ruminal fluid were sampled. Animals had access to water at all times. Animals were weighed without feed or water restriction weekly on two consecutive days.
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Feces and urine were collected daily between d 16 and 20 before the 0800 feeding and weighed to determine output. A 1% subsample of urine and a 3% subsample of feces were collected, composited by period, and stored at -20°C until later analysis. Urine was collected from the bladder with a 22-gauge Foley catheter (Bard Medical Division, Covington, GA) into a plastic container with enough 50% (vol/vol) H2SO4 to reduce the pH to less than 2.5. An additional urine subsample was collected daily, diluted, and stored at -20°C for analysis of purine derivatives (PD).
On d 21, blood, saliva, and ruminal fluid were sampled 10, 50, and 90 min after the 10:00 a.m. meal in order to portray a 2-h feeding period. Blood was collected into Vacutainers containing heparin (Becton Dickinson, Rutherford, NJ), placed on ice, and then centrifuged at 1,500 x g for 15 min to obtain plasma. Saliva was collected by suction from the oral cavity and centrifuged at 1,500 x g for 15 min to eliminate feed particles. Approximately 200 mL of ruminal fluid was collected at each time. An aliquot (10 mL) was acidified with 50% H2SO4 to prevent the loss of ammonia and centrifuged at 1,500 x g for 15 min. All samples were frozen at -20°C after centrifugation until analysis. The remaining ruminal fluid was stored at 4°C, pooled within animal, and centrifuged at 800 x g for 10 min in order to remove feed particles and protozoa. The supernatant was then centrifuged at 10,000 x g for 30 min, the pellet resuspended in saline solution (9 g/L of NaCl), and then centrifuged a second time at 10,000 x g for 30 min. The pellet was then stored at -20°C until freeze-dried.
At the end of the experiment and while the animals were still being fed the treatment diets, ruminal mucosa was biopsied by clipping papillae from the ventral sac of the rumen. These were frozen in liquid N and stored at -80°C. Because we were concerned with possible side effects of the biopsy procedure, the samples were taken at the end of the experiment, and these data lack replication.
Sample Analyses
Urinary urea was isolated using a cation exchange resin (AG 50W-X8, 100 to 200 mesh hydrogen form, Bio-Rad, Richmond, CA) and diluted to a final concentration of 6 mmol/L. The solution was then reacted under vacuum with lithium hypobromite, leading to a monomolecular degradation of urea into N2 (Sarraseca et al., 1998). This reaction has been found to be sensitive to the concentration of urea with more 15N14 N gas molecules at higher concentrations due to the cross-reaction of 14N14N and 15N15N urea molecules (Sarraseca et al., 1998). The concentration of urea used in the analysis ensured suitable amounts of gas, but from a low urea concentration to reduce the nonmonomolecular degradation of urea. The occurrence of nonmonomolecular reaction was calculated from the increase in 15 N14N gas molecules when enriched urea standards were run alongside the samples, and under these conditions was 5%, which is similar to the value found by Sarraseca et al. (1998). The N2 obtained was transferred into 6-mm glass tubes, captured with silica gel, and the tubes were sealed. The tubes were then cracked by a Finnigan delta plus multiport (Finnigan, San Jose, CA) and read with a NC2500 Carlo Erba dual inlet elemental analyzer (Thermoquest, Milan, Italy) as a mass:charge ratio of 28, 29, and 30 corresponding to the 14N14N, 14N15N, and 15N15N parent urea molecule, respectively. The model of Lobley et al. (2000) was used to calculate urea kinetics. Bacterial and fecal 15N enrichments were analyzed in continuous-flow mode using the NC2500 Carlo Erba elemental analyzer mentioned earlier.
Plasma urea N (PUN), salivary urea N (SUN), and urinary urea N (UUN) were determined by the diacetyl monoxime method of Marsh et al. (1957) using a Technicon autoanalyzer (Technicon Instruments Corporation, Tarrytown, NY). Feed, fecal (fresh feces), and urinary N was measured by a macro Kjeldahl (AOAC, 1990) method that was modified to include the use of boric acid and steam distillation (Pierce and Haenisch, 1940). Ruminal ammonia N (RAN) was measured using the Berthelot reaction (Chaney and Marbach, 1962). Feed and fecal NDF were analyzed using the method of Van Soest et al. (1991) with the addition of sodium sulfite. Plasma and urine creatinine were determined by the Jaffe reaction with a commercial kit (Sigma 555-A, Sigma, St. Louis, MO). Purine derivatives were measured in urine by reverse-phase HPLC (Beckman Instruments, Palo Alto, CA) according to the method of Shingfield and Offer (1999).
Ruminal mucosa was homogenized in isolation buffer (10 mM triethanolamine, 250 mM sucrose, 1 µg/mL of leupeptin, 0.1 mg/mL of phenylmethanesulfonyl fluoride [Sigma, St. Louis, MO], pH 7.6, 0.025 to 0.1 g of tissue/mL isolation buffer). Concentrated sodium dodecyl sulfate (SDS) was added to achieve a final concentration of 1%, and then samples were sheared by passage through a 28-gauge needle and centrifuged for 15 min at 14,000 x g. Protein was determined using the BioRad DC protein assay kit (BioRad, Richmond, CA). Proteins (10 µg/lane) were separated on 10% SDS-polyacrylamide gels, and then transferred to a polyvinylidene difluoride membrane. Membranes were probed with two antibodies as follows: 1) affinity-purified polyclonal antibody to UT-A (3.8 mg/mL used at 1:5,000 for Western blot); this antibody was prepared against the C-terminal portion of UT-A1 and will also detect UT-A2 and UT-A4, or 2) affinity purified polyclonal anti-UT-B (0.94 mg/mL diluted 1:3,000 for Western blot). The UT-B1 antibody was prepared against the carboxyl-terminal 19 AA of hUT-B1. The antibodies were diluted in Tris-buffered saline (TBS) with 0.5% Tween-20 overnight at 4°C, and then washed three times in TBS/Tween. Blots were then incubated with horseradish peroxidase-linked goat anti-rabbit IgG diluted 1:5,000 (Amersham, Arlington Heights, IL) for 2 h at room temperature, and then washed twice with TBS/Tween. The immunoreactive proteins were visualized by enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL).
Statistical Analyses
Data were analyzed using the MIXED procedure of SAS (SAS Inst., Inc., Cary, NC). The Youden square design had treatment and period as fixed effects and animal as a random effect. Degrees of freedom were calculated with the Satterthwaite option and used to calculate the standard error of the mean. Least squares means were obtained for each treatment, and linear and quadratic orthogonal polynomial contrasts on equally spaced treatments were analyzed to determine whether there was a trend in the response variables as N levels increased. Post-hoc pairwise comparisons, using the Tukey adjustment for multiple comparisons, were conducted when data suggested (by departure from linearity) that a breakpoint was reached. The overall means obtained were least square means and were assessed for significant differences at P < 0.05.
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Results and Discussion |
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). Nitrogen balance increased (P < 0.01) from 20 g/d (1.45% diet), reaching a plateau at 30 to 32 g/d (2.50, 2.97, and 3.40% N diets; Table 2). The 2.50% N diet provided N below, and the 2.97% diet above, the estimated requirements for dairy heifers maintained under the conditions of the experiment (Fox et al., 2000). Weight gains were consistent with the N balances observed, but only a trend in BW gain was detected (P = 0.07) among the treatments (Table 2).
Nitrogen intake increased PUN and SUN concentrations linearly (P < 0.001, Table 2). Urea concentration in saliva was, on average, 73% of the plasma concentration. Plasma urea values encompassed the range of values reported in the literature for young growing animals (Elrod and Butler, 1993; Lammers et al., 1999).
Urea entry rate (UER, urea production) and UUN increased linearly (P < 0.001, Table 2) with N intake but GI entry rate (GER, urea recycled to the GI tract) remained similar among diets (P = 0.24, Table 2). The UER was equivalent to 35 and 66% of the N ingested for the 1.45 and 3.40% N diet, respectively. This value can vary considerably depending on the N and energy content of the diet and physiological state of the animal. In steers weighing 380 to 420 kg, Huntington et al. (1996) and Lapierre et al. (2000) observed higher UER values as a proportion of N intake (60 to 98%) consistent with more mature animals fed at approximately maintenance. In growing animals (approximately 230 kg), Bunting et al. (1989a) and Archibeque et al. (2001) reported that the ratio of UER to N intake was 32:43% and 52:60%, respectively. Alternatively, UER can be expressed as a percentage of the N apparently digested, and in this experiment was not different among treatments (86.4 ± 7.0%, P = 0.32). This ratio decreased from 98% (Archibeque et al., 2001) to 49% (Bunting et al., 1989a) with increasing DM digestibility (62, 70, and 83%; Archibeque et al., 2001; present experiment; Bunting et al., 1989a, respectively) suggesting that the relationship between urea production and digestible N could be modulated by the fermentability of the carbohydrates and the demands of the animal for N.
Excess N was excreted in the urine as urea N, and for heifers on the 3.40% N diet (95.8 g/d), it was 25 times greater than for animals on the 1.45% N diet (3.8 g/d), whereas the urinary nonurea fraction remained relatively constant among diets (17 to 25 g/d). It appears that N intake had little or no effect on the nonurea component of the urine, whereas urea-N accounted for 92% of the additional N in the urine. These results are similar to those reported by Ciszuk and Gebregziabhur (1994) in dairy cows, where urea accounted for 99% of the additional N excreted in the urine. Of the urea produced, only 15% was excreted in the urine of animals receiving the 1.45% N diet, compared with 71% for animals on the 3.40% N diet. Huntington et al. (1996) fed isocaloric and isonitrogenous diets to steers and observed a decrease in the ratio of urea excreted to urea produced from 50 to 9% as NDF increased from 15 to 76%. In growing animals, Bunting et al. (1989a) and Archibeque et al. (2001) reported that 13 to 35% of the UER was excreted in the urine, whereas Lapierre et al. (2000) observed higher values (67 to 74%) in older animals.
The amount of urea entering the rumen with saliva can be calculated based on the estimated saliva production and measured concentrations (Table 2). Because most of the dietary NDF was provided by the brome grass hay, and diets were similar in NDF content (34 and 31% for the 1.45 and 3.40% N diets, respectively), the volume of saliva produced by the heifers was assumed to be similar among diets (20% of BW daily; Yarns et al., 1965). The amount transferred in saliva was negligible for the 1.45 and 1.89% N diets (3 and 4% of the total GER), but increased to approximately 20% for the 3.40% N diet. Similarly, according to the calculations of Bunting et al. (1989a) and Huntington et al. (1996), only 4 to 10% and 14 to 16% of the urea degraded in the GI tract entered the rumen with saliva, respectively.
In sheep fed high-roughage diets (Nolan and Leng, 1972; Norton et al., 1982) most of the urea was recycled to the hindgut, and saliva accounted for most of the urea recycled to the rumen. This contrasts with the findings of Huntington et al. (1996), where most of the urea recycled entered the rumen independently of the amount of concentrate fed. However, it is believed that an increase in the amount of carbohydrates degraded in the rumen shifts the transfer of urea from the hindgut to the rumen (Kennedy and Milligan, 1980; Huntington, 1989).
Increasing N intake decreased the quantity of N excreted in the feces that originated in plasma urea, but it did not affect the total amount used for anabolic purposes; and the fraction of GER used for anabolism remained fairly constant across diets, from 62 to 58% for heifers on the 1.45 and 3.40% N diets, respectively (Table 2). Very few data are available on the partitioning of the urea N recycled due to the recent development and application of the double-labeled urea technique in ruminants (Sarraseca et al., 1998). Archibeque et al. (2001) observed that 65 to 70% of the GER was used either for anabolic purposes or excreted in the feces, a value similar to those reported in this paper. Lobley et al. (2000) reported that the N enrichment in feces did not reach a plateau when animals were infused for 96 h. Because the N used for anabolic purposes is calculated by difference, the double-labeled urea technique overestimates the amount of N used for anabolism, while at the same time underestimating the N excreted in the feces.
Although no linear effect (P = 0.11) was detected for the N recycled to the ornithine cycle, pairwise comparison indicated that less N (P < 0.05) was reutilized to make a new urea molecule in heifers fed the 1.45% diet than those on the other diets, whereas similar amounts were recycled for 1.89 through 3.40% N diets (Table 2). Archibeque et al. (2001) in beef steers and Sarraseca et al. (1998) in sheep observed that similar percentages of N were returned to the ornithine cycle (26 to 41% of GER), which are similar to the values reported in this experiment, whereas Lobley et al. (2000) and Marini et al. (2002) reported higher values in sheep (50%). What seems a futile and costly cycle of urea synthesis in the liver and degradation in the GI tract might be needed in order to provide ammonia for microbial synthesis and be an adaptive mechanism to retain N within the system.
Ruminal ammonia concentration increased quadratically with increasing levels of nitrogen intake (P < 0.006, Table 3). At the lowest N intake level, the 1.45% N diet, RAN was barely detectable. According to current recommendations (Satter and Slyter, 1974), RAN values below 3.5 mM limit microbial growth. Based on the urinary PD excretion analysis and the equations derived by Chen and Gomes (1992), the bacterial yield was estimated (Table 3). No differences in bacterial N reaching the duodenum could be detected (Table 3, P = 0.31) among the treatments due to the large SEM, suggesting that RAN concentration probably did not reflect the flux of RAN. Russell and Strobel (1987) found that in in vitro studies, ammonia could be transported into microbes, reaching high intracellular ammonia concentrations despite the fact that the ammonia concentration used in the media was very close to zero. This mechanism of active ammonia transport is likely to be present in vivo, allowing ruminal microbes to obtain ammonia from the medium at low RAN concentrations. Cellulolytic ruminal bacteria require ammonia as a N source and are unable to ferment fiber when ammonia is depleted (Bryant, 1973) resulting in a depression in fiber digestibility. In the current experiment, total-tract NDF digestibility was depressed in heifers on the 1.45% diet (Table 3), and this was likely due to the low RAN measured for animals on this diet and not to a low ruminal pH, because pH did not fall below 6.0 (data not shown).
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< 0.001). Although some recycling of the label is likely to occur due to bacterial turnover within the rumen, this ratio suggested that approximately 18.7% of the bacterial N in animals on the 1.45% N diet originated in plasma urea N, whereas this value was only 4.3% in the 3.40% N diet. Animals fed low-N diets incorporated more recycled N into microbial products at low N intakes (P < 0.003). Approximately 43% of the N recycled to the total GI tract was used for microbial protein synthesis by the ruminal bacteria of animals offered the 1.45% N diet (Table 3), intermediate values were observed for animals in 1.89 and 2.50% N diets (26 to 25%), and lower values were seen for the 2.97 and 3.40% N diets (9 and 6%, respectively). Because only a fraction of the N recycled to the total tract enters the rumen, the actual efficiencies of urea N capture are likely to be higher, although it would probably not affect all the treatments similarly (Bunting et al., 1989a). Additional observations suggest that when ruminal ammonia concentration was limiting, bacteria at or near the rumen wall were able to scavenge ammonia derived from the blood before it mixed with the ruminal ammonia pool (Bunting et al., 1989b). Because the bacterial isolation protocol employed does not include the bacteria attached to the rumen wall, the difference observed might underestimate the real utilization of N originating from the blood for certain bacteria species in animals fed low-N diets.
Assuming that urea space is a constant fraction of BW (approximately 55%, Preston and Kock, 1973) and that PUN represents the concentration of the urea pool, the urea pool size and turnover time can be calculated (Table 4). Increasing dietary N increased urea pool size (P < 0.001) and turnover time of the pool (P < 0.004, Table 4). Animals fed low-N diets showed a smaller urea pool turning over faster than when the animals were fed high-N diets.
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). The creatinine clearance remained constant across diets, suggesting that this effect was due to an increase in urea reabsorption by the kidney (Table 4). Based on these clearances it was calculated that approximately 47% of the urea filtered by the kidney was reabsorbed in heifers fed the 1.45% N diet, whereas animals on the 3.40% N diet reabsorbed only 8%. Because creatinine clearance overestimates true clearance (Vander, 1995), these values probably overestimate the true urea reabsorption. Similar increases in urea reabsorption to low-N diets were reported by Phillips et al. (1977) in cattle and by Cirio and Boivin (1990) and Leng et al. (1985) in sheep. The increase in the reabsorption of urea is consistent with the overexpression of UT in the inner medullary collecting duct found by Isozaki et al. (1994) in rats fed low-N diets.
The same concept of clearance has been applied to the GI tract (Ford and Milligan, 1970; Kennedy and Milligan, 1980). Approximately 7.1 times more plasma was cleared by the GI tract than by the kidney for animals on the 1.45% N diet, whereas for heifers on the 3.40% N diet, the plasma cleared by the kidney was twice the volume of plasma cleared by the GI tract (Table 4). This suggested that a mechanism redirecting urea into the GI tract was present in heifers fed low-N diets. The discovery of UT in the GI tract of ruminants (Ritzhaupt et al., 1998) could provide an explanation for the increase in urea transfer when ruminants are fed low-N diets. Contrary to our initial hypothesis, the results of western blots of UT-B in ruminal mucosa biopsies (Figure 1) demonstrated that UT were expressed more when animals were fed high-N diets. Ritzhaup et al. (1997) reported a carrier-mediated (facilitated) transport of urea from the mucosal to the serosal side of the ruminal epithelium. The possibility of urea diffusing into the GI tract via the paracellular space and/or leaking across the lipid bilayer, and then returning to the blood through UT in animals fed high N diets has to be considered. Urease activity is known to be reduced by high ammonia concentrations (Bunting et al., 1989b), a condition that arises when high-N diets are fed. If urease activity is depressed, and more UT are expressed in the ruminal mucosa, more urea could then return to the blood before being hydrolyzed. This could prove beneficial to the animal because when high N diets are fed, urea recycled to the GI tract contributes little to the N economy of the rumen.
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Implications |
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Footnotes |
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2 The authors gratefully acknowledge J. Sands and J. Klein (Renal Division, Emory University) for the analysis of urea transporters. We also wish to thank L. Lintault for her assistance with data collection and analysis.
Received for publication January 21, 2002. Accepted for publication October 14, 2002.
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