Ruminal Urea Research Articles

Mammary gland, kidney and rumen urea and uric acid transporters of dairy cows differing in milk urea concentration

The milk urea concentration (MUC) serves as indicator of urinary nitrogen emissions, but at comparable crude protein (CP) intake, cows with high (HMU) and low (LMU) MUC excrete equal urea amounts. We hypothesized that urea and uric acid transporters and sizes of the kidney, mammary gland, and rumen account for these phenotypes. Eighteen HMU and 18 LMU Holstein dairy cows fed a low (LP) and normal (NP) CP diet were studied. Milk, plasma and urinary urea concentrations were greater with NP feeding, while plasma and urinary urea concentrations were comparable between phenotypes. Milk and plasma uric acid concentrations were higher with LP feeding but not affected by phenotype. The milk-urine uric acid ratio was greater in HMU cows. The mRNA expressions of the ruminal urea transporter SLC14A1 and AQP10, the mammary gland and rumen AQP3, and the mammary gland uric acid transporter ABCG2 were not affected by group or diet. Renal AQP10, but not AQP3, AQP7, and SLC14A2 expressions, and the kidney weights were lower in HMU cows. These data indicate that renal size and AQP10 limit the urea transfer from blood to urine, and that MUC determines if uric acid is more released with milk or urine.

Effect of Post-Ruminal Urea Supply on Growth Performance of Grazing Nellore Young Bulls at Dry Season.

The objective of this study was to evaluate the effect of the use of post-ruminal urea on performance, nitrogen metabolism and the ruminal environment of Nellore cattle reared on pasture during the dry season. In experiment 1 (Exp. 1), nine ruminal-cannulated Nellore steers, 30 ± 2 months old (651 ± 45 kg body weight (BW)), were allotted to a 3 × 3 Latin triple square. In experiment 2 (Exp. 2), 84 Nellore bulls, 18 ± 3 months old (315 ± 84 kg BW), were distributed in complete randomized blocks, by initial BW. Protein supplements were supplied daily, in the amount of 2 g/kg BW, and consisted of either CONT: protein + conventional urea (50% CP), PRU: protein + post ruminal urea (50% CP) and U + PRU: protein + urea conventional + post-ruminal urea (70% CP). The paddocks were composed of Urochloa brizantha cv. Marandu grass. In Exp. 1, there was no treatment effect for DM, OM, NDF, forage intake, and CP, but there was a higher intake for PRU (p < 0.005) and a higher digestibility for U+ PRU (p = 0.001). There was no effect on ruminal pH or NH3-N concentration (p ≥ 0.232), but there was an interaction between treatment and time for them (p < 0.039). Furthermore, there was a treatment effect on the total SCFA concentration, with CONT being higher than the others. A difference in the acetate:propionate ratio was found (p < 0.027), with a greater relationship for PRU and U + PRU. A treatment effect (p = 0.049) was found for the propionate proportion, with a higher proportion in the CONT. Nitrogen intake was consequently lower for the CONT and higher urinary excretion for the U + PRU (p = 0.002). Animals supplemented with CONT showed a tendency to have more Bacteria and fewer Archaea (p = 0.086). In Exp. 2, there was a treatment effect for the disappearance rate of the supplement (p < 0.001). Intake was faster for PRU and CONT, but performance was not affected by PRU (p = 0.311). The use of post-ruminal urea alters the microbial population, but does not affect performance. Therefore, supplementation with post-ruminal urea presented similar results compared to conventional urea. Ruminal and blood parameters and animal performance were not influenced by treatments.

Localization of urea transporter B in the developing bovine rumen.

Urea nitrogen secreted from blood to rumen is a crucial factor shaping the symbiotic relationship between host ruminants and their microbial populations. Passage of urea across rumen epithelia is facilitated by urea transporter B (UT-B), but the long-term regulation of these proteins remains unclear. As ruminal function develops over a period of months, the developing rumen is an excellent model with which to investigate this regulation. Using rumen epithelium samples of calves from birth to 96 d of age, this study performed immunolocalization studies to localize and semi-quantify UT-B protein development. As expected, preliminary experiments confirmed that ruminal monocarboxylate transporter 1 (MCT1) short chain fatty acid transporter protein abundance increased with age (P < 0.01, n = 4). Further investigation revealed that ruminal UT-B was present in the first few weeks of life and initially detected in the basolateral membrane of stratum basale cells. Over the next 2 months, UT-B staining spread to other epithelial layers and semi-quantification indicated that UT-B abundance significantly increased with age (P < 0.01, n = 4 or 6). These changes were in line with the development of rumen function after the advent of solid feed intake and weaning, exhibiting a similar pattern to both MCT1 transporters and papillae growth. This study therefore confirmed age-dependent changes of in situ ruminal UT-B protein, adding to our understanding of the long-term regulation of ruminal urea transporters.

The role of rumen epithelial urea transport proteins in urea nitrogen salvage: A review.

The symbiotic relationship between the host and the rumen microbiome plays a crucial role in ruminant physiology. One of the most important processes enabling this relationship is urea nitrogen salvaging (UNS). This process is important for both maintaining ruminant nitrogen balance and supporting production of their major energy supply, bacterially-derived short chain fatty acids (SCFA). The key step in UNS is the trans-epithelial movement of urea across the ruminal wall and this is a highly regulated process. At the molecular level, the key transport route is via the facilitative urea transporter-B2, localized to ruminal papillae epithelial layers. Additional urea transport through aquaporins (AQP), such as AQP3, is now also viewed as important. Long-term regulation of these ruminal urea transport proteins appears to mainly involve dietary fermentable carbohydrates; whereas, transepithelial urea transport is finely regulated by local conditions, such as CO2 levels, pH and SCFA concentration. Although the key principles of ruminal urea transport physiology are now understood, there remains much that is unknown regarding the regulatory pathways. One reason for this is the limited number of techniques currently used in many studies in the field. Therefore, future research in this area that combines a greater range of techniques could facilitate improvements to livestock efficiency, and potentially, reductions in the levels of waste nitrogen entering the environment.

Sex-Related Differences in UT-B Urea Transporter Abundance in Fallow Deer Rumen.

Rumen studies have focused almost exclusively on livestock species under strictly regimented diets. This means that the ruminal condition of free-living and free-feeding wildlife remains practically unstudied. Urea nitrogen salvaging, a process by which urea is passed into the rumen, to both provide a valuable source of nitrogen for bacterial growth and to buffer the potentially harmful acidic effects of bacterial short chain fatty acids, has remained unexplored in wild ruminants, such as deer. UT-B2 transporters are the key proteins reported to facilitate the transepithelial ruminal urea transport. In this study, we investigate the expression, abundance and localisation of urea transporters in the rumen of a semi-wild fallow deer (Dama dama) population. Physical measurements confirmed that males had larger rumen than females, while adults had longer papillae than juveniles. Initial RT-PCR experiments confirmed the expression of UT-B2, while immunolocalisation studies revealed that strong UT-B staining was present in the stratum basale of deer rumen. Western blotting analysis demonstrated that a 50 kDa UT-B2 protein was significantly more abundant in adult females compared to adult males. This study confirms the presence of UT-B2 urea transporters in deer rumen and suggests that sex-related differences occur, bringing new insight into our understanding of rumen physiology.

A Novel Urease Inhibitor of Ruminal Microbiota Screened through Molecular Docking.

Inhibition of the urease activity of ruminal microbiota is not only beneficial for increasing dietary and endogenic urea-N utilization efficiency in ruminants but also might be applicable for the preservation of nitrogen fertilizer in soil and treatment of gastrointestinal and urinary tract infections caused by ureolytic bacteria. To discover urease inhibitors to efficiently target ruminal microbiota, the identified ruminal microbial metagenomic urease gene was used to construct a homology model to virtually screen urease inhibitors from the ChemDiv database by molecular docking. The GMQE and QMEAN values of the homology model were 0.85 and −0.37, respectively, indicating a good model quality. The inhibition effect of the screened urease inhibitor for ruminal urea degradation was assessed by ruminal microbial fermentation in vitro. The toxic effect of the candidate inhibitor was performed using gut Caco-2 cells in vitro. The results showed that compound 3-[1-[(aminocarbonyl)amino]-5-(4-methoxyphenyl)-1H-pyrrol-2-yl] propanoic acid (ChemDiv_ID: 6238-0047, IC50 = 65.86 μM) was found to be the most effective urease inhibitor among the candidate compounds. Compound 6238-0047 significantly lowered the amount of urea degradation and ammonia production in ruminal microbial fermentation. The 24 h degradation rate of compound 6238-0047 in ruminal microbial fermentation was 3.32%–16.00%. In addition, compound 6238-0047 (10–100 μM) had no significant adverse effect on the cell viability of Caco-2 cells. Molecular docking showed that compound 6238-0047 could interact with Asp359 in the active site and Cys318 in the flap region by the hydrogen bond and Pi-Alkyl interaction, respectively. Compound 6238-0047 could be used as a novel inhibitor for decreasing the urease activity of ruminal microbiota.

Localization of aquaporin-3 proteins in the bovine rumen

Urea nitrogen salvaging is a crucial mechanism that ruminants have evolved to conserve nitrogen. Facilitative urea transporter-B proteins are known to be involved in urea transport across the rumen epithelium and thus efficiently facilitate the urea nitrogen salvaging process. Recently, functional studies have suggested that aquaglyceroporin transporters might also play a significant role in ruminal urea transport and aquaporin-3 (AQP3) protein has previously been detected in rumen tissue. In this current study, we investigated the specific localization of AQP3 transporters in the bovine rumen. First, end-point reverse-transcription PCR experiments confirmed strong AQP3 expression in both bovine rumen and kidney. Immunoblotting analysis using 2 separate anti-AQP3 antibodies detected AQP3 protein signals at 25, 32, and 42-45 kDa. Further immunolocalization studies showed AQP3 protein located in all the layers of rumen epithelium, especially in the stratum basale, and in the basolateral membranes of kidney collecting duct cells. These data confirm that AQP3 transporters are highly abundant within the bovine rumen and appear to be located throughout the ruminal epithelial layers. The physiological significance of the multiple AQP3 proteins detected and their location is not yet clear, hence further investigation is required to determine their exact contribution to ruminal urea transport.

A revised representation of urea and ammonia nitrogen recycling and use in the Molly cow model

Accurately predicting nitrogen (N) digestion, absorption, and metabolism will allow formulation of diets that more closely match true animal needs from a broad range of feeds, thereby allowing efficiency of N utilization and profit to be maximized. The objectives of this study were to advance representations of N recycling between blood and the gut and urinary N excretion in the Molly cow model. The current work includes enhancements (1) representing ammonia passage to the small intestine; (2) deriving parameters defining urea synthesis and ruminal urea entry rates; (3) adding representations of intestinal urea entry, microbial protein synthesis in the hindgut, and fecal urea-N excretion; and (4) altering existing urinary N excretion equations to scale with body weight and adding purine derivatives as a component of urinary N excretion. After the modifications, prediction errors for ruminal outflows of total N, microbial N, and nonammonia, nonmicrobial N were 29.8, 32.3, and 26.2% of the respective observed mean values. Prediction errors of each were approximately 7 percentage units lower than the corresponding values before model modifications and fitting due primarily to decreased slope bias. The revised model predicted ruminal ammonia and blood urea concentrations with substantially decreased overall error and reductions in slope and mean bias. Prediction errors for gut urea-N entry were decreased from 70.5 to 26.7%, which was also a substantial improvement. Adding purine derivatives to urinary N predictions improved the accuracy of predictions of urinary N output. However, urinary urea-N excretion remains poorly predicted with 69.0% prediction errors, due mostly to overestimated urea-N entry rates. Adding representations of undigested microbial nucleic acids, microbial protein synthesized in the hindgut, and urea-N excretion in feces decreased prediction errors for fecal N excretion from 21.1 to 17.1%. The revised model predicts that urea-N entry into blood accounts for approximately 64% of dietary N intake, of which 64% is recycled to the gut lumen. Between 48 and 67% of the urea recycled to the gut flows into the rumen largely depending on diet, which accounts for 29 to 54% of total ruminal ammonia production, and 65 to 76% of this ammonia-N is captured in microbial protein, which represents 17% of N intake. Based on model simulations, feeding a diet with moderately low crude protein and high rumen-undegradable protein could increase apparent ruminal N efficiency by 20%.

An evaluation of Molly cow model predictions of ruminal metabolism and nutrient digestion for dairy and beef diets

Model evaluation, as a critical process of model advancement, is necessary to identify adequacy and consistency of model predictions. The objectives of this study were (1) to evaluate the accuracy of Molly cow model predictions of ruminal metabolism and nutrient digestion when simulating dairy and beef cattle diets; and (2) to identify deficiencies in representations of the biology that could be used to direct further model improvements. A total of 229 studies (n = 938 treatments) including dairy and beef cattle data, published from 1972 through 2016, were collected from the literature. Root mean squared errors (RMSE) and concordance correlation coefficients (CCC) were calculated to assess model accuracy and precision. Ruminal pH was very poorly represented in the model with a RMSE of 4.6% and a CCC of 0.0. Although volatile fatty acid concentrations had negligible mean (2.5% of mean squared error) and slope (6.8% of mean squared error) bias, the CCC was 0.28, implying that further modifications with respect to volatile fatty acid production and absorption are required to improve model precision. The RMSE was greater than 50% for ruminal ammonia and blood urea-N concentrations with high proportions of error as slope bias, indicating that mechanisms driving ruminal urea N recycling are not properly simulated in the model. Only slight mean and slope bias were exhibited for ruminal outflow of neutral detergent fiber, starch, lipid, total N, and nonammonia N, and for fecal output of protein, neutral detergent fiber, lipid, and starch, indicating the mechanisms encoded in the model relative to ruminal and total-tract nutrient digestion are properly represented. All variables related to ruminal metabolism and nutrient digestion were more precisely predicted for dairy cattle than for beef cattle. This difference in precision was mostly related to the model's inability to simulate low forage diets included in the beef studies. Overall, ruminal pH was poorly simulated and contributed to problems in ruminal nutrient degradation and volatile fatty acid production predictions. Residual analyses suggested ruminal ammonia concentrations need to be considered in the ruminal pH equation, and therefore the inaccuracies in predicting ruminal urea N recycling must also be addressed. These modifications to model structure will likely improve model performance across a wider array of dietary inputs and cattle type.

Modulation of sheep ruminal urea transport by ammonia and pH.

Ruminal fermentation products such as short-chain fatty acids (SCFA) and CO2 acutely stimulate urea transport across the ruminal epithelium in vivo, whereas ammonia has inhibitory effects. Uptake and signaling pathways remain obscure. The ruminal expression of SLC14a1 (UT-B) was studied using polymerase chain reaction (PCR). The functional short-term effects of ammonia on cytosolic pH (pHi) and ruminal urea transport across native epithelia were investigated using pH-sensitive microelectrodes and via flux measurements in Ussing chambers. Two variants (UT-B1 and UT-B2) could be fully sequenced from ovine ruminal cDNA. Functionally, transport was passive and modulated by luminal pH in the presence of SCFA and CO2, rising in response to luminal acidification to a peak value at pH 5.8 and dropping with further acidification, resulting in a bell-shaped curve. Presence of ammonia reduced the amplitude, but not the shape of the relationship between urea flux and pH, so that urea flux remained maximal at pH 5.8. Effects of ammonia were concentration dependent, with saturation at 5 mmol/l. Clamping the transepithelial potential altered the inhibitory potential of ammonia on urea flux. Ammonia depolarized the apical membrane and acidified pHi, suggesting that, at physiological pH (< 7), uptake of NH4 (+) into the cytosol may be a key signaling event regulating ruminal urea transport. We conclude that transport of urea across the ruminal epithelium involves proteins subject to rapid modulation by manipulations that alter pHi and the cytosolic concentration of NH4 (+). Implications for epithelial and ruminal homeostasis are discussed.

Short communication: Urea transporter protein UT-B in the bovine parotid gland

Ruminant nutrition relies upon the symbiotic relationship that exists with microbial populations in the rumen. Urea transported across the ruminal epithelia and secreted by the salivary glands is a key source of nitrogen for microbial growth in the rumen. As ruminal urea transport can be mediated by specific UT-B urea transporters, this study investigated whether UT-B urea transporters were also present in the bovine salivary gland. Western blotting experiments detected only small amounts of UT-B protein in whole-cell lysate from the bovine parotid gland. In contrast, strong 32 to 34 and 40kDa UT-B proteins were detected in parotid plasma membrane-enriched protein, showing the importance of using enriched samples. These signals were also detected in rumen and correspond to bovine UT-B1 and UT-B2 urea transporters, respectively. Further immunolocalization studies identified that these proteins were located in the ductal system of the parotid gland. This study, therefore, confirmed the presence of UT-B urea transporter protein in the bovine parotid salivary gland.

Effect of time duration of ruminal urea infusions on ruminal ammonia concentrations and portal-drained visceral extraction of arterial urea-N in lactating Holstein cows

The effects of a 6 versus 24h ruminal urea infusion in lactating dairy cows fed a basal diet deficient in N on ruminal ammonia concentration, arterial urea-N concentration, net portal-drained viscera (PDV) urea-N flux, arterial urea-N extraction across the PDV, and renal urea-N kinetics were investigated. Three Danish Holstein cows fitted with ruminal cannulas and permanent indwelling catheters in major splanchnic blood vessels were randomly allocated to a 3 × 3 Latin square design with 21-d periods. Treatments were ventral ruminal infusion of water for 24h (water INF), 24-h infusion of 15g of urea/kg of dry matter intake (DMI; 24-h INF), and 6-h infusion of 15g of urea/kg of DMI (6-h INF). The 6-h INF was initiated 0.5h after the afternoon feeding, and ran until 2230h. Eight sample sets of arterial, portal, and hepatic blood, ruminal fluid, and urine were obtained at 0.5h before the morning feeding and 0.5, 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5h after feeding (i.e., 9 to 15.5h after the 6h infusion was terminated). A substantial decrease in DMI for 6-h INF compared with 24-h INF and water INF was observed, and it has to be recognized that DMI may have confounding effects. However, the experimental setting plan was met (i.e., to cause changes in the daily pattern of ruminal ammonia and blood urea-N concentrations). The arterial urea-N concentration for 24-h INF and 6-h INF were greater than the arterial urea-N concentration with water INF throughout the sampling window. However, the arterial urea-N concentration for 6-h INF decreased steadily with sampling time reflecting a carryover effect from the ruminal urea infusion. The ruminal ammonia concentration and net portal flux of ammonia for 6-h INF were not different from water INF; hence, no carryover effect on ruminal ammonia concentration was observed. The portal flux of urea-N was not affected by treatment (i.e., even the combination of low ruminal ammonia and high arterial urea-N concentration with 6-h INF was not used by the cow to increase the uptake of urea-N across the PDV). Arterial urea-N extraction across the PDV was increased with water INF especially from 0.5 to 3.5h postprandial relative to the urea infusion treatments, reflecting increased epithelial permeability for urea-N. This indicates that daily ruminal peak of ammonia or blood urea-N concentrations overruled potential signals from low ruminal ammonia concentration observed during the sampling window. In conclusion, dairy cows appear unable to increase transport of urea-N from blood to gut in periods with low ruminal ammonia concentrations, even in a situation with infrequent N supply and apparent carryover effects on blood urea-N. It is speculated that mechanisms responsible for downregulation of epithelial urea-N transport based on daily maximum concentrations of ammonia in the rumen or urea-N in the blood suppresses any short-term signal from low ruminal ammonia during periods with low ruminal N supply.

Effects of nitrogen supply on inter-organ fluxes of urea-N and renal urea-N kinetics in lactating Holstein cows

The effects of decreasing ruminal urea infusion in lactating dairy cows fed a basal diet deficient in rumen degradable protein on inter-organ urea-N fluxes, epithelial urea-N extraction, and renal urea-N kinetics were investigated. Eight Danish Holstein cows fitted with a ruminal cannula and permanent indwelling catheters in the major splanchnic blood vessels and the gastrosplenic vein were used. The cows were randomly allocated to a triplicate incomplete 3×3 Latin square design with 14-d periods. Treatments were continuous ventral ruminal infusion of water, 4.1g of feed urea/kg of dry matter intake, and 8.5g of feed urea/kg of dry matter intake. Dry matter intake and milk yield decreased linearly with decreasing urea infusion. Arterial blood urea-N and ruminal ammonia concentrations decreased linearly with decreasing urea infusion. In absolute amounts, the urea-N recycling did not increase when urea infusion was decreased. Arterial urea-N extraction across the portal-drained viscera and rumen wall increased linearly with decreasing urea infusion (2.46, 3.65, and 4.32±0.31% and 7.5, 11.5, and 16.9±0.9%, respectively), indicating that cows responded to the changes in N supply. The relative urea-N extraction across the ruminal wall increased compared with the total portal-drained viscera extraction. We observed a postprandial decrease in ruminal extraction of arterial urea-N that might reflect that the activity of the protein, presumably facilitating urea-N transport, is regulated by ruminal ammonia. The urea-N clearance by the kidneys decreased (35, 30, and 25±2L/h) and the urea-N reabsorbed by the kidney increased (42, 51 and 56±3%) with decreasing urea infusion, indicating that the kidneys salvaged urea-N with low-N supply. The urea transporter B mRNA abundance in rumen papillae (papillae harvested at sampling days) was not affected by dietary N supply. The study showed, that rumen wall extraction of arterial urea-N is subjected to both long- and short-term regulation. Extraction increases with decreasing N supply long-term; however, a short-term postprandial decrease in extraction was observed. No association between long-term adaptation of urea-N extraction across the rumen wall and urea transporter B mRNA abundance could be demonstrated.

UT-B is expressed in bovine rumen: potential role in ruminal urea transport

The UT-A (SLC14a2) and UT-B (SLC14a1) genes encode a family of specialized urea transporter proteins that regulate urea movement across plasma membranes. In this report, we describe the structure of the bovine UT-B (bUT-B) gene and characterize UT-B expression in bovine rumen. Northern analysis using a full-length bUT-B probe detected a 3.7-kb UT-B signal in rumen. RT-PCR of bovine mRNA revealed the presence of two UT-B splice variants, bUT-B1 and bUT-B2, with bUT-B2 the predominant variant in rumen. Immunoblotting studies of bovine rumen tissue, using an antibody targeted to the NH2-terminus of mouse UT-B, confirmed the presence of 43- to 54-kDa UT-B proteins. Immunolocalization studies showed that UT-B was mainly located on cell plasma membranes in epithelial layers of the bovine rumen. Ussing chamber measurements of ruminal transepithelial transport of (14)C-labeled urea indicated that urea flux was characteristically inhibited by phloretin. We conclude that bUT-B is expressed in the bovine rumen and may function to transport urea into the rumen as part of the ruminant urea nitrogen salvaging process.

Influence of the novel urease inhibitor N-(n-butyl) thiophosphoric triamide on ruminant nitrogen metabolism: II. Ruminal nitrogen metabolism, diet digestibility, and nitrogen balance in lambs.

Three lamb metabolism experiments were conducted to investigate the effects of chronic administration of the novel urease inhibitor N (n-butyl) thiophosphoric triamide (NBPT) on ruminal N metabolism, fermentation, and N balance. In Exp. 1, ruminally cannulated wethers (n = 28; 45.0 +/- .9 kg) were administered one of seven doses of NBPT (0 [control], .125, .25, .5, 1, 2, or 4 g of NBPT daily) and fed a common cracked corn/cottonseed hull-based diet twice daily containing 2% urea at 2.5% of initial BW for the duration of the 15-d experiment. Overall, NBPT decreased (linear P < .0001; quadratic P < .001) ruminal urease activity, resulting in linear increases (P < .0001) in ruminal urea and decreases in ruminal NH3 N concentrations. However, the detection of an NBPT x day interaction (d 2 vs 15; P < .01) indicated that this depression in urea degradation diminished as the experiment progressed. Increasing NBPT linearly decreased (P < .01) total VFA concentrations on d 2 of the experiment, but it had no effect (P > .10) on d 15. Increasing NBPT had no effect (P > .10) on DM or ADF digestibilities, but it linearly decreased (P < .01) N digestibility. Supplementing NBPT produced a linear increase (P < .05) in urinary N excretion and a linear decrease (P < .01) in N retention. In Exp. 2, ruminally cannulated wethers (n = 30; 46.8 +/- .6 kg) were fed one of two basal diets (2.0 vs 1.1% dietary urea) at 2.5% of initial BW and dosed with either 0 (control), .25, or 2 g of NBPT daily for the duration of the 15-d experiment. There were no NBPT x dietary urea interactions (P > .10) for Exp. 2. Increasing NBPT depressed (linear and quadratic P < .0001) ruminal urease activity, producing linear (P < .0001) increases in urea N and linear decreases in NH3 N in the rumen. As in Exp. 1, an NBPT x day interaction (P < .05) was noted for urea, NH3 N, and total VFA concentrations; the maximum response to NBPT occurred on d 2 but diminished by d 15 of the experiment. Administration of NBPT did not influence (P > .10) DM, ADF, or N digestibilities in Exp. 2. In Exp. 3, wether lambs (n = 30; 26.4 +/- .7 kg) were subjected to the same treatment regimen as in Exp. 2 for a 14-d N balance experiment. Although several NBPT x dietary urea interactions (P < .05) were noted, increasing NBPT did not affect (P > .10) N digestibility. Administration of NBPT quadratically increased (P < .10) urinary N excretion, producing a linear decrease (P < .05) in N retention. These results suggest that although NBPT is capable of inhibiting ruminal urease short-term, the ruminal microflora may be capable of adapting to chronic NBPT administration, thereby limiting its practical use in improving the utilization of dietary urea.