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ANIMAL NUTRITION |
* Department of Animal Sciences and Industry, and
Department of Clinical Sciences, Kansas State University, Manhattan 66506-1600
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: cattle • protein • recycling • urea
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Lee et al., 1985; Köster et al., 1996) and improves animal performance (Mathis et al., 1999). Protein supplementation accomplishes this by providing a source of ruminally available N (RAN). Ruminally available N is used along with fermentable OM by ruminal microbes to synthesize nitrogenous compounds, allowing the microbes to grow. Increased microbial activity improves the energy status of the animal via increased VFA production and improves the protein status by increasing microbial N flow to the duodenum (Scott and Hibberd, 1990; Köster et al., 1996). The contribution of a protein supplement to the RAN pool is typically accounted for as the amount of rumen-degradable intake protein (DIP) contained in the supplement. However, the ability of N recycling to contribute to RAN is documented not only by experiments designed to measure N recycling (Houpt, 1959; Norton et al., 1982; Huntington, 1989), but also by the net appearance of N between the mouth and the duodenum in animals fed low-quality forage (Kropp et al., 1977; Hunter and Siebert, 1980; Currier et al., 2004). Recent advances in methodologies to measure urea kinetics (Sarraseca et al., 1998; Lobley et al., 2000) allow for the determination of urea kinetics in a relatively noninvasive manner. This method, however, fails to provide a satisfactory measure of how much urea recycling contributes to meeting RAN requirements, because estimates of urea recycling are for urea recycled not only to the rumen but to the entire gut. By combining a urea kinetics determination with a duodenal flow study, the incorporation of recycled urea-N into microbial N can be measured directly (Wickersham, 2006). This study was designed to measure the contribution of N recycling to meeting microbial N requirements when graded amounts of supplemental DIP were provided. |
MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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This study evaluated the effects of increasing amounts of supplemental DIP on urea kinetics and recycled urea-N use by ruminal bacteria in steers consuming low-quality forage. Five duodenally and ruminally fistulated Angus x Hereford steers (278 ± 19 kg of initial BW) were used in a 4 x 4 balanced Latin square with an additional column (steer) included. All data from 1 observation were lost because of problems unrelated to treatment. Steers were provided ad libitum access to fresh water and a trace mineral-salt block (composition: 
96.0% NaCl, 0.16% Fe, 0.40% Zn, 0.32% Mn, 0.01% I, 0.04% Cu, and 0.004% Co; American Stockman, Overland Park, KS) and were offered tallgrass-prairie hay—composed primarily of big bluestem (Andropogon gerardii Vitman), little bluestem [Schizachyrium scoparium (Michx.) Nash], and Indiangrass [Sorghastrum nutans (L.) Nash]—at 0630 h each day (Table 1) at 130% of average voluntary intake for the preceding 4 d. The tallgrass-prairie hay was baled in late summer and processed through a small bale slicer.
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) dosed ruminally once daily at 0620 h to provide the supplemental DIP. Casein was dosed by removing approximately 1 kg of ruminal contents, placing the dry casein in the rumen, and then returning the ruminal contents to the rumen. Casein was selected as a protein supplement because of its high protein content, high ruminal degradability, and the absence of other components (i.e., carbohydrates) that are found in conventional protein supplements. Supplemental protein amounts were based on previous research that used forage of a similar type and quality, and the amount of 177 mg of N/kg of BW daily was near the DIP requirement for maximum forage utilization (Köster et al., 1996; Klevesahl et al., 2003; Wickersham et al., 2004).
Experimental procedures used general methodologies and adaptation periods validated by Wickersham (2006). Experimental periods were 13 d long, with 7 d for adaptation to treatments and 6 d for collection. For the first 4 d of adaptation, steers were housed in individual pens (1.5 x 3.1 m). For the remainder of the adaptation and throughout the collection period, steers were placed in metabolism crates to facilitate the total collection of urine and feces and the jugular infusion of 15N15N-urea. Metabolism crates were designed such that urine was collected into a funnel directly beneath the midportion of the steer and subsequently diverted into a bucket by using gravity, whereas feces were collected into a pan placed directly behind the steer. Water was poured periodically into the funnel to aid in the complete collection of urine and to remove any impediments to flow.
On d 9 an indwelling catheter was placed in the jugular vein of each steer to infuse 15N15N-urea for the determination of urea kinetics. The 15N15N-urea solution was prepared by combining 1.20 g of 15N15N-urea (Medical Isotopes Inc., Pelham, NH) with 1 L of sterile saline solution (0.9% NaCl) and was then filtered (0.22 µm filter unit, Sterivex, Millipore Corporation, Billerica, MA). Saline solution was infused continuously from the time the catheter was placed until 0600 h on d 10, when infusion of the 15N15N-urea solution began. The 15N15N-urea solution was infused continuously until the end of the experimental period on d 13 at approximately 4 mL/h, which delivered 0.154 mmol of urea-N/h via a syringe infusion pump (Harvard Apparatus, South Natick, MA). Total collections of urine and feces from d 9 were used to determine background enrichments of 15N. Total collections of urine and feces from d 12 were used to measure enrichments for calculating urea kinetics. On d 13 ruminal fluid samples were collected by suction strainer (Raun and Burroughs, 1962; 19 mm diameter, 1.5 mm mesh) just before treatments were administered (0 h) and at 4, 8, 12, 16, and 20 h after supplements were dosed. Ruminal fluid (4 mL) from each collection was combined with 1 mL of 1 M HCl and frozen for NH3 and VFA analysis. On d 13, whole ruminal contents (1 kg) and duodenal samples (300 mL) were collected before feeding (0 h) and at 4, 8, 12, 16, and 20 h after supplements were dosed to determine duodenal flows and incorporation of recycled urea-N into microbial protein. To isolate ruminal bacteria from the whole ruminal contents, 0.5 L of 0.9% NaCl solution was added immediately after the sample was collected, blended (5 min; NuBlend, Waring Commercial, Torrington, CT), and strained through 2 layers of cheesecloth. The liquid fraction was frozen immediately and the material remaining in the cheesecloth was placed in the rumen. Blood was collected from the jugular vein opposite the catheter into heparinized Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) 12 h after feeding on d 13. Samples were placed in ice water immediately after collection and centrifuged at 5,000 x g for 15 min within 1 h after collection. Plasma was frozen for subsequent determination of plasma urea-N concentration.
Calculations of intake, digestion, and N balance were made from observations on d 8 through 12. Feed and ort samples were collected on d 8 through 11 to correspond with fecal and urine samples collected on d 9 through 12. Duodenal flows were based on samples from d 13, with acid detergent insoluble ash (ADIA) serving as an internal marker. Hay was sampled as it was being fed, with 400 g of hay retained each day for later analysis. Orts were removed at 0600 h daily, and approximately 200 g was retained for analysis. Fecal bins and urine buckets were removed and contents were weighed at 0615 h daily. Feces collected over each 24-h period were mixed thoroughly, and 3% was sampled and frozen (–20°C) for subsequent analysis. Urine collected over each 24-h period was mixed thoroughly and 2% was retained as a sample and subsequently frozen (–20°C). Urine pH was maintained below 3 by adding 400 mL of 6 M HCl to urine containers before collection.
Laboratory Analyses
Hay, ort, and fecal (used for fecal output determination) samples were dried at 55°C in a forced-air oven for 96 h, air-equilibrated, and weighed to determine partial DM. Duodenal samples were frozen and lyophilized. Subsequently, all dried samples were ground with a Wiley mill to pass a 1-mm screen. Hay samples collected during the measurement period were pooled across days on an equal weight basis. Ort and fecal samples were composited by steer across days. The DM of hay, casein, ort, fecal, and duodenal samples was determined by drying for 24 h at 105°C in a forced-air oven, and OM was determined as loss in dry weight upon combustion for 8 h at 450°C in a muffle furnace. Nitrogen content of hay, casein, wet feces, urine, and duodenal digesta was determined by combustion (Nitrogen Analyzer Model FP-2000, Leco Corporation St. Joseph, MI). Crude protein was calculated as N x 6.25. All hay, ort, fecal, and duodenal samples were analyzed for NDF and ADF with a fiber analyzer (model 200, Ankom Technology, Fairport, NY), with sodium sulfite and amylase omitted and without correction for residual ash. To determine ADIA of hay, ort, fecal, and duodenal samples, the Ankom bags containing the ADF residues were combusted for 8 h at 450°C in a muffle furnace. To isolate ruminal bacteria, samples of ruminal contents were thawed and feed particles were removed from the sample by centrifugation at 500 x g for 20 min. Supernatants were then centrifuged at 20,000 x g for 20 min to pellet the bacteria. The pellet was resuspended with 0.9% NaCl and centrifuged at 20,000 x g for 20 min. The bacterial pellets were frozen and lyophilized. Dry fecal, duodenal, and bacterial samples were analyzed for 15N by using a stable isotope elemental analyzer (ThermoFinnigan Delta Plus, Thermo Electron Corporation, Waltham, MA). Ruminal VFA were determined by GLC as described by Vanzant and Cochran (1994). Colorimetric determinations of ruminal ammonia (Broderick and Kang, 1980) and plasma urea (Marsh et al., 1965) were made with an autoanalyzer (Technicon Analyzer II, Technicon Industrial Systems, Buffalo Grove, IL).
For determination of urea kinetics, urinary urea and ammonia concentrations were determined colorimetrically with an AutoAnalyzer (Technicon Analyzer II) by using the methods of Marsh et al. (1965) and Broderick and Kang (1980). To initially remove ammonia from the urine samples, 2 mL of a strong cation exchange resin (Dowex 50W-X8, 100 to 200 mesh, H+ form, Sigma Chemical, St. Louis, MO) was mixed with 10 mL of urine, vortexed for 15 s, and allowed to stand for 15 min. From the tube, 31 µmol of urea was pipetted onto a column containing 2 mL of the cation resin to remove any remaining ammonia, and the effluent was discarded. Deionized water (20 mL) was added to the column, and the effluent was discarded. An additional 10 mL of deionized water was added to the column, and the effluent was retained. Urea and ammonia concentrations of the retained effluent were qualitatively assessed by visual appraisal, using the methods described previously by Marsh et al. (1965) and Broderick and Kang (1980). If no ammonia was present, then 3 µmol of urea was transferred into an Exetainer (Labco International, Houston, TX) tube, and the sample was brought to 4 mL with deionized water and subsequently frozen. If ammonia was present, the initial step of mixing the urine with the Dowex resin was repeated until no ammonia was present in the final effluent. To prepare samples for Hoffman degradation, He was bubbled through the samples for approximately 10 min, and samples were frozen quickly in liquid N2. After freezing, 0.5 mL of hypobromite [27 g of bromine/100 mL of 40% (wt/wt) NaOH] was added and the lid was screwed on. With the tube remaining in liquid N2, a vacuum pump was used to remove the gas from the tube and He was added; this process was repeated three times. After the final addition of He, the Exetainer was then removed from liquid N2 and allowed to thaw at room temperature. The thawed sample was then placed in a 60°C water bath for 15 min to allow the Hoffman degradation reaction to occur more rapidly. Samples were then analyzed for 28N2, 29N2, and 30N2 by using a stable isotope gas bench (ThermoFinnigan Delta Plus).
Calculations
Urea kinetics were calculated as outlined by Lobley et al. (2000). Duodenal flow was calculated by dividing fecal ADIA output by the ADIA concentration of duodenal digesta. Bacterial N flow was calculated by multiplying duodenal N flow by the ratio of duodenal 15N enrichment to bacterial 15N enrichment. The flow of bacterial N derived from recycled urea-N was calculated by multiplying bacterial N flow by the ratio of bacterial 15N enrichment to urinary 15N enrichment. Ruminally undegraded intake protein flow was determined by subtracting microbial N flow from total duodenal N flow. Therefore, our estimates of ruminally undegraded intake protein flow include endogenous secretions that did not contribute to microbial N.
Statistical Analyses
Intake, digestion, N balance, urea kinetics, duodenal flows, and plasma urea-N concentration were analyzed by using the MIXED procedure (SAS Inst. Inc., Cary, NC). Terms in the model were treatment and period, with steer included as a random effect. Fermentation profile variables were analyzed by using the MIXED procedure. Terms in the model were treatment, period, hour, and hour x treatment, with steer and treatment x period x steer included as random terms. The repeated term was hour, with treatment x steer serving as the subject. Compound symmetry was used for the covariance structure. The LSMEANS option was used to calculate treatment means. Orthogonal polynomial contrasts (linear, quadratic, and cubic) were used to partition treatment sums of squares.
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) from 4.47 to 6.90 kg/d. Rumen-degradable intake protein increased total tract OM and NDF digestibilities (linear, P 
0.009). Total digestible OM intake, an estimate of overall forage utilization, was increased by DIP supplementation (linear, P < 0.001). The greatest level of total digestible OM intake was 4.11 kg/d and occurred at the greatest amount of supplementation, 177 mg of N/kg of BW daily; this was 184% of total digestible OM intake of the unsupplemented steers. Nitrogen intake increased linearly (P < 0.001) with increasing provision of supplemental N, both as a direct result of the N provided by the casein and as an indirect result of the increases in forage intake. Corresponding with increased N intake, fecal and urinary N excretion increased (linear, P < 0.001) in response to increasing DIP. Apparent N absorption and N retention increased (linear, P < 0.001) with DIP provision. Retention of N increased (linear, P < 0.001) from –2.7 to 35.2 g of N/d. Nitrogen retention as a percentage of N intake and as a percentage of apparently absorbed N increased quadratically (P 0.003), with the most pronounced increases occurring with the first increment of supplementation.
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0.02; Table 3) only modestly from 0 to 118 mg of N/kg of BW daily (1.1 to 2.3% of urinary N excretion) and then increased more between 118 and 177 mg of N/kg of BW daily to 7.1% of urinary N excretion. Urinary ammonia excretion was not affected by supplemental DIP (P 0.35).
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Duodenal N flow, microbial N flow, and ruminally undegraded intake protein flow increased (linear, P 0.002; Table 4) with increasing provision of supplemental protein. Incorporation of recycled urea-N by ruminal microbes was increased (linear, P = 0.02) by supplemental DIP. However, the contribution of recycled urea-N as a percentage of total microbial N was not altered by supplemental DIP (P 0.27). Additionally, the percentage of urea production and gut entry that was incorporated into microbial N was not changed by supplemental DIP. Microbial efficiency increased (linear, P < 0.03) with increasing supplemental protein. Plasma urea-N concentration increased quadratically (P = 0.02) in response to increasing DIP, with greater increases for the greater level of DIP than for the lesser levels (Table 5).
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0.003) for ruminal ammonia, acetate, isobutyrate, is-ovalerate, and valerate. These interactions were largely due to treatment differences at times slightly after supplementation being greater than at times far from supplementation, as would be expected, rather than to changes in treatment rankings; thus, the overall treatment means are presented because they simplify presentation without affecting interpretation (data not shown). Therefore, to facilitate discussion of treatment effects, means averaged across time are presented and discussed. Ruminal ammonia concentration increased linearly (P < 0.001; Table 5) with increasing supplemental protein. Total VFA concentration increased (linear, P = 0.002) with increasing DIP supplementation. The molar proportion of acetate decreased (linear, P < 0.001) and the proportion of propionate increased (linear, P < 0.001) with increasing protein supplementation. Butyrate concentration tended to be less for 177 mg of N/kg of BW daily than for the other treatments (quadratic, P = 0.06). Isobutyrate, isovalerate, and valerate were increased linearly (P 0.03) with supplemental DIP. |
DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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; Klevesahl et al., 2003; Wickersham et al., 2004). Although others have observed a plateauing in intake with greater levels of DIP supplementation (Köster et al., 1996; Klevesahl et al., 2003; Wickersham et al., 2004), our greatest level of DIP supplementation was designed to be near the DIP requirement, not above it (Klevesahl et al., 2003; Wickersham et al., 2004), such that a plateau in response was not expected. Our greatest treatment was likely near the DIP requirement, which is supported by the fact that DIP intake at the greatest level of DIP supplementation (96 g of DIP/kg of digestible OM) was slightly below the DIP requirement of 110 g of DIP/kg of digestible OM suggested by Köster et al. (1996) and Wickersham et al. (2004) and slightly above the requirement of 90 g of DIP/kg of digestible OM reported by Klevesahl et al. (2003) when similar forages were fed.
In cattle consuming low-quality forage, protein supplementation has consistently increased N retention (Hunter and Siebert, 1980; Hennessy and Nolan, 1988; Farmer et al., 2004). This increase is the result of addressing N deficiencies at the ruminal level and energy deficiencies at both the ruminal and animal levels (i.e., greater intake of fermentable OM). Supplying the ruminal microbes with a source of N, casein in our study, increases forage intake and microbial synthesis of CP, thus increasing duodenal flows of microbial N and ruminally undegraded intake protein (Köster et al. 1996), both of which contribute to an increased supply of MP. More importantly, the increase in forage intake increases the supply of energy to the animal, which allows for greater N retention. Given that the majority of the cattle maintained on low-quality forage have relatively low MP requirements, the improvement in energy intake is likely the response of greatest importance.
Urinary N increased linearly with increasing supplemental protein, but when compared with other experiments (Archibeque et al., 2001; Marini and Van Amburgh, 2003) in which increased N intake produced dramatic increases in urinary N excretion, the increases in our study were relatively small. In accordance with our study, Hunter and Siebert (1980) observed that when intake of N increased from 21 to 54 g of N/d with the provision of cottonseed meal, urinary N excretion increased from only 14 to 16 g of N/d. Fecal N excretion increased in our study, which is in accordance with Hunter and Siebert (1980), who observed dramatic increases in fecal N excretion with supplementation. In contrast, Marini and Van Amburgh (2003) and Archibeque et al. (2001, 2002) did not observe increases in fecal N excretion as N intake increased. The reasons for the increases in fecal N and only small increases in urinary N, despite dramatic increases in N intake with supplemental DIP, are linked. As more supplemental DIP was provided in our study, microbial incorporation of N increased by 68.5 g of N/d (from 0 to 177 mg of N/kg of BW daily). If 20% of the microbial N was indigestible (NRC, 1996), an additional 13.7 g of N/d would be excreted in the feces, which accounts for much of our observed increase in fecal N excretion. Furthermore, increases in forage intake with DIP supplementation increased fecal N losses from indigestible forage residues and endogenous protein loss. Increases in urinary N loss in response to supplemental protein were relatively small because much of the additional N consumed (supplement plus increased forage) was retained by the animal consequent to increases in supplies of MP and energy. In contrast, when Marini and Van Amburgh (2003) increased N intake, there were no significant increases in microbial N, so more ammonia was absorbed ruminally and subsequently excreted in the urine as urea.
Quantifying urea kinetics and the incorporation of recycled urea-N into microbial N were the primary objectives of this study. Production of urea consistently increases as N intake increases (Cocimano and Leng, 1967; Bunting et al., 1989; Marini and Van Amburgh, 2003). In our study, provision of supplemental DIP increased urea production similarly to that observed in steers consuming carpetgrass hay, where supplementation with 101 mg of N/kg of BW daily increased urea production from 16 g of urea-N/d for unsupplemented steers to 44 g of urea-N/d for supplemented steers (Hennessy and Nolan, 1988). Norton et al. (1979) also observed low rates of urea production (9 g of urea-N/d) in unsupplemented steers eating low-quality sorghum stubble hay. Urea production was observed by Archibeque et al. (2002) to range from 52 to 64 g of urea-N/d for N intakes of 88 to 112 g of N/d, whereas steers in our study supplemented with 177 mg of N/kg of BW daily produced slightly less urea (51 g of urea-N/d) despite a similar N intake (107.5 g of N/d). For steers at the least N intake (62 g of N/d), Archibeque et al. (2001) measured urea production of 33 g of urea-N/d, whereas our DIP treatment of 59 mg of N/kg of BW daily had a N intake of 63 g of N/d and urea production of 25 g of urea-N/d. When N intakes were 88 and 110 g of N/d, Marini and Van Amburgh (2003) reported urea production rates of 31 and 56 g of urea-N/d, which are very similar to the observations in our study when steers received 118 and 177 mg of N/kg of BW daily. An important difference between the studies of Archibeque et al. (2001, 2002) and of Marini and Van Amburgh (2003) and our study is the low quality of our basal diet (i.e., the low CP content) relative to the diets used in their experiments.
Urea produced by the ornithine cycle has 2 fates: urinary excretion and recycling to the gastrointestinal tract (gut entry). As supplemental DIP was increased, there was an increase in urinary urea excretion, but the excretion at all levels was quite small and urinary urea excretion ranged from only 1.1 to 7.1% of urinary N. In light of the low plasma urea-N concentrations observed in our study and the relationship between plasma urea-N concentration and urinary urea excretion reported by Cocimano and Leng (1967) in sheep, urinary urea excretion would be expected to be low. Although our greatest level of DIP supplementation increased plasma urea-N concentrations to more than 4 times that of unsupplemented steers, our greatest concentrations of plasma urea-N were approximately half the concentration at which Cocimano and Leng (1967) observed dramatic increases in urinary excretion rate in sheep. In accordance with our observations, steers fed carpetgrass hay (4.9% CP) excreted only 0.41 g of urea-N/d when N intake was 29.5 g of N/d (Hennessy and Nolan, 1988). Similarly, Thornton (1970) reported that steers consuming oat straw (1.9% CP) excreted 1.6 g of urea-N/d, which was approximately 7% of urinary N excretion, when N intake was 40.0 g/d. With a greater quality forage (switchgrass, 8.8% CP), Archibeque et al. (2001) observed that 4.3 g of urea-N/d was excreted, which accounted for 32% of urinary N excretion. In contrast to our work, when sheep were unsupplemented and fed a low-quality forage ration, 35% of urinary N excretion was urea-N (McIntyre, 1971). The low urinary urea excretion when low-quality forage is fed is the result of a severe N deficiency, low plasma urea concentrations, and the conservation of urea-N for recycling to the gut.
In our study, gut entry of urea as a percentage of urea production was extremely high, pointing to the remarkable ability of cattle facing a N deficiency to conserve N when plasma urea and ruminal ammonia concentrations are low. Gut entry increased from 19.8 g of urea-N/d (98.9% of urea production) for unsupplemented steers to 48.6 g of urea-N/d (95.9% of urea production) for 177 mg of N/kg of BW daily. Increasing plasma urea-N concentrations (Houpt and Houpt, 1968; Ford and Milligan, 1970) and increasing intakes of fermentable OM (Houpt, 1959; Norton et al., 1982) with increasing provision of DIP would support the increased recycling of urea-N that we observed (Kennedy and Milligan, 1980). Additionally, the low concentrations of ruminal ammonia observed in our study would favor the recycling of urea (Kennedy and Milligan, 1980). In comparison, the greatest rates of gut entry reported by Marini and Van Amburgh (2003) and Archibeque et al. (2001, 2002) were 84.1, 86.6, and 53.1% of urea production, respectively. Supporting our observation of extremely high gut entry when low-quality forages are fed are the observations of Hennessy and Nolan (1988), in which urea transfer to the gut was 97.5% of urea production for unsupplemented steers fed carpetgrass hay and 79.5% for supplemented steers.
As stated previously, the fate of urea synthesized by the liver is the balance between urinary excretion and gut entry. The conditions created by feeding a low-quality forage (e.g., low ruminal ammonia and low plasma urea concentrations) and the increasing supply of fermentable OM (increased forage intake) favor the transfer of urea to the gut rather than the excretion of urea in the urine. In essence, the low-quality forage diet creates an ideal situation for the entry of urea into the gut to be maximized as a percentage of urea synthesis. However, because urea synthesis increases with N intake, the absolute amount of urea transferred to the gut may continue to increase even though the percentage of urea transferred to the gut may decrease.
After urea enters the gut and is hydrolyzed to ammonia, the N can either be absorbed from the gut or incorporated into microbial CP. Ammonia-N absorbed from the gut can be returned to the ornithine cycle, used in mammalian amination or amidation reactions, or excreted in the urine in a form other than urea. If incorporated into microbial CP, the N can subsequently be excreted in the feces as undigested microbial residues (fecal excretion); deposited in the body, most prevalently as tissue protein (anabolic utilization); excreted in the urine as something other than urea; or returned to the ornithine cycle. In our study, the percentage and amount of gut entry urea-N returned to the ornithine cycle increased as N intake increased, and correspondingly as gut entry of urea (grams of N daily) increased. The percentage of gut entry urea-N captured by microbes was not affected by an increasing DIP supply; therefore, it is likely that the increases in the amount returned to the ornithine cycle were not the result of increased absorption of ammonia from the rumen, but rather increases in the catabolism of nitrogenous compounds of microbial origin (i.e., predominantly AA) and subsequent conversion of the N to urea. In accordance, Marini and Van Amburgh (2003) observed that between their 2 least quantities of N intake (88 and 110 g/d), the percentage of urea-N returned to the ornithine cycle increased from 17 to 32% of gut entry. In contrast with our results, the capture of gut entry urea-N by ruminal microbes decreased from 42 to 26% in their study, indicating that greater absorption of ammonia-N from the rumen was driving the increases in the percentage of gut entry urea-N returned to the ornithine cycle in their study.
The contribution of recycled urea-N to microbial N was between 25 and 32% of the N in microbes flowing to the duodenum and was not affected by treatment. In contrast, Neutze et al. (1986) found that in sheep fed alkali-treated wheat straw, 31% of microbial N came from blood urea, but the proportion of microbial N from recycled urea-N decreased to 12% with increasing urea supplementation. Similarly, Bunting et al. (1989) found that recycled urea-N contributed 40% of microbial N to cattle consuming a low-protein diet and decreased to 13% in cattle consuming high-protein diets. Marini and Van Amburgh (2003) reported that at the least level of N intake, 19% of microbial N was derived from recycled urea-N, whereas it was only 3% for the greatest level of N intake. In our study, the relative constancy in the fractional contribution of recycled urea to microbial N reflects the simultaneous increase both in dietary N directly available to the microbes from dietary DIP and in recycled urea-N available to the microbes as supplemental DIP increased. Additionally, it reinforces the degree of ruminal N deficiency that was associated with our low-quality forage diets and the importance of N recycling mechanisms in enabling ruminants to survive on such forage resources.
Anabolic utilization of recycled urea-N is defined by Lobley et al. (2000) as the portion of gut entry of urea-N that is used to support anabolism. It was calculated as gut entry of urea minus urea returned to the ornithine cycle and fecal excretion of recycled urea-N, and, in theory, would predominantly represent recycled urea-N that was incorporated into microbial AA, intestinally absorbed, and used for net deposition of body protein. In practice, however, it will include urea-N that, subsequent to gut entry, is excreted in the urine in forms other than urea, because the urinary excretion of label in forms other than urea was not measured. Moreover, body pools of N (predominantly protein) will not reach isotopic plateaus over the relatively short infusion periods, which leads to overestimation of anabolic use, because, under that condition, incorporation of labeled AA by the animal reflects protein synthesis rather than net protein deposition, and uptake of labeled N into AA via mammalian amination or amidation reactions is likely to merely displace unlabeled N in the body.
Our measures of anabolic utilization as a percentage of gut entry decreased linearly with increasing supplementation, although the amount (grams of N daily) retained for anabolic use increased with supplementation. A portion of the decrease in anabolic utilization as a percentage of gut entry can be explained by the aforementioned apparent increase in the amount of urea-N being returned to the ornithine cycle originating from microbial AA.
In our study, anabolic utilization of recycled urea-N was greater than microbial incorporation of recycled urea-N for unsupplemented steers and was less than microbial incorporation of recycled urea-N for all steers provided supplemental DIP. It is expected that microbial incorporation of recycled urea should exceed anabolic utilization, because anabolic use of urea, by definition, should be largely dependent on microbial synthesis of AA that could subsequently be used for protein deposition. Additionally, excretion of indigestible microbial residues and the catabolism of AA of microbial origin by the cattle would decrease the nitrogenous compounds available for anabolic use. For the unsupplemented steers, the opposite was true, and this is most likely explained by the overestimation of anabolic utilization discussed above.
The increase in the amount of recycled urea-N used for anabolic purposes in response to the greatest level of supplementation (8.0 g/d = 21.7 – 13.7 g/d) was 9% of the increase in N intake when the cattle received that supplement (69.0 g/d = 107.5 – 38.5 g/d), whereas the increase in the amount of recycled urea-N that was captured by ruminal microbes (16.6 g/d = 28.9 – 12.3 g/d) was 24% of the increase in N intake. The lesser percentage for anabolic use than for microbial capture would be due to excretion in the feces of a portion of microbial N as undigested microbial residues and catabolism by the animal of a portion of microbial AA absorbed from the small intestine.
Provision of supplemental DIP increased forage utilization and N retention in cattle consuming low-quality forage. Cattle maintained on low-quality forage are remarkably efficient at conserving N through urea recycling mechanisms. The transfer of urea from the blood to the rumen contributes between one-fourth and one-third of the N utilized by ruminal microbes for the synthesis of microbial protein. Further investigations into the contribution of urea recycling to meeting ruminal N demands is warranted, with the ultimate goal of incorporating estimates of N recycling into ruminant feeding systems.
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Footnotes |
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2 Present address: Texas A&M University, College Station, TX 77843
3 Corresponding author: etitgeme{at}ksu.edu
Received for publication June 4, 2007. Accepted for publication June 5, 2008.
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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T. A. Wickersham, E. C. Titgemeyer, R. C. Cochran, E. E. Wickersham, and E. S. Moore Effect of frequency and amount of rumen-degradable intake protein supplementation on urea kinetics and microbial use of recycled urea in steers consuming low-quality forage J Anim Sci, November 1, 2008; 86(11): 3089 - 3099. [Abstract] [Full Text] [PDF]
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