J. Anim Sci. 2008. 86:E293-E305. doi:10.2527/jas.2007-0475
© 2008 American Society of Animal Science
ALPHARMA BEEF CATTLE NUTRITION |
Nitrogen recycling through the gut and the nitrogen economy of ruminants: An asynchronous symbiosis1
C. K. Reynolds*,2 and
N. B. Kristensen
* School of Agriculture, Policy, and Development, University of Reading, Earley Gate, Reading, RG6 6AR, United Kingdom; and and
University of Aarhus, Faculty of Agricultural Sciences, Tjele, DK-8830, Denmark
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Abstract
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The extensive development of the ruminant forestomach sets apart their N economy from that of nonruminants in a number of respects. Extensive pregastric fermentation alters the profile of protein reaching the small intestine, largely through the transformation of nitrogenous compounds into microbial protein. This process is fueled primarily by carbohydrate fermentation and includes extensive recycling of N between the body and gut lumen pools. Nitrogen recycling occurs via blood and gut lumen exchanges of urea and NH3, as well as endogenous gut and secretory N entry into the gut lumen, and the subsequent digestion and absorption of microbial and endogenous protein. Factors controlling urea transfer to the gut from blood, including the contributions of urea transporters, remain equivocal. Ammonia produced by microbial degradation of urea and dietary and endogenous AA is utilized by microbial fermentation or absorbed and primarily converted to urea. Therefore, microbial growth and carbohydrate fermentation affect the extent of NH3 absorption and urea N recycling and excretion. The extensive recycling of N to the rumen represents an evolutionary advantage of the ruminant in terms of absorbable protein supply during periods of dietary protein deficiency, or asynchronous carbohydrate and protein supply, but incurs a cost of greater N intakes, especially in terms of excess N excretion. Efforts to improve the efficiency of N utilization in ruminants by synchronizing fermentable energy and N availability have generally met with limited success with regards to production responses. In contrast, imposing asynchrony through oscillating dietary protein concentration, or infrequent supplementation, surprisingly has not negatively affected production responses unless the frequency of supplementation is less than once every 3 d. In some cases, oscillation of dietary protein concentration has improved N retention compared with animals fed an equal amount of dietary protein on a daily basis. This may reflect benefits of Orn cycle adaptations and sustained recycling of urea to the gut. The microbial symbiosis of the ruminant is inherently adaptable to asynchronous N and energy supply. Recycling of urea to the gut buffers the effect of irregular dietary N supply such that intuitive benefits of rumen synchrony in terms of the efficiency of N utilization are typically not observed in practice.
Key Words: ammonia • liver • nitrogen • rumen • synchrony • urea
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INTRODUCTION
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There is increasing concern regarding the contributions of ruminant milk and meat production to emissions of environmental pollutants (FAO, 2006
), including emissions of N generally and ammonia and nitrous oxide specifically. Concerns include the contribution of manure to the eutrophication of aquatic environments, effects of ammonia on air quality, and the contribution of nitrous oxide to global greenhouse gas concentrations. Recent metaanalyses of experimental data from lactating cows have concluded that the major factor determining total N excretion as manure (feces plus urine) in lactating dairy cows is total dietary N intake (Castillo et al., 2000; Yan et al., 2006). However, there is some variation in the excretion measured at a given level of N intake, which may arise due to effects of experimental variation, animal variation (genetic differences in digestive or metabolic efficiency), or dietary and other environmental variables. Thus, there is opportunity for reducing N excretion by reducing the amount fed but also through other dietary management and breeding strategies.
Nutritional management of ruminants for improved utilization of absorbed nitrogenous compounds will reduce not only the amount of manure N excreted but also the portion excreted as more volatile urinary urea N. Strategies to reduce the excretion of N in lactating dairy cows include reducing the amount fed in excess of requirements (so-called precision feeding), improving the efficiency of capture of recycled N as absorbable microbial protein, and improving the efficiency of utilization of absorbed AA. Strategies to improve the utilization of ammonia for microbial protein synthesis include changes in the amount, type, and degradability of dietary carbohydrates and proteins (Lapierre and Lobley, 2001). In this regard, there has been considerable interest in the potential beneficial effects of synchronizing rates of carbohydrate and protein degradation in the rumen to optimize the postprandial availability of energy and N for microbial protein synthesis (Cabrita et al., 2006). As restrictions on N losses from animal production facilities increase, so too will the need to more precisely formulate diets to meet the requirements for specific AA and minimize N excretion. The recycling of urea N to the gastrointestinal tract may well be central to the success of these approaches by virtue of the buffering effect of N recycling on the pattern of N supply to the rumen microbes and subsequent microbial protein supply to the animal.
In light of these considerations, our objective was to review the quantification and regulation of NPN flux between the gastrointestinal tract and body of ruminants. We considered the effects of rumen synchrony, or asynchrony, on NPN cycling and productive responses and the potential role of labile protein reserves, or undetermined losses of N, on the response to variations in dietary protein supply.
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AMMONIA N ABSORPTION
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It has long been known that ruminants typically absorb substantial amounts of ammonia from the rumen, primarily as a consequence of microbial degradation of protein (McDonald, 1948; Lewis et al., 1957). Some of the ammonia released is used for microbial protein synthesis, but a portion is absorbed into the portal vein from which virtually all that is absorbed is removed by the liver, on a net basis (Reynolds, 1995). Ammonia removed by the liver is largely converted to urea or used to synthesize Gln from glutamate (Parker et al., 1995; Reynolds, 1995; Nieto et al., 2002). Ammonia is absorbed across the epithelium of the rumen, as well as other sections of the gastrointestinal tract, as both NH3 and NH4+, although the exact mechanisms by which these ions are transferred from the gut lumen to venous blood are equivocal. In a recent review, Abdoun et al. (2007) concluded from data available that NH3 is absorbed in the lipophilic NH3 form by simple diffusion at rates linearly related to pH at 7 or greater. At pH of 6.5 or lower, which would be expected in the rumen, NH3 is predominantly absorbed as NH4+, probably via a K+ channel. Thus, NH3 absorption is influenced by pH and interacts with the transport of K, Na, and Mg across the apical membrane. However, less is known about transport across the basolateral membrane (Abdoun et al., 2007).
Ammonia absorption into the portal vein is substantial and includes N from both dietary and endogenous sources, including urea. Measurements of net absorption of NH3 across the portal-drained viscera (PDV) and net release of urea by the liver can be obtained using chronic blood-sampling catheters and measurements of venous-arterial concentration difference and blood flow. Across 315 measurements in growing and lactating cattle (Firkins and Reynolds, 2005), net absorption of NH3-N into the portal vein increased with increasing N intake, and the linear regression had a slope of 0.42, indicating that on an incremental basis, across a variety of diet formulations and physiological states, net NH3 absorption accounted for 42% of dietary N intake. These measurements of NH3 absorption include NH3 absorbed from the entire gastrointestinal tract and NH3 generated from tissue metabolism of AA and other nitrogenous compounds. In cattle, NH3 absorption across the mesenteric-drained viscera (MDV; i.e., the small and large intestines and cecum) accounted for from 28 to 53% of NH3 absorption across the entire PDV (Huntington, 1989; Parker et al., 1995; Theurer et al., 2002). Differences in the proportion of net PDV NH3 absorption attributable to MDV tissues among these studies could be attributable to both technical considerations (e.g., site of mesenteric vein sampling) and diet composition effects, but it is notable that within specific studies, effects of diet composition on the ratio of MDV to PDV NH3 absorption have been small (e.g., Huntington, 1989). Dietary factors that are known to influence relative amounts of NH3 absorption from the rumen have been reviewed previously (e.g., Parker et al., 1995; Tan and Murphy, 2004) and include amounts and degradability of dietary and endogenous sources of N in the gastrointestinal tract, as well as dietary carbohydrate sources. The use of NH3 for microbial protein synthesis is energy-dependent; therefore, the amount and degradability of dietary carbohydrate can influence NH3 absorption. This is illustrated by the reduction in net NH3 absorption across the PDV of lactating dairy cows caused by steam-flaking of sorghum grain (Delgado-Elorduy et al., 2002) and both ruminal and abomasal starch infusion (Reynolds, 2006). In the case of abomasal starch infusion, the reduction in NH3 absorption may be attributable to an increase in microbial protein synthesis in the hindgut (Reynolds, 2006).
In the multicatheterization studies in cattle summarized by Firkins and Reynolds (2005), the linear regression for the relationship between total N intake and net urea release by the liver indicates that liver production of urea accounts for 65% of increments in N intake. The absolute and incremental difference between liver urea production and net NH3 absorption (42% of incremental N intake) and removal by the liver represents the net contribution of AA and other nitrogenous compounds to liver urea synthesis. Based on the divergent slopes observed, the contribution of compounds other than NH3 increases with greater intakes, which in the case of these studies, also represents measurements in lactating dairy cows.
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GUT UREA N ENTRY
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Of the urea produced by the liver, a part is excreted in the urine, and the remainder is recycled back to the gut through either direct transfer from blood across the epithelial tissue or via saliva. In the gut lumen, the urea is degraded to NH3 through the action of microbial urease, and the N thus recycled can be used for microbial protein synthesis or absorbed as NH3. This recycling of N via NH3 and urea transfers provides a source of N for microbial protein synthesis when N from the diet is insufficient and could be considered an evolutionary advantage of ruminants that enables them to survive when protein supplies are sporadic or inadequate. Called the protein regeneration cycle by Houpt (1959), NPN use for microbial protein synthesis in the rumen enables ruminants to survive, and produce milk and meat, on diets that are based solely on NPN sources (Virtanen, 1966).
Blood Urea Concentration
Houpt (1959) showed that in addition to urea recycling to the gut via saliva, blood urea was transferred directly to the rumen across epithelial tissues and degraded to NH3 that could be used for AA synthesis or absorbed into the portal vein. However, despite considerable research in the succeeding 48 yr, the factors regulating urea transfer to the gut lumen are still not totally understood. The transfer of N from blood urea to rumen NH3 was shown to be positively related to blood urea concentration in 2 cattle fed a low-protein hay (7% CP) and receiving blood urea infusions (Vercoe, 1969). Rumen NH3 concentration increased with increasing plasma urea concentration, to a plateau of 12 mg of urea N/100 mL of plasma (8.6 mM). More recently, Sunny et al. (2007) reported that in sheep fed low-protein diets, the increase in rumen NH3 concentration with blood urea infusion was due to an increase in the amount of urea recycled to the gut, measured using dual-labeled urea. These studies indicate a relationship between blood urea concentration and the transfer of urea to the gut lumen, at least in animals fed low-protein diets. However, in a review of published studies using cattle, Lapierre and Lobley (2001) found no evidence of a relationship between arterial urea N concentration and net PDV removal of urea directly from blood across a wide range of blood urea N concentrations. Although these measurements do not include salivary urea transfer, this indicates that other factors in addition to arterial urea N concentration determine the amount of blood urea N transferred to the lumen of the gastrointestinal tract.
Dietary Carbohydrate and Rumen NH3 Concentration
Numerous reviews of factors affecting urea transfer to the rumen have highlighted the importance of microbial urease and the positive effect of carbohydrate fermentation on urease activity, as well as the potential negative effect of rumen NH3 concentration on urea transfer to the rumen (Houpt, 1970; Kennedy and Milligan, 1980; Abdoun et al., 2007). In addition to negative effects on NH3 absorption, in some cases, steam-flaking cereal grains to increase rumen starch degradability has increased net PDV removal of urea (Theurer et al., 1999, 2002). As stated previously, these measurements of net PDV removal of urea exclude recycling of urea via saliva and represent direct transfer of urea to the entire PDV, including the small intestine and hindgut. To distinguish between these routes of transfer, measurements of net PDV, MDV, and liver flux of urea can be combined with measurements of urea excretion in urine and salivary transfer estimated as the difference between liver production and net PDV removal plus urine excretion. Using this approach, Huntington (1989) reported marked effects of dietary carbohydrate and protein type and intake on urea transfers in growing steers. In that study, feeding a 12% CP high-starch diet increased the amount of urea transferred directly to the rumen from blood. Compared with the high-concentrate diet, feeding a 17% CP alfalfa hay diet increased the amount of urea transferred to the MDV, presumably as a consequence of dietary fiber fermentation in the hindgut, and decreased the direct transfer of urea to the stomach. However, this decrease in direct transfer to the rumen was associated with an increase in transfer via saliva such that the total urea transfer to the rumen was similar on the 2 diets, which were fed at similar ME intakes. In that study, the decrease in direct transfer of urea to the rumen was associated with a more than 2-fold increase in net PDV absorption of NH3, and presumably rumen NH3 concentration (Huntington, 1989). The effects of rumen NH3 concentration on urea transfer are not certain (Marini et al., 2006), but Kennedy and Milligan (1980) showed that when urea was infused into the rumen, plasma urea transfer to ruminal NH3, measured on the basis of 15N transfers, decreased as ruminal NH3 concentration increased. The hypothesized negative effect of ruminal NH3 concentration on urea transfer has been attributed to direct effects of NH3 absorption on urea transport across the ruminal epithelium, or inhibitory effects on adherent microbial urease (Kennedy and Milligan, 1980; Abdoun et al., 2007).
Urea Transporters
Studies in the last decade have identified specific transporters for urea in the kidney and have shown that these transporters are also expressed in other tissues, including the epithelium of gut tissues of various species (Stewart et al., 2005; Marini et al., 2006; Abdoun et al., 2007). Two major variants of the urea transporter (UT) gene have been reported, UT-A and UT-B. At present, it appears that although mRNA for UT-A was found in the sheep duodenum, UT-B was the variant transcribed in the rumen of both sheep and cattle (Marini et al., 2006; Abdoun et al., 2007). In the rumen, UT-B has been identified in all the epithelial layers except the stratum corneum (Stewart et al., 2005), and the amount present was altered by dietary protein level in 1 study (Marini and Van Amburgh, 2003) but not in a subsequent study (Marini et al., 2004). Increasing dietary protein increased the expression of UT-B in ruminal epithelium, which was contrary to expectations (Marini and Van Amburgh, 2003) but indicates that the transporter may play a role in regulating urea transfer to the gut, possibly via cycling across the gut epithelium under conditions of elevated blood urea concentrations (Marini and Van Amburgh, 2003). Regulators of UT function and the role, or not, of the UT in mediating known effects of dietary carbohydrate, NH3, and other factors on urea transfer from blood to specific sections of the gut lumen remain to be determined (Abdoun et al., 2007).
Microbial Capture of Recycled Urea N
Based on measurements obtained by isotopically labeling the ruminal NH3-N pool, ruminal NH3 accounts for 23 to 95% of bacterial N incorporation, depending on the diet fed and the predominant bacterial species (Nolan and Dobos, 2005; Firkins et al., 2007). Although the potential contribution of NH3 to bacterial N incorporation can be high, the enrichment of bacterial N by endogenous urea N from the blood pool is typically low. For example, based on measurements of 15N transfers from Leu infused into the jugular vein, Ouellet et al. (2002) estimated that only 12% of bacterial N flow to the duodenum was derived from endogenous urea in lactating dairy cows. This may reflect a reduced probability of endogenous N capture relative to the supply of NH3-N from proteolysis of feed proteins and differences in the use of urea N between bacteria associated with the solid and liquid phases of the rumen. As suggested by Firkins et al. (2007), the efficiency of endogenous N utilization for microbial protein synthesis would be expected to be greater when dietary protein and thus ruminal NH3 concentrations are reduced. However, in beef steers fed very low protein prairie hay, when the salvage of urea N for protein synthesis in the rumen would be expected to be critical, only one-third of microbial N flow to the duodenum was derived from endogenous urea entry to the gut (Wickersham, 2006). This may be due, in part, to a limitation of microbial growth and, thus, NH3 capture by fermentable energy supply (Nolan and Dobos, 2005). In addition, the effect of proto-zoa on intraruminal N cycling, and the net capture of endogenous urea N as bacterial protein, needs clarification (Firkins et al., 2007).
Quantification of Urea Transfers
As indicated previously, in addition to the use of multicatheterization procedures, urea transfer to the gut can be quantified using the dual-labeled urea technique used previously in humans and adapted for use in ruminants by Sarraseca et al. (1998) and Lobley et al. (2000). A tracer dose of double-labeled (15N15N) urea is used to determine the flux of urea and analysis of single-labeled (15N14N) urea used as an indicator of urea N recycling to the Orn cycle, which will largely represent NH3 derived from urea metabolism in the gut. Total urea transfer to the gut can then be estimated as the difference between urea entry to the blood pool and measured excretion in the urine, whereas the difference between urea transfer to the gut and the sum of measured fecal excretion and return to the Orn cycle provides an estimate of the recycled urea N used for anabolic purposes, which will largely represent microbial protein synthesis. Based on available measurements obtained using this methodology, Lapierre and Lobley (2001) concluded that on average in sheep and cattle, two-thirds of urea entry was recycled to the gut, from which 40% was returned to the Orn cycle and 10% was excreted in feces, leaving 50% used for anabolic purposes. Since that time, there have been a number of studies conducted in cattle fed a variety of dietary protein levels for which similar measurements of urea flux and transfers to the gut have been obtained using dual-labeled urea tracer. Across these studies, the fraction of urea entry transferred to the gut, or excreted in the urine, varied with dietary protein concentration in a reciprocal manner (Figure 1). When growing cattle were fed prairie hay with very low protein concentrations (Wickersham, 2006), virtually all (98%) urea entering the blood pool was returned to the gut, and little (2%) was excreted in urine. Similarly, high portions of urea entry were recycled to the gut in dairy cows (Ruiz et al., 2002) and growing steers (Archibeque et al., 2001) fed low-protein diets. Urinary N excretion was still occurring in these animals, but the N was excreted primarily in forms other than urea. These measurements in animals fed low-protein diets emphasize the inherent ability of ruminants to salvage urea N for recycling and anabolic purposes when dietary protein is in short supply. Because virtually all of urea entry was transferred to the gut lumen in cattle fed low-CP diets (<12%), the fraction of urea entry used for anabolic purposes (Figure 2) was typically greater (28 to 72%) than for higher-protein diets (17 to 26%), but other dietary factors also influence the fate of urea N in the gut lumen. Across these studies, the fraction of total urea entry that returned to the Orn cycle (Figure 2) varied from 10 to 50%, but as for the fraction used for anabolic purposes, was consistently less at greater dietary protein concentrations. For total gut urea entry, the fractions used for anabolic purposes or returned to the Orn cycle were not consistently affected by diet CP level (Figure 3) or N intake (data not shown) and averaged 54 and 34%, respectively, which is comparable to the values of 50 and 40% reported by Lapierre and Lobley (2001). The data in Figures 1, 2, and 3 are based on portions of total urea entry, not amounts of urea flux. Marini and Van Amburgh (2003) fed heifers increasing amounts of soybean meal and urea, which linearly increased N intake and urine urea N output and at the greatest dietary N concentration, reduced the fraction of urea entry recycled to the gut to 29%. However, at dietary CP concentrations of 12% and above, the total amount of urea N transferred to the gut (g/d) was relatively constant. In a summarization of this and 2 other studies in growing cattle (Figure 4), increasing N intake was associated with linear increases in urea entry rate that accounted for 70% of the increment in total N intake, but the effect of N intake on total gut urea transfer was small. The observations reported for lactating dairy cows also are shown. For dairy cows, the slope of the regressions were similar to those for the data from growing cattle, but the intercepts were lower and higher for total and gut urea entry, respectively, even after correcting for BW0.75. This difference between growing and lactating cattle may reflect differences in maintenance requirements as well as differences in the dynamics of rumen N metabolism between growing and lactating animals, but more data are needed to substantiate this comparison.
If urea transfer is determined by fermentable energy supply, and the use of urea N for anabolic purposes (e.g., microbial protein synthesis), then it is logical to expect that at dietary protein levels above a fermentation requirement set by intake and diet composition, additional dietary N would be absorbed as NH3 and excreted in urine, without a concomitant increase in the amount of urea N transfer to the gut. This was the case when multicatheterized steers fed a higher-protein alfalfa hay-based diet were fed supplemental urea (Maltby et al., 2005). Increases in dietary N intake (as urea) were matched by similar increases in net PDV absorption of NH3-N, net liver release of urea N, and urine N excretion, but the total amount of urea transferred to the gut, measured as net PDV flux and estimated salivary excretion, was similar for the 2 diets (Table 1). Net PDV removal of urea increased slightly when urea was fed, but this was partially countered by a numerical decrease in salivary urea. In this case, marked increases in NH3 absorption, and presumably ruminal NH3 concentration, were not associated with a decrease in total removal of urea N by the PDV, but the amount removed by the MDV tissues was not determined. In addition, the diet was fed every 2 h to minimize post-prandial responses.
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RUMEN SYNCHRONY OR ASYNCHRONY
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As mentioned previously, there has been considerable interest in the potential benefits of synchronizing rates of carbohydrate and protein degradation in the rumen to optimize the postprandial availability of energy and N for microbial protein synthesis. Synchrony refers to events occurring at the same time, or simultaneously, and thus the strict interpretation of the term rumen synchrony is that degradation of energy- and N-yielding substrates required for microbial growth occur simultaneously. In practical usage, rumen synchrony typically refers to the postprandial, or diurnal, availability of energy from carbohydrate fermentation and NH3 for microbial protein synthesis and growth (Cabrita et al., 2006). It is logical to expect that maximizing the efficiency of dietary energy and protein utilization for microbial growth using this approach will improve feed conversion efficiency, reduce NH3 absorption, and reduce N excretion into the environment. However, in a recent review, Cabrita et al. (2006) reported that across a large number of studies in lactating dairy cows, there was no consistent effect of attempts to synchronize carbohydrate fermentation and N availability on ruminal NH3 concentration or microbial N supply in lactating dairy cows. Similarly, in these studies, there was no indication of a positive effect of diets that attempted to synchronize rumen energy and N supply on DM intake or milk yield. Similar attempts to synchronize postprandial carbohydrate degradation and N supply to the rumen of beef cattle have had little effect on growth rate or the efficiency of dietary energy or protein utilization (Valkeners et al., 2004). Cabrita et al. (2006) emphasized that when interpreting these studies and designing experiments to evaluate effects of rumen synchrony vs. asynchrony, it is important to be cognizant of the fact that changing dietary carbohydrate or protein sources changes much more than just synchrony and that effects of the changes in specific substrates (e.g., starch vs. NDF, protein vs. NPN) may be just as important as effects of synchrony per se. As emphasized by Valkeners et al. (2004) and Cabrita et al. (2006), the recycling of N via NH3 and urea N enables rumen microbes to overcome any short-term effects of asynchrony.
Effects of Oscillating or Infrequent Dietary Protein Supply
In addition to interest in improving rumen synchrony, there also has been considerable research interest in the use of oscillating dietary protein concentration or infrequent protein supplementation as a management tool to reduce labor costs of feeding sheep and cattle but also as a potential dietary strategy to improve N retention and reduce N excretion to the environment (Archibeque et al., 2007b). This dietary strategy represents an example of rumen energy and N asynchrony over periods of days, rather than hours, and emphasizes the importance of N recycling, and perhaps labile N reserves, to the N economy of the ruminant.
To our knowledge, the first full report of the use of infrequent protein supplementation for cattle was published by McIlvain and Shoop (1962). Cottonseed meal was fed to steers grazing rangeland in equal weekly amounts on a daily, every third day, or weekly basis, and over the course of 3 winters, no effect on ADG was measured. Similarly, Coleman and Wyatt (1982) reported no effect of supplementation frequency (24-, 48-, or 96-h basis) on N digestion and retention when the same amount of cottonseed meal was provided per 96 h to beef steers. Since these early reports, there have been a number of studies in which dietary protein concentration has been oscillated at intervals ranging from 1 to 2 d or a basal diet has been supplemented with protein at intervals ranging from 1 to 6 d in growing beef cattle (Figure 4
; Coleman and Wyatt, 1982; Cole et al., 2003; Farmer et al., 2004; Archibeque et al., 2007c) or lambs (Figure 5; Cole, 1999; Bohnert et al., 2002; Ludden et al., 2002; Currier et al., 2004; Archibeque et al., 2007b). Except when supplementation frequency was extended to more than once every 3 d (Bohnert et al., 2002; Farmer et al., 2004), compared with a daily supplementation control, there were surprisingly few deleterious effects of supplementation frequency, or oscillation, on N utilization and excretion (Figures 5 and 6). Similarly, in a feedlot trial with beef steers fed diets with oscillating protein concentration, there was no effect on ADG or carcass composition, but oscillating protein did increase volatile losses from manure (Archibeque et al., 2007a).
It is noteworthy that in 2 cases (e.g., Cole, 1999; Archibeque et al., 2007b), oscillating dietary protein concentration increased N retention compared with feeding a diet providing the same amount of protein on a daily basis as the average protein content of the oscillating diets. In the study by Archibeque et al. (2007b), this increase in N retention was attributable to an increase in the amount of N digested.
The lack of a negative effect of oscillating or infrequent protein supplementation on N utilization has been attributed to the recycling of urea to the rumen, which provides N for microbial protein synthesis on days when protein is not supplemented or dietary protein concentration is low (Krehbiel et al., 1998; Archibeque et al., 2007b). This concept is supported by increases in arterial concentration and net PDV removal of urea N in sheep fed oscillating dietary protein (Krehbiel et al., 1998; Archibeque et al., 2007b) and sustained elevations in plasma urea concentrations measured in sheep supplemented with sources of rumen-degradable protein or ruminally undegraded feed CP at 3- or 6-d intervals (Bohnert et al., 2002). Increases in plasma urea concentration and urea transfer to the gut after protein supplementation would be enhanced if kidney urea clearance is downregulated during the period without protein supplementation, and there is a lag in the increase in urea excretion in the urine after protein is consumed (Marini et al., 2006).
The lack of a negative effect of oscillating or infrequent dietary protein on N balance in ruminants may be due to more than just a sustained elevation of urea and NH3 pool size and recycling. Deposition of N in nitrogenous compounds absorbed in excess of requirements on days when protein is provided into pools other than urea and NH3, which are subsequently catabolized to generate N for urea synthesis, may also buffer the effects of infrequent protein supply. In this regard, Nolan and Leng (1972) reported that in sheep, a large proportion (62%) of the endogenous urea N degraded in the digestive tract and absorbed as NH3 does not reappear in urea but is incorporated into pools of nitrogenous compounds that turn over more slowly, providing a more continuous supply of N for synthetic processes. These pools have often been referred to as labile proteins or protein reserves (Paquay et al., 1972; Waterlow, 1999). The identification of these labile nitrogenous compounds is not certain (Waterlow, 1999), but it has long been known that in humans, and other nonruminants, a large short-term increase in body N retention is typically measured after an abrupt increase in dietary protein concentration. Over time, measured N balance then decreases as urinary N increases, until a new steady state is achieved (Oddoye and Margen, 1979). Similarly, there is a dramatic decrease in measured body N balance (N loss) when N intake is abruptly reduced in humans (Oddoye and Margen, 1979), until an adaptation of urinary N excretion occurs that, depending on the magnitude of the change in protein intake, can take weeks to occur. The deposition of N in labile pools on days when protein is fed, which are then degraded to produce urea that can be recycled to the gut on days when protein is not fed, would provide a convenient explanation for the lack of a negative effect of oscillating dietary protein supplementation in ruminants. Previous studies have described a pool of N in cattle that is more rapidly degraded after abrupt reductions in dietary protein (Paquay et al., 1972; Biddle et al., 1975). Biddle et al. (1975) reported that plasma proteins and urea were components of the labile N reserves in growing cattle, and their total labile N pool was equal to 5.6% of body N. Paquay et al. (1972) suggested that in addition to plasma proteins, the liver and other viscera, as well as newly synthesized muscle protein, contributed to the more labile pool of N in the dairy cow. In this regard, Ludden et al. (2002) reported that mass of the small intestine and total gastrointestinal tract plus liver was increased in sheep fed oscillating dietary protein. Finally, the microbial population of the rumen and hindgut may also serve as a reservoir for N, especially if urea transfer to the gut lumen is upregulated when protein supply is reduced.
The large increases in N balance measured in nonruminants abruptly changed from low- to high-protein diets has been attributed to a lag in the adaptation of Orn cycle enzymes observed in the classic study of Schimke (1962). Although the total capacity of the Orn cycle probably responds much more quickly than the activity of the component enzymes (Waterlow, 1999), this lag in urea cycle activity, and resulting deposition of absorbed AA N in labile reserves, would reduce the amount of excess N consumed subsequently excreted in the urine, providing a source of N for urea recycled to the gut on days when dietary protein is insufficient. In addition, if the capacity of the urea cycle is reduced by days of not feeding protein, absorbed AA may be spared for anabolic uses on days when the protein is fed. Depending on the diet fed and the resulting dietary and MP requirements of the animals, this may explain the positive effects of protein oscillation on N balance reported (Cole, 1999). However, Waterlow (1999) suggested that factors other than labile proteins and the capacity of the Orn cycle must contribute to the constancy of N balance in humans, who often have sporadic dietary protein intake.
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ERRORS OF MEASUREMENT OF N BALANCE
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In considering the responses to energy and protein asynchrony in ruminants, it is perhaps relevant that measured N balance (body N retention) in both ruminants and nonruminants is typically greater than expected, based on changes in body mass or measured protein accretion (Young, 1986; Manatt and Garcia, 1992; MacRae et al., 1993). Some have proposed that this is due to cumulative errors of measurement or the loss of N through routes that are not measured (Hegsted, 1976), such as dermal and gaseous losses (Young, 1986), which may explain the excessively high N balances often measured. Unexplained losses may also contribute to the excessively high measurements of N retention during the transition from low- to high-protein diets, rather than a mysterious unexplained pool of labile N. In their review of published measurements of N balance in lactating dairy cows, Sphanghero and Kowalski (1997) concluded that most published measurements of N retention in dairy cows are too high, in part, due to NH3 losses from urine and feces during collection and analysis and unaccounted for hair and scurf losses. However, even after correction for estimates of these losses, in most cases, measurements of N retention are too high. They also suggested that there may be nitrate and nitrite formation from dietary N by microbes in the gut, which would not be measured by normal Kjeldahl analysis (Sphanghero and Kowalski, 1997).
Martin (1966) quantified potential errors of measurement of N balance in sheep using an exhaustive and detailed series of measurements and found that the loss of N as NH3 from the lungs, rumen, skin, and fleece was minor. There was a negligible loss of NH3 from feces over the course of a 24-h period, but NH3 loss from urine was substantial unless acid was added. It is standard practice to acidify urine during collection for N balance measurements, but the potential losses of NH3 during collection, storage, processing, and analysis of feces can be substantial and should be carefully considered (Manatt and Garcia, 1992), especially when bulk or composite samples of daily fecal collections are analyzed. The study of Martin (1966) in sheep and detailed studies in humans (e.g., Calloway et al., 1971; Oddoye and Margen, 1979) have failed to identify any large losses of N that are not accounted for in standard measurements of N balance but do highlight multiple minor losses that, in total, may be of consequence. These unaccounted for losses, such as gaseous forms of N or nitrate formation (Oddoye and Margen, 1979), may be especially important if they are greater during periods of adaptation after an abrupt change in N intake or if the losses are differentially affected by treatments being compared in a specific study. These mysteries of N balance have perplexed nutritionists for much of the last century (Waterlow, 1999) but need to be addressed as we enter an age of environmental budgeting at the farm or feedlot level based on estimates of N balance.
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CONCLUSIONS
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The cycling of N between body pools and the gut in ruminants has been studied for more than 50 yr, but the factors regulating urea transfer to the gut are not certain. Ammonia absorption into the portal vein is substantial and influenced by dietary protein and carbohydrate composition. Similarly, dietary composition, and particularly fermentable energy supply, influences urea transfer to the gut. It is known that factors such as dietary starch and rumen NH3 concentration influence transfer of urea to the gut, and recent evidence suggests that these effects on urea transfer may be mediated in part by urea transporters in the gut epithelium. The transfer of urea to the gut accounts for increasing fractions of total urea production as dietary protein is reduced, but the total amount ultimately utilized for anabolic purposes is likely determined by microbial requirements. This salvage of urea N when low-protein diets are fed provides a source of N for microbial protein synthesis and buffers effects of asynchronous energy and N supply to the rumen, which may explain the general lack of positive effects of attempts to synchronize rumen energy and N supply reported in the literature as well as the general lack of negative effects of oscillating or infrequent protein supplementation. As environmental concerns force reductions in total dietary protein concentrations, the role of urea salvage in the rumen for microbial protein synthesis may be critical for the maintenance of acceptable levels of milk and meat production.
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Footnotes
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1 Presented at the Alpharma Beef Cattle Nutrition symposium at the annual meeting of the American Society of Animal Science, San Antonio, TX, July 8 to 12, 2007. 
2 Corresponding author: c.k.reynolds{at}reading.ac.uk
Received for publication July 30, 2007.
Accepted for publication October 6, 2007.
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Abstract
INTRODUCTION
