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ANIMAL NUTRITION |
* Department of Animal and Food Sciences, University of Kentucky, Lexington 40546-0215;
and
Agri-Nutrients Technology Group, Petersburg, VA 23803; and
University of Aarhus, Faculty of Agricultural Sciences, Tjele, DK-8830, Denmark
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: metabolism • nitrogen • nonprotein nitrogen • ruminant • steer • urea
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Increased blood ammonia concentrations alter hepatic metabolism by increasing ureagenesis and may also affect glucose metabolism in the liver and peripheral tissues (Spires and Clark, 1979; Fernandez et al., 1990; Huntington et al., 2006).
A potential way to minimize excess ammonia reaching the liver is to increase microbial utilization of ammonia by modulating its appearance in the rumen. To achieve this goal, some researchers have used microbial urease inhibitors, with mixed results (Whitelaw et al., 1991; Ludden et al., 2000). An alternate approach is to use slow-release urea (SRU) compounds such as biuret, urea phosphate, or urea bound to substrates like calcium chloride (Oltjen et al., 1968; Huntington et al., 2006). More recently, slow-release properties have been achieved by using coatings based on oil (Garrett et al., 2005) or polymers (Tedeschi et al., 2002; Galo et al., 2003) to control the release rate of ammonia from urea. Polymer-coated urea has been demonstrated to reduce ruminal ammonia concentrations compared with feedgrade urea (Taylor-Edwards et al., 2009). However, no research has examined the effects of a polymer-coated SRU on nutrient flux across the portal-drained viscera (PDV). Furthermore, more information regarding the effects of polymer-based SRU on N balance and total tract digestibility is needed.
Therefore, 2 experiments were designed to determine the behavior of SRU for ruminants. The objective of Exp. 1 was to determine the effects of ruminal SRU addition on ruminal fermentation and nutrient flux across the PDV. The objective of Exp. 2 was to determine the effect of feeding SRU on whole-body N balance and nutrient flux across the PDV in steers.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Experiment 1
Animals and Treatments.
Four Holstein steers (319 ± 5 kg of BW) were surgically prepared with ruminal cannulas approximately 6 mo before the experiment. Additionally, permanent indwelling catheters were inserted into the hepatic portal vein, hepatic vein, mesenteric vein, and mesenteric artery by procedures adapted from Katz and Bergman (1969) and Huntington et al. (1989) as described by McLeod et al. (1997). Catheters were prepared and maintained as described by Huntington et al. (1989). Steers were individually housed in stalls (2.4 x 2.4 m) during diet adaptation periods or metabolism tie stalls (1.2 x 2.4 m) during collection periods. Rooms were temperature (20°C) and light-controlled (12 h light: 12 h dark), and water was available for ad libitum consumption.
Steers were randomly assigned to treatment sequence within a crossover design with 2 treatments, urea or SRU (Agri-Nutrients Technology Group, Petersburg, VA). Treatment periods were 21 d in length with 20 d of diet adaptation and sampling on d 21. The basal diet (Table 1) contained corn silage (89.1% of diet DM) and ground corn-based vitamin and mineral supplement (9.0% of diet DM). Experimental diets (basal diet + treatment) were offered once daily at 1.5% of BW and were adjusted weekly for changes in BW and ingredient DM. Treatments were top-dressed onto the diet on d 1 to 20 and were intraruminally dosed on d 21.
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On d 21, p-aminohippuric acid (250 mM, pH 7.4; pAH) was infused continuously into the mesenteric vein catheter (approximately 1.3 mL/min) starting 1 h before feeding and continuing throughout the sampling period. After 1 h of infusion, blood was sampled to obtain a time 0 sample. Each blood sampling consisted of simultaneously collecting arterial, portal venous, and hepatic venous blood samples (10 mL each) into heparinized syringes. Immediately following the time 0 sample, steers were offered the basal diet and their daily aliquot of urea or SRU was dosed intraruminally and mixed thoroughly by hand. Blood samples were collected 0.5, 1, 2, 4, 6, 8, and 10 h after dosing. Samples were transferred to centrifuge tubes and placed on ice. Additionally, ruminal fluid (100 mL) was collected by suction strainer (Raun and Burroughs, 1962; 19-mm diameter, 1.6-mm mesh) at 0.5, 1, 2, 4, 6, 8, and 10 h after dosing.
Blood was centrifuged at 3,000 x g for 15 min at 4°C to obtain plasma, and an aliquot was immediately analyzed by membrane-immobilized enzymes (YSI Inc., Yellow Springs, OH) for L-lactate (coupled to L-lactate oxidase), D-glucose (coupled to glucose oxidase), L-glutamate (coupled to L-glutamate oxidase), and lglutamine (coupled to glutaminase and L-glutamate oxidase). Plasma urea-N concentrations were determined by automated analysis (AutoAnalyzer II, Technicon Industrial Systems, Tarrytown, NY) as described by Marsh et al. (1965). Remaining plasma was stored at –20°C for later analysis. For ruminal fluid samples, pH was measured immediately before aliquots (5 mL) were acidified with 1 mL of 25% (wt/vol) metaphosphoric acid and stored (–20°C).
Frozen plasma samples were thawed and assayed for pAH concentrations using procedures of Harvey and Brothers (1962) adapted for use on a Cobas Fara II centrifugal analyzer (Roche Diagnostic Systems, Montclair, NJ). The standard curve (4 to 125 mM) was developed from the pAH infusion solution for the corresponding sampling day. Frozen rumen fluid samples were thawed, centrifuged at 39,000 x g for 20 min at 4°C, and supernatant was collected for analysis of ammonia-N. Ammonia- N concentrations of plasma and ruminal fluid were determined enzymatically (coupled with glutamate dehydrogenase) using procedures adapted for use on a Cobas Fara II centrifugal analyzer (Roche Diagnostic Systems, Montclair, NJ).
Calculations and Statistical Analysis.
Means were generated for the 8 sampling times for pAH and metabolite concentrations in portal and hepatic venous and arterial blood samples. Blood flow was calculated as pAH infusion rate divided by the venoarterial pAH concentration difference across the respective vascular beds (Krehbiel et al., 1992). Net flux of metabolites across the PDV was calculated as the product of portal plasma flow and portal-arterial concentration difference. Net total splanchnic flux of metabolites was calculated as the product of hepatic plasma flow and hepatic-arterial concentration difference, and net hepatic flux of metabolites was calculated as the difference between net total splanchnic flux and net PDV flux. Hepatic extraction ratios were calculated as [1 – (hepatic output/hepatic input)], where hepatic output was (hepatic venous concentration x hepatic plasma flow), and hepatic input was [(portal plasma flow x portal nutrient concentration) + (arterial plasma flow x arterial nutrient concentration)]. A positive ratio indicates extraction or uptake by the liver, and a negative ratio indicates production by the liver.
Data were analyzed using the MIXED procedure (SAS Inst. Inc., Cary, NC). The statistical model for analyzing rumen variables and nutrient flux data included treatment, time, and the interaction between treatment and time as fixed effects and steer within period as a random effect, with time used as a repeated measure with steer within period as the subject. Treatment effects and treatment x time interactions were declared significant at P < 0.05, and tendencies were declared at P < 0.10.
Experiment 2
Animals and Treatments.
Four Holstein steers (236 ± 43 kg of BW) and 6 Angus steers (367 ± 46 kg of BW) were surgically fitted with permanent indwelling catheters in the hepatic portal vein, mesenteric vein, and mesenteric artery as described previously. Steers were housed as described for Exp. 1. Steers were randomly assigned to treatment sequence in a replicated crossover design with 2 treatments, urea or SRU (Agri-Nutrients Technology Group). Periods were 21 d, consisting of 14 d of diet adaptation followed by 7 d of collection. Total fecal and urine output was collected from d 15 to 20, and blood was sampled for nutrient flux measurements on d 21. Treatments were the sole source of supplemental N and were mixed into the basal diet at 1.6% of diet DM. The basal diet contained corn silage (88.4% of diet DM) and ground corn-based vitamin and mineral supplement (10% of diet DM). Experimental diets (basal diet + treatment) were offered as a total mixed ration twice daily (every 12 h) at 2.0% of BW daily and were adjusted weekly for changes in BW and ingredient DM. Ingredient and nutrient compositions of the experimental diet are shown in Table 1
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Sample Collection and Analysis. During the experiment, DM of all dietary ingredients was determined weekly for adjustment of amount of diet offered. Dry matters were determined in a forced-air oven at 55°C for 48 h. Feed ingredients were sampled daily, composited by week, and frozen for later analysis. Any orts were weighed, subsampled (12.5%), and frozen for later analysis.
Urine and feces were collected for five 24-h periods from d 15 to 20 for determination of N balance. Total urine output was collected by aspiration into aluminum kegs from a urine collection funnel as described by Archibeque et al. (2001). Urine kegs contained 500 mL of 11 N H3PO4 daily to maintain urine pH <3 in the keg (pH measured daily before sampling). Urine was collected daily, weighed, and a representative aliquot (1% by weight) was retained. The urine aliquot was pooled for each steer in a given period as a composite urine sample and was stored at –20°C. Feces were collected daily, weighed, and mixed well by hand before obtaining representative aliquots; one fecal aliquot (1% by weight) was pooled for each steer in a given period as a composite fecal sample and was stored at –20°C. Another aliquot of feces was dried with forced air at 55°C for 48 h for DM determination.
On d 21, steers were continuously infused with pAH (250 mM, pH 7.4) into the mesenteric vein catheter (approximately 1.1 mL/min) starting 1 h before the first sample and continuing throughout the sampling period. After 1 h of infusion, blood was sampled to obtain a time 0 sample. Each blood sampling consisted of simultaneously collecting 2 sets of arterial and hepatic portal blood samples into heparinized syringes; one set (25 mL each) for pAH and metabolite analysis and another set (3 mL each) for oxygen saturation and hemoglobin analysis. Immediately following the time 0 sample, steers were offered their respective experimental diet containing either urea or SRU. Blood samples were collected 2, 4, 6, 8, and 10 h after feeding.
Whole blood from sealed 3-mL syringes was immediately analyzed for O2 saturation and hemoglobin (Model OSM2 Hemoximeter, Radiometer America, Westlake, OH). Whole blood was subsampled for pAH analysis before centrifugation at 3,000 x g for 15 min at 4°C to obtain plasma. Whole blood and plasma were analyzed for pAH concentration by automated analysis (AutoAnalyzer II, Technicon Industrial Systems, Tarrytown, NY) according to Harvey and Brothers (1962). The standard curve for pAH assay was developed from the pAH infusion solution used that day. A plasma aliquot was immediately analyzed for L-lactate, D-glucose, L-glutamate, and L-glutamine by membraneimmobilized enzymes as described previously (YSI Inc., Yellow Springs, OH). Plasma was analyzed for urea-N by automated analysis (AutoAnalyzer II, Technicon Industrial Systems, Tarrytown, NY) as described previously. Remaining plasma was stored at –20°C for later analysis.
Composite samples of diet ingredients and orts were dried in a forced-air oven at 55°C for 48 h to determine DM concentration. Corn silage and orts samples were ground with a Wiley mill (1-mm screen; Authur H. Thomas, Philadelphia, PA), and samples of supplement, urea, and SRU were ground through a 1-mm screen (Cyclotec 1093 Sample Mill; Tecator, Hoganas, Sweden). Dried fecal samples were ground to pass a 2-mm screen and were pooled for each steer in a given period. Dried diet ingredient and ort samples were analyzed for DM, ash, CP, NDF, and ADF. Ash concentration was determined after 5 h of oxidation at 500°C in a muffle furnace. Crude protein (N x 6.25) was determined by analyzing N content with a Leco FP-2000 (Leco Corp., St. Joseph, MI) N analyzer (AOAC, 1995). Concentrations of ADF and NDF were determined according to Van Soest et al. (1991; method A) using an Ankom200 Fiber Analyzer (Ankom Technology, Macedon, NY). Dried fecal composites were analyzed for DM, ash, NDF, and ADF as described previously. Concentrations of all nutrients except DM were expressed as percentages of DM determined by drying at 70°C in a vacuum oven for more than 8 h.
Frozen fecal and urine composite samples were mixed well and analyzed for N content with a Leco FP-2000 (Leco Corp., St. Joseph, MI) N analyzer (AOAC, 1995). Urinary urea concentrations were determined on composite urine samples by automated analysis as described previously (AutoAnalyzer II, Technicon Industrial Systems, Tarrytown, NY). Plasma samples were assayed for ammonia-N (coupled with glutamate dehydrogenase) and β-hydroxybutyrate (coupled with β-hydroxybutyrate dehydrogenase) concentrations using procedures adapted for use on a Konelab 20XTi Analyzer (Thermo Electron Corporation, Waltham, MA).
Calculations and Statistical Analysis. Nutrient intake was calculated using the amounts and compositions of feed offered and refused and total tract digestibility was calculated. Blood flow and nutrient fluxes were calculated as described for Exp. 1.
Data were analyzed using the MIXED procedure (SAS Inst. Inc.). The statistical model for analyzing nutrient intake and total tract digestibility included treatment as a fixed effect and square, steer within square, and period within square as random effects. The statistical model for analyzing N balance and flux data included treatment, time, and the interaction between treatment and time as fixed effects and square, steer within square, and period within square as random effects, with time used as a repeated measure with steer within period and square as the subject. Treatment effects and treatment x time interactions were declared significant at P < 0.05, and tendencies were declared at P < 0.10.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Intraruminal dosing of urea resulted in a rapid (within 0.5 h) and marked increase in ruminal ammonia concentrations, whereas dosing with SRU did not substantially affect ruminal ammonia concentrations (treatment x time P = 0.001; Figure 1A), which resulted in greater mean ruminal ammonia concentrations for urea compared with SRU (12.9 vs. 3.6 mM; P = 0.01). Ruminal pH tended to increase 4 h after dosing for the urea treatment compared with SRU (treatment x time P = 0.07; Figure 1B), likely as a result of the greater ruminal ammonia concentrations for the urea treatment because pH was positively correlated with ruminal ammonia concentrations (r = 0.88; P = 0.0001) across treatment x time means.
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. Dosing steers with SRU reduced mean arterial urea concentrations by 32% compared with urea treatment (2.15 vs. 3.18 mM; P = 0.004) because arterial urea concentrations increased more rapidly and to a greater extent for steers dosed with urea vs. SRU (treatment x time P < 0.001). Arterial ammonia concentrations rose rapidly (within 0.5 h) for steers dosed with urea and did not return to baseline until 8 h after dosing, but arterial ammonia concentrations for steers dosed with SRU were similar to 0 h across the entire 10-h sampling period (treatment x time P < 0.001), which resulted in 75% less mean arterial ammonia concentrations for SRU compared with urea (P = 0.009). Arterial glutamate and glutamine concentrations were not affected by treatment (P > 0.16), although there was a tendency (P = 0.10) for a treatment x time interaction for arterial glutamate concentrations; arterial glutamate concentration increased slightly through 4 h after dosing, then decreased to below baseline by 10 h for steers dosed with SRU, whereas arterial glutamate concentrations slowly decreased over the entire 10-h sampling period for steers dosed with urea. Although mean arterial glucose concentrations were not affected by treatment (P = 0.22), urea treatment numerically increased arterial glucose concentrations at 1, 2, and 4 h after dosing compared with SRU, but concentrations were not different at other time points (treatment x time P = 0.14). Compared with SRU, urea treatment increased (P = 0.03) arterial lactate concentrations.
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. There were no differences in portal or hepatic plasma flow (P > 0.83). Net portal urea flux of steers dosed with urea rose sharply at 0.5 h after dosing so that urea was released by the PDV before returning to baseline by 2 h after dosing; in contrast, SRU-dosed steers maintained a consistent uptake of urea by the PDV over the 10-h sampling period (treatment x time P = 0.005; Figure 2A). Hepatic urea flux did not differ between treatments (P = 0.12), although urea treatment numerically increased hepatic urea release by 50% compared with SRU treatment (Figure 2B). Therefore, mean net total net splanchnic urea release was greater for urea- versus SRU-dosed steers (124 vs. 56 mmol/h; P = 0.007). In addition, total net splanchnic urea release exhibited a spike similar to that for net portal urea flux at 0.5 h after dosing for the urea treatment but not the SRU treatment, although this effect was not significant (treatment x time P = 0.42; Figure 2C). Hepatic extraction ratio of urea did not differ between treatments (P = 0.77; Figure 2D).
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). Intraruminal dosing of urea increased net hepatic ammonia uptake rapidly (within 0.5 h) and extensively (maximum 358 mmol/h); in contrast, intraruminal dosing of SRU gradually increased net hepatic ammonia uptake, reaching a maximum uptake 2 h after dosing of 224 mmol/h (treatment x time P = 0.02; Figure 2F). Total splanchnic ammonia flux varied little from the overall mean of –2 mmol/h over the 10-h sampling period for SRU treatment, but for urea treatment total splanchnic ammonia flux exhibited a substantial peak at 0.5 h (treatment x time P = 0.006; Figure 2G). Hepatic extraction ratio of ammonia was greater (0.83 vs. 0.70; P = 0.008) for SRU than urea treatment because urea treatment markedly decreased hepatic ammonia extraction ratio at 0.5, 1, 2, and 4 h after dosing (treatment x time P = 0.003; Figure 2H).
There was no effect of treatment on the net portal, hepatic, or total splanchnic fluxes (Table 3) or hepatic extraction ratios (data not shown) of glutamate, glutamine, or glucose (P > 0.24). Likewise, net portal flux of lactate was not affected by treatment (P = 0.87). Net hepatic lactate uptake was 41% greater for urea than SRU treatments because of a substantial increase in hepatic lactate uptake at 1 and 2 h after dosing (treatment x time P = 0.02). This is also reflected in a treatment x time effect (P < 0.001) for hepatic extraction ratio of lactate because urea treatment increased hepatic extraction of lactate 1 and 2 h after dosing but was not different from SRU treatment at other time points (overall mean of 0.17 and 0.14 for urea and SRU, respectively). There was a tendency for total splanchnic lactate uptake to be up to 44 times greater for urea treatment than SRU (treatment x time P = 0.07) because of substantial lactate uptake by the splanchnic tissue bed, especially in the first 2 h after dosing.
Experiment 2
Arterial metabolite concentrations are shown in Table 2. Mean arterial urea concentrations were 41.6% greater for steers consuming urea compared with SRU (P = 0.03). Additionally, although the peak in arterial urea concentrations occurred 4 h after feeding for both treatments, peak arterial concentrations were only 23% above baseline for SRU but 51% above baseline for urea treatment (treatment x time P = 0.007). Arterial ammonia concentrations did not differ among treatments (P = 0.41). Relative to urea, SRU tended to increase arterial concentrations of glutamate by 15.2% (P = 0.10) but decreased arterial concentrations of glutamine by 16.1% (P = 0.01). Treatment did not affect arterial concentrations of O2, glucose, lactate, or β-hydroxybutyrate (P > 0.16).
Portal blood and plasma flow and metabolite flux data are shown in Table 4. There was a tendency for blood flow to increase more following feeding immediately after the 0 time sample for the SRU versus urea treatment (treatment x time P = 0.06); however, this effect is likely due differences in feed intake patterns and not treatment per se. Portal plasma flow was not affected by treatment (P = 0.85).
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Nutrient intakes and apparent total tract digestibilities are presented in Table 5. Intake of DM, OM, NDF, and ADF did not differ among treatments (P > 0.61). Additionally, apparent total tract digestibility of DM, OM, NDF, and ADF was not affected by treatment (P > 0.53). However, apparent total tract digestibility of N was less for SRU compared with urea treatment (61.7 vs. 66.0%; P = 0.003). Greater N excretion for steers consuming SRU is also reflected in the N balance data presented in Table 5. Steers fed SRU excreted more N in the feces than steers fed urea (49.6 vs. 45.6 g/d, P = 0.04). There were no differences among treatments for urinary N excretion (g/d or % N intake; P > 0.56) or urinary urea excretion (mean 61.7 g/d, P = 0.19, data not shown). Nitrogen retention (g/d) tended to be greater (P = 0.08) for urea vs. SRU treatment; however, because N intake (g/d) was numerically greater for urea treatment, there were no differences when N retention was expressed as a % of N intake (P = 0.18).
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Urea undergoes rapid hydrolysis in the rumen to ammonia as demonstrated in Exp. 1; mean ruminal ammonia concentrations were 263% greater for steers dosed intraruminally with urea than steers dosed with SRU primarily because ruminal ammonia concentrations for urea treatment rose markedly within 0.5 h of dosing. This rapid rise in ammonia concentrations for urea treatment was substantial enough to increase ruminal pH by over 0.5 units within 0.5 h of dosing. Indeed, ruminal pH and ruminal ammonia concentrations were positively related (r = 0.88), an effect that has been observed previously (Bartley et al., 1976; Puga et al., 2001). Additionally, ruminal ammonia concentrations remained greater for steers dosed with urea than those dosed with SRU until 8 to 10 h after dosing. These results demonstrate that in vivo SRU does indeed have a slower release rate of ammonia than urea and can effectively modulate ruminal ammonia concentrations when substituted for urea.
Apparent total tract digestibilities of DM, OM, NDF, and ADF were not affected by treatment, which is consistent with several experiments that found that substitution of SRU products for urea did not affect DM or OM digestibility (Oltjen et al., 1968; Owens et al., 1980; Currier et al., 2004). In contrast to our findings, another polymer-coated SRU (Optigen; CPG Nutrients, Syracuse, NY) has been demonstrated to increase total tract DM and CP digestibilities when fed to lactating dairy cows (Galo et al., 2003). In our experiment, SRU reduced apparent total tract N digestibility compared with urea. Despite the increase in fecal N excretion with SRU treatment, overall N retention was not affected by treatment.
In Exp. 2, increased fecal N excretion for steers fed SRU suggests that the coating may hinder full release of urea and some SRU may pass through the digestive tract. Another possible explanation is that the slowrelease nature of the SRU product may increase fecal N excretion by supporting hindgut microbial growth. This could be the result of greater nutrient availability (including N from SRU) in the lower gastrointestinal tract, especially if ruminal OM digestion were reduced with SRU. Greater hindgut nutrient supply has been associated with greater fecal N excretion and numerically greater fecal bacterial counts and urea irreversible loss rate, suggesting a transfer of urea to the gastrointestinal tract with increasing hindgut microbial fermentation (Oncuer et al., 1990). The results from Exp. 2 observing increased urea transfer to the gastrointestinal tract from blood in combination with greater fecal N excretion with SRU could support the concept of increased hindgut fermentation.
Replacement of urea with SRU did not affect ruminal VFA patterns or concentrations in a similar experiment (Taylor-Edwards et al., 2009), suggesting that SRU does not negatively affect ruminal fermentation. Additionally, the ability of SRU to modulate ruminal ammonia release is possibly beneficial to more effectively match availability of other nutrients necessary for microbial growth. Conversely, despite the lack of treatment effect on DM, OM, NDF, and ADF total tract digestibilities, there is a possibility that if SRU limits ruminal N availability, some nutrient digestion could be shifted from the rumen to the hindgut without affecting total tract digestibilities or ruminal VFA patterns or concentrations, as observed by Herrera-Saldana et al. (1990). Finally, another unknown aspect is how the coating on SRU affects ruminal fermentation of nutrients. Therefore, research investigating the effects of SRU on site of nutrient digestion would seem warranted.
Blood Nutrient Concentrations and Portal, Hepatic, and Total Splanchnic Nutrient Fluxes
The reduction in ruminal ammonia concentrations when SRU replaced urea was also reflected in treatment effects on net portal flux of ammonia, which was reduced by SRU in both Exp. 1 and 2. In Exp. 1, urea dosing increased net portal ammonia flux by 336, 184, and 79% at 0.5, 1, and 2 h, respectively, compared with steers dosed with SRU. A similar (albeit smaller) treatment effect was observed 2 h after feeding in Exp. 2, and for both experiments net portal ammonia flux was similar between treatments by 4 h after treatment administration. Portal ammonia flux was positively correlated with ruminal ammonia concentrations (r = 0.83), in agreement with the correlation of 0.82 observed by Huntington et al. (1983) for net absorption of ammonia and ruminal ammonia concentrations. Greater portal flux and arterial concentrations of ammonia after intraruminal dosing with urea are also likely at least partly related to the increased ruminal pH that occurred in response to ruminal dosing, as portal ammonia flux was also positively correlated with ruminal pH (r = 0.76). At greater ruminal pH, more ammonia exists in the lipophilic form that can penetrate biological membranes via simple diffusion, rather than the predominant (membrane impermeable) ammonium form that exists at decreased ruminal pH and requires absorption via a channel (Abdoun et al., 2006). Therefore the greater absorption of ammonia that occurred as ammonia concentrations in the rumen increased may be related to both pH and concentration gradient.
Despite the increase in hepatic ammonia uptake (especially for the first 4 h after dosing) for urea treatment, increased liver uptake did not compensate for the increased portal ammonia flux so that net splanchnic flux of ammonia was still substantially greater for urea compared with SRU. This difference is significant because net splanchnic flux of ammonia is rarely positive (Huntington and Archibeque, 1999). Additionally, urea dosing numerically increased hepatic urea flux compared with SRU, and in combination with the substantial increase in portal urea flux at 0.5 h after dosing with urea, total splanchnic urea flux was significantly greater for urea treatment than SRU and exhibited the same spike as portal urea flux at 0.5 h after dosing. Although urinary excretion was not measured in Exp. 1, increased urea output from the PDV and liver would also be expected to increase urea excretion by the kidneys.
One interesting observation was the relationship between hepatic urea flux and portal ammonia flux. Hepatic urea flux was positively related (r = 0.50) with portal ammonia flux until portal ammonia flux reached approximately 300 mmol/h, at which point hepatic urea flux appeared to plateau despite even greater flux of ammonia to the liver (r = 0.01). This relationship may suggest that when portal ammonia flux exceeds 300 mmol/h (which occurred at 0.5, 1, and 2 h after dosing of urea but not SRU treatment), the capacity of the liver to remove ammonia for urea production was surpassed, thus contributing to greater plasma ammonia concentrations. Likewise, total splanchnic ammonia flux increased only slightly (from –23.6 to 15.5 mmol/h) as portal ammonia flux increased from 73.9 to 295 mmol/h (r = 0.30); however, when portal ammonia flux increased above approximately 300 mmol/h, total splanchnic ammonia flux markedly and rapidly increased (r = 0.91). Furthermore, there was a marked reduction in hepatic extraction ratio of ammonia for the urea treatment during these same time points.
Hepatic extraction ratio differences between treatments and the relationships of portal ammonia flux with total splanchnic ammonia flux and hepatic urea flux suggest that at 0.5, 1, and 2 h after dosing of urea treatment, ammonia entering the liver may have exceeded the removal capacity of the liver. The observed 300 mmol/h breakpoint is equivalent to 0.94 mmol·kg of BW–1·h–1 or 67.2 mmol·kg of wet liver wt–1·h–1 (if it is assumed that the liver is 1.4% of BW). In examining hepatic ammonia extraction rates reported in the literature, Baird et al. (1975) reported hepatic ammonia clearance rates of 1.55 mmol·kg of BW–1·h–1 and Symonds et al. (1981) reported that the maximum capacity of the liver to remove first-pass ammonia was approximately 110.4 mmol·h–1·kg of wet liver weight–1. Although these estimates are substantially greater than the breakpoint that we observed in Exp. 1, their estimated clearance rates were obtained in adult lactating dairy cows versus the young steers in our experiment. Because the liver of lactating cows is approximately twice as large as a steer when expressed on a metabolic BW basis (g/BW0.75; Johnson et al., 1990), the potential maximal clearance rate of ammonia in a lactating cow is likely greater than in the steers of our experiment. Hepatic ammonia clearance estimates of 46 to 64 mmol·kg of wet liver wt–1·h–1 were reported by Lobley et al. (1995) during NH4Cl infusion in sheep and are similar to our observations. Although the maximum capacity for ammonia clearance by the liver likely changes with physiological state, diet, and other factors, our observation that hepatic clearance of ammonia is exceeded at a portal ammonia flux of approximately 300 mmol/h is plausible. However, it is important to note that although portal ammonia flux may have temporarily exceeded hepatic capacity for removal, recovery occurred relatively rapidly; indeed, Symonds et al. (1981) remarked that rate (rather than amount) of ammonia input to the liver was the major determinant of hepatic ammonia clearance capacity.
The escape of ammonia past the liver for the first 2 h after urea treatment dosing certainly contributed to greater plasma ammonia concentrations. Intraruminal dosing of SRU reduced mean arterial ammonia concentrations by 75% in Exp. 1 compared with urea treatment because dosing of urea elevated arterial ammonia concentrations for 4 h after dosing with a peak of 0.46 mM at 1 h after dosing. Greater plasma ammonia concentrations observed with urea treatment may be the result of ammonia flux exceeding liver capacity for removal, as discussed previously, but may also be the result of greater diffusion of ammonia from the gastrointestinal tract directly into peripheral blood, thus bypassing the liver, especially at high ruminal ammonia concentrations (Chalmers et al., 1971). The concern regarding plasma ammonia concentrations relates to the rapidly toxic effects that ammonia can have on physiological processes, including central nervous system aberrations and death. In nonadapted steers, ammonia toxicity occurred when blood ammonia concentrations reached 0.47 to 0.52 mM within 1 h of dosing (Bartley et al., 1976). The peak plasma ammonia concentration of 0.46 mM observed 1 h after dosing of urea treatment in Exp. 1 would be considered toxic if sustained.
Although elevated plasma ammonia concentrations can cause significant health problems at toxic values, subclinical ammonia toxicity may still cause physiological aberrations in intermediary metabolism, especially of glucose. Infusion of ammonium chloride to achieve subclinical toxicity resulted in plasma ammonia concentrations ranging from 271 to 327 µg/dL during a 240-min infusion period and was associated with an increase in plasma glucose concentrations (Fernandez et al., 1990). Likewise, greater glucose concentrations have been positively associated with ammonia concentrations (Chalupa and Opliger, 1969; Spires and Clark, 1979). This increase in plasma glucose concentrations that occurs within 2 h in response to ammonia has been attributed at least partially to a reduction in glucose utilization rate (Spires and Clark, 1979) or increased net hepatic glucose production (Huntington et al., 2006) or both, possibly because of an increased rate of hepatic glycogenolysis (Spires and Clark, 1979). Although toxic effects of urea feeding were not observed in either Exp. 1 or 2, plasma glucose concentrations numerically increased 1 to 4 h after dosing for urea treatment in Exp. 1 and may indicate that in the 4 h after dosing of urea treatment steers were experiencing subacute ammonia toxicity. These results are in agreement with another experiment in which urea-calcium, a slow-release form of urea, prevented the marked increase in plasma glucose observed with dosing of urea treatment (Huntington et al., 2006). Slow-release urea forms may diminish or abolish the aberrations in glucose homeostasis observed under conditions in which plasma ammonia concentrations are elevated.
However, in normal urea feeding practices, peripheral plasma ammonia concentrations do not demonstrate a rapid peak. Feeding diets varying in hay and casein hydrolysate increased ruminal ammonia concentrations up to 50 mM and increased portal ammonia concentrations up to 0.8 mM but did not increase jugular ammonia concentrations (Lewis et al., 1957). Results from Exp. 2 are in agreement; feeding either urea or SRU did not affect either arterial ammonia or glucose concentrations over the entire sampling period. However, in the same experiment by Lewis et al. (1957), addition of ammonium acetate in the rumen led to a rapid and parallel increase in carotid arterial ammonia concentrations once portal blood concentrations exceeded 0.8 mM. Also in agreement, we observed in Exp. 1 that arterial ammonia concentrations averaged 0.06 mM when portal ammonia concentrations were less than 0.53 mM, but when portal ammonia concentrations increased above this point arterial ammonia concentrations increased to an average of 0.44 mM (range of 0.14 to 0.68 mM); these increased concentrations occurred only at 0.5, 1, 2, and 4 h after dosing of urea but not SRU in Exp. 1. Although exposure to a urea dosage such as administered in Exp. 1 (intraruminal full day aliquot) would be a rare occurrence, it is possible in cases of mixing errors or extremely rapid consumption of a supplement. In this case SRU provides a margin of safety to avoid the potentially toxic concentrations of plasma ammonia because our results demonstrate the ability of coated urea to release ammonia slowly and without peaks in ammonia and urea concentrations associated with feeding feed-grade urea.
Recycling of N in the ruminant is an important mechanism to provide degradable N to the gastrointestinal microbes. Thus, portal urea flux is usually negative because of urea transfer from the blood to the gastrointestinal tract. However, intraruminal dosing of urea caused a large positive spike in portal urea flux 0.5 h after dosing, indicating that urea was actually being removed from the gastrointestinal tract by the blood. Slow-release urea completely prevented this peak in portal urea flux and maintained a consistent PDV uptake of urea throughout the 10 h sampling period. The transient transfer of urea from the gastrointestinal tract to the blood with urea treatment has also been observed in steers dosed with either urea or urea-calcium and occurs within the first 0.5 h after dosing (Huntington et al., 2006). In contrast to Exp. 1, PDV urea uptake occurred in Exp. 2 for both urea and SRU treatments over the 10-h sampling period; however, SRU numerically increased urea transfer to the gastrointestinal tract from 4 to 10 h after feeding by 18.9%, whereas in Exp. 1 PDV urea uptake did not differ among treatments during the same time period.
Recycling of N from the blood to the gastrointestinal tract varies from 10 to 42% of N intake (Huntington, 1986) and depends on many factors. Rate of recycling is negatively related to ruminal ammonia concentrations and positively related to ruminal OM digestibility and plasma urea concentrations (Kennedy and Milligan, 1980). Substitution of urea with SRU reduced arterial urea concentrations by 32 and 29% in Exp. 1 and 2, respectively, by reducing the rate and extent of urea appearance in arterial blood. Therefore, it would be expected that N recycling would be decreased for SRU because of decreased arterial urea concentrations. However, the inhibitory effect of high ruminal ammonia concentrations on urea transfer to the gastrointestinal tract may be removed when SRU replaces urea. The decreased and more consistent ruminal ammonia concentrations with SRU, as demonstrated in Exp. 1, probably allowed an increase in transfer of urea to the gastrointestinal tract. Also, transfer of urea to the postruminal gastrointestinal tract may be more significant that previously recognized (Nolan and Leng, 1972; Huntington, 1986) and may support greater postruminal fermentation, as discussed previously.
In conclusion, SRU reduces the rapidity of ammonia- N release and hepatic processing of ammonia and urea. These experiments demonstrate that available technology effectively controls the release of ammonia from urea in the rumen. In the present experiments there were no detrimental effects on DM and fiber digestibility associated with feeding a SRU. However, increased fecal N excretion and increased urea transfer to the gastrointestinal tract suggest SRU may not be fully hydrolyzed in vivo or may affect digestion patterns in the gastrointestinal tract. Despite reducing ruminal ammonia fluctuations, release of N from SRU may not be optimal for ruminal utilization. Further research is needed to determine if site of nutrient digestion and ruminal N availability are affected by SRU products. Any negative effects of urea that may occur with mixing errors or rapid urea consumption can be completely prevented by SRU, thus providing the producer with insurance against ammonia toxicity when feeding nonprotein N supplements.
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2 Corresponding author: dharmon{at}uky.edu
Received for publication January 29, 2008. Accepted for publication August 31, 2008.
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) on ruminal ammonia concentrations (A) and ruminal pH (B) of steers in Exp. 1 (n = 4). Treatment differences (P < 0.05) are indicated by *.