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J. Anim Sci. 2008. 86:3089-3099. doi:10.2527/jas.2007-0326
© 2008 American Society of Animal Science
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ANIMAL NUTRITION

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 forage1

T. A. Wickersham*,2, E. C. Titgemeyer*,3, R. C. Cochran*, E. E. Wickersham* and E. S. Moore{dagger}

* Department of Animal Sciences and Industry, and {dagger} Department of Clinical Sciences, Kansas State University, Manhattan 66506-1600


   Abstract
Top
 Abstract
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
We evaluated the effect of frequency and amount of rumen-degradable intake protein (DIP) on urea kinetics in steers consuming prairie hay. Five ruminally and duodenally fistulated steers (366 kg of BW) were used in a 5 x 5 Latin square and provided ad libitum access to low-quality prairie hay (4.7% CP). Casein was provided daily in amounts of 61 and 183 mg of N/kg of BW (61/d and 183/d) and every third day in amounts of 61, 183, and 549 mg of N/kg of BW per supplementation event (61/3d, 183/3d, and 549/3d). Periods were 18-d long with 9 d for adaptation and 9 d for collection. Steers were in metabolism crates for total collection of urine and feces. Jugular infusion of 15N15N-urea followed by determination of urinary enrichment of 15N15N-urea and 14N15N-urea was used to determine urea kinetics. Treatment means were separated to evaluate the effects of increasing DIP supplementation and the effects of frequency at the low (61/d vs. 183/3d) and at the high (183/d vs. 549/3d) amounts of DIP provision. Forage OM and total digestible OM intakes were linearly (P ≤ 0.05) increased by increasing DIP provision but were not affected by frequency of supplementation at either the low or high amounts. Production and gut entry of urea linearly (P ≤ 0.006) increased with DIP provision and tended to be greater (P ≤ 0.07) for 549/3d than 183/d but were not different between 61/d and 183/3d. Microbial N flow to the duodenum was linearly (P < 0.001) increased by increasing DIP provision. Additionally, 183/d resulted in greater (P = 0.05) microbial N flow than 549/3d. Incorporation of recycled urea-N into microbial N linearly (P = 0.04) increased with increasing DIP. Microbial incorporation of recycled urea-N was greater for 549/3d than 183/d, with 42 and 23% of microbial N coming from recycled urea-N, respectively. In contrast, there was no difference due to frequency in the incorporation of recycled urea-N by ruminal microbes at the low level of supplementation (i.e., 61/d vs. 183/3d). This study demonstrates that urea recycling plays a substantial role in the N supply to the rumen and to the animal, particularly in steers supplemented infrequently with high levels of protein.

Key Words: cattle • frequency • protein • recycling • urea


   INTRODUCTION
Top
 Abstract
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Protein supplementation to cattle consuming low-quality forage makes up a significant cost in the cow-calf segment of the beef industry. To decrease the cost of supplementation, producers can decrease the labor and equipment (e.g., vehicle depreciation) inputs by decreasing supplementation frequency. Typically, when supplementation frequency is decreased, the amount of supplement offered per week remains the same, but the amount offered per supplementation event is increased. Performance studies have shown small reductions in BCS and BW as supplementation frequency decreases (Beaty et al., 1994Go; Farmer et al., 2001Go; Currier et al., 2004Go). Intake and digestion studies have demonstrated that when large amounts of supplements are provided infrequently, there is a reduction in forage intake on the day supplements are provided (Farmer et al., 2001Go; Bohnert et al., 2002aGo). Nitrogen retention has been shown to decrease with less frequent supplementation (Farmer et al., 2004Go). Additionally, infrequent supplementation significantly alters microbial populations. For example, when a large dose of supplemental protein was provided, there was a lag in the ruminal degradation of the protein contained in the supplement because of inadequate protein fermentation (Farmer et al., 2004Go). Although observations of animal performance, intake and digestion, and microbial adaptations have been made in infrequently supplemented ruminants, the role of N recycling has not been well investigated. Based on the fluctuating, but typically low, ruminal ammonia and plasma urea concentrations observed with infrequent supplementation, it is likely that N recycling in infrequently supplemented cattle plays a significant role in the N economy of the rumen and the ruminant. Our objective was to determine the importance of urea recycling in providing ruminally available N (RAN) to cattle supplemented with different amounts of rumen degradable intake protein (DIP) at varying frequencies.


   MATERIALS AND METHODS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
The experimental protocol was approved by the Institutional Animal Care and Use Committee at Kansas State University.

Five duodenally and ruminally fistulated Angus x Hereford steers (366 ± 43 kg of initial BW) were used in a 5 x 5 Latin square to evaluate the effects of amount and frequency of supplemental DIP on urea kinetics and recycled urea-N use by ruminal microbes in steers consuming low-quality forage. All data from 1 observation were lost due to problems not related to treatment. Additionally, data were not collected for microbial flow measurement in 1 steer, because the catheter fell out during collection. 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) while being offered tallgrass-prairie hay [comprised primarily of big bluestem (Andropogon gerardii Vitman), little bluestem (Schizachyrium scoparium (Michx.) Nash), and Indiangrass (Sorghastrum nutans (L.) Nash); Table 1Go] 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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  		Table 1. Composition of forage and casein
 
Three amounts of protein (61, 183, and 549 mg of N/ kg of BW) were provided as casein (Alanate 180, New Zealand Milk Products Inc., Santa Rosa, CA; Table 1Go) dosed ruminally 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 provided daily in amounts of 61 or 183 mg of N/kg of BW (61/d and 183/d) or every third day in amounts of 61, 183, or 549 mg of N/kg of BW per supplementation event (61/3d, 183/3d, and 549/3d). The DIP requirement for maximum forage utilization in previously conducted experiments was near 183/d (Klevesahl et al., 2003Go; Wickersham et al., 2004Go). Casein was selected as the protein supplement because of its high protein content and high ruminal degradability. Treatment 183/3d provided as much DIP every 3 d as did 61/d and 549/3d as much as 183/d. The 61/3d, 183/3d, and 549/3d supplements were provided on d 1, 4, 7, 10, 13, and 16 of each period.

Experimental procedures used general methodologies and adaptation periods consistent with Wickersham (2006)Go with modifications to account for the effects of infrequent supplementation on nutrient metabolism. Experimental periods were 18-d long, with 9 d for adaptation to treatments and 9 d for collection. For the first 6 d of adaptation, steers were housed in individual pens (1.5 x 3.1 m). For the remainder of the adaptation and during 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 mid portion of the steers and subsequently diverted into a bucket using gravity, whereas feces were collected into a pan placed directly behind the steer. Water was poured periodically on the funnel to aid complete collection of urine and to remove any impediments to flow.

On d 9, an indwelling catheter was placed in a 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 then it was filtered (0.22-µm filter unit Sterivex, Millipore Corporation, Billeric, MA). Saline solution was infused continuously from the time the catheter was placed in the jugular vein until 0600 h on d 10 when infusion of the 15N15N-urea solution began. The 15N15N-urea solution was infused continuously at approximately 4 mL/h, which delivered 0.154 mmol of urea-N/h until the end (d 18) of the experimental period using a syringe infusion pump (Harvard Apparatus, South Natick, MA). Total collections of urine and feces on d 9 were used to determine background enrichments of 15N. Total collections of urine and feces on d 13, 14, and 15 were composited in proportion to their daily excretion and used to determine urinary enrichment with 15N15N-urea and 15N14N-urea and fecal enrichment of 15N for calculation of urea kinetics. Beginning on d 16 and extending through d 18, ruminal fluid samples were collected by suction strainer (Raun and Burroughs, 1962Go; 19-mm diameter, 1.5-mm mesh) just before treatments were administered (0 h) and at 2, 3, 4, 6, 9, 12, 15, 21, 27, 33, 39, 45, 51, 57, 63, and 69 h after dosing supplements on d 16. At each collection, pH was measured, and 10 mL of ruminal fluid was combined with 2 mL of 25% (wt/ vol) metaphosphoric acid and frozen for ammonia and VFA analysis. Additionally, on d 16 through 18, whole ruminal contents (1 kg) and duodenal samples (300 mL) were collected before feeding (0 h) and at 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, and 69 h after dosing supplements on d 16 to determine incorporation of recycled urea-N into microbial protein and duodenal flows. To isolate ruminal bacteria from whole ruminal contents, 0.5 L of 0.9% NaCl solution was added immediately after the sample was collected, and then it was 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. Before analysis, ruminal bacteria samples and duodenal samples were pooled across time, resulting in 1 ruminal bacteria sample and 1 duodenal sample per steer for each period. 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 16 through 18. 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 urea concentration.

Calculations of intake, digestion, and N balance were made from observations on d 9 through 15. Feed and ort samples were collected on d 9 through 14 to correspond with fecal and urine samples collected on d 10 through 15. Duodenal flows were based on samples from d 16 through 18 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 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). Urinary 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 total 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. Dry fecal and duodenal samples were analyzed for 15N using a stable isotope elemental analyzer (Thermofinnigan Delta Plus, Thermo Electron Corporation, Waltham, MA). To isolate ruminal bacteria, ruminal contents were thawed and feed particles were removed 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. Bacteria were analyzed for 15N enrichment. Ruminal VFA were determined by GLC as described by Vanzant and Cochran (1994)Go. Colorimetric determinations of ruminal ammonia (Broderick and Kang, 1980Go) and plasma urea (Marsh et al., 1965Go) concentrations were made using 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) using methods of Marsh et al. (1965)Go and Broderick and Kang (1980)Go. 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 that 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 methods described previously by Marsh et al. (1965)Go and Broderick and Kang (1980)Go. 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 cation exchange resin was repeated until there was no ammonia present. To prepare samples for Hoffman degradation, He was bubbled through the samples, and samples were frozen quickly in liquid N2. After freezing, 0.5 mL of hypobromite [27 g of bromine per 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 thrice. After the final addition of He, the tube 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 rapidly. Samples were then analyzed for 28N2, 29N2, and 30N2 using a stable isotope gas bench (Thermofinnigan Delta Plus).

Calculations

Urea kinetics were calculated as outlined by Lobley et al. (2000)Go. 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 Analysis

Intake, digestion, N balance, urea kinetics, and duodenal flows were analyzed using PROC MIXED (SAS Inst. Inc., Cary, NC). Terms in the model were treatment and period with steer included as the random term. Fermentation profile variables were analyzed using PROC MIXED. 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. Plasma urea concentrations were analyzed using the same SAS code as the fermentation profile variables with the exception of hour being replaced by day. Treatment means were calculated using the LSMEANS option and were separated using linear and quadratic contrasts for amount of supplemental protein provided per 3 d by all 5 treatments and contrasts between 61/d and 183/3d (effect of frequency within the low level of supplementation) and between 183/d and 549/3d (effect of frequency within the high level of supplementation).


   RESULTS
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Forage OM intake, total OM intake, and total digestible OM intake increased linearly (P ≤ 0.05; Table 2Go) in response to increased amounts of DIP. There were no differences (P ≥ 0.11) in any of the intake variables between frequencies when DIP was provided in either low (61/d and 183/3d) or high (183/d and 549/3d) amounts. Ruminal NDF digestion tended (P = 0.06) to be linearly decreased by increasing amounts of supplemental DIP, although there was no difference in true OM digestion in the rumen. Total tract digestibilities of OM and NDF were not different among treatments, although there was a tendency (P = 0.06) for 183/3d to have greater NDF digestion than 61/d. Intake of N was increased linearly (P < 0.001) by increasing amounts of supplemental protein. Intake of N was not altered by frequency of supplementation at either the low or high level of DIP supplementation. Fecal excretion of N was increased linearly (P < 0.001) by increasing amounts of supplemental protein. Additionally, fecal excretion of N was greater when supplemental DIP was provided more frequently at the low level of supplementation (37.9 g/d for 61/d vs. 32.8 g/d for 183/3d). Fecal N excretion was not affected by frequency of supplementation when the greater amounts of supplemental DIP (183/d and 549/3d) were provided. Urinary N excretion increased linearly (P < 0.001) when increasing amounts of supplemental DIP were provided, and there was no effect of frequency at either the low or high level of DIP supplementation. Apparent absorption of N was increased (linear, P < 0.001) with increasing level of DIP provision. At the low level of supplementation (61/d and 183/3d), decreasing frequency from daily to every third day resulted in 12 g/d greater apparent absorption of N, but this effect was not observed at the high level of supplementation (183/d and 549/3d). Retention of N was increased (linear, P < 0.001) with increasing provision of DIP. There was a tendency (P = 0.07) for N retention to be greater for steers supplemented less frequently (21.8 g/d for 183/3d) than more frequently (12.9 g/d for 61/d) at the low level. Nitrogen retention as a percentage of N intake (P = 0.06) and as a percentage of N apparently absorbed increased with supplemental DIP, with greater responses between the lowest (61/3d) and intermediate (61/d and 183/3d) levels than between the intermediate and greatest (183/d and 549/3d) levels (quadratic, P = 0.02). There was a tendency (P = 0.10) for N retention as a percentage of N intake to be greater for 183/3d (26.7%) than for 61/d (17.0%)


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  		Table 2. Effects of frequency and level of protein supplementation on intake, digestion, and N balance in steers fed tallgrass-prairie hay
 
Urinary excretion of urea as well as the percentage of total urinary N as urea were increased by the greatest level of supplementation (183/d and 549/3d) but not by the intermediate (61/d and 183/3d) level (quadratic, P = 0.006; Table 3Go). There was a tendency (P = 0.07) for the percentage of total urinary N excretion coming from urea to be greater with less frequent supplementation at the high level of DIP supplementation. The amount of ammonia-N excreted in the urine was increased linearly (P = 0.009) by increasing supplemental DIP, but it was not affected by frequency at either level of supplementation. Ammonia-N as a percentage of urinary N excretion was unchanged by amount of supplementation. Urea production, gut entry, return to ornithine cycle, and anabolic utilization of recycled urea (grams of urea-N daily) were increased linearly (P ≤ 0.03) by increasing amounts of supplemental DIP. Urea production and gut entry (grams of urea-N daily) tended to be greater (P ≤ 0.07) when steers were supplemented the greatest amount of DIP every third day (549/3d) rather than every day (183/d). Anabolic utilization of recycled urea was greater (P = 0.02) for 549/3d than for 183/d. Urinary excretion of urea as a percentage of urea production was increased, and the percentage of urea production transferred to the gut was decreased correspondingly, by the greatest level of supplementation (183/d and 549/3d) but not by the intermediate (61/d and 183/3d) level (quadratic, P = 0.009). Urea returned to the ornithine cycle as a percentage of gut entry linearly (P < 0.001) increased with additional supplemental DIP. Fecal excretion of recycled urea-N as a percentage of gut entry was not affected by supplemental DIP, whereas anabolic utilization of recycled urea-N as a percentage of gut entry was decreased linearly (P = 0.005) by supplemental DIP. Gut entry of urea as a percentage of urea production was greater (P = 0.02) for 549/3d than 183/d. There was a tendency (P = 0.08) for the percentage of gut entry returned to the ornithine cycle to be greater for 183/d than for 549/3d.


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  		Table 3. Effects of frequency and level of protein supplementation on urinary urea excretion and ammonia excretion and urea kinetics in steers fed tallgrass-prairie hay
 
Total duodenal N and microbial N flow (g of N/d) were increased linearly (P < 0.001; Table 4Go) by supplemental DIP. More frequent provision of the greatest amount of supplemental DIP (183/d) resulted in greater (P = 0.05) microbial N flow (133.3 vs. 114.7 g/d) than did less frequent supplementation (549/3d). There were no changes in ruminally undegraded intake protein flow at the duodenum in response to supplemental DIP. The incorporation of recycled urea-N into microbial N (g of N/d) was increased (linear, P = 0.04) with increasing supplemental DIP. Increasing provision of supplemental DIP did not alter the percentage of microbial N coming from recycled urea-N. Microbial incorporation of recycled urea was greater (P = 0.03) in amount and as a percentage of total microbial N for 549/3d than for 183/d. Microbial incorporation of recycled urea-N as a percentage of urea production and gut entry was decreased linearly (P < 0.001) with increasing supplemental DIP. Microbial efficiency was not affected by increasing amounts of supplemental DIP or frequency of supplementation at high or low levels of DIP.


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  		Table 4. Effects of frequency and level of protein supplementation on duodenal N flows and recycled urea-N incorporation into microbial N in steers fed tallgrass-prairie hay
 
The treatment x time interaction was significant (P ≤ 0.01) for total VFA concentration, and for molar percentages of acetate, propionate, isobutyrate, isovalerate, and valerate, but the interactions were largely due to the magnitude of the difference existent between treatments at different times rather than to changes in treatment rankings (data not shown). Therefore, to facilitate discussion, only treatment means are presented (Table 5Go). Total ruminal VFA concentration was increased (linear, P = 0.005) by supplemental DIP. There was no effect of frequency at either high or low amounts of DIP. There was a linear (P < 0.001) reduction in the proportion of acetate with increasing DIP amounts. Propionate proportion, in contrast, was increased linearly (P < 0.001) with additional DIP. Butyrate proportion was not affected by treatment. Isobutyrate, isovalerate, and valerate were increased linearly (P < 0.001) as DIP amount increased. Ruminal pH decreased linearly (P = 0.008) with increasing provision of DIP.


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  		Table 5. Effects of frequency and level of protein supplementation on ruminal fermentation characteristics in steers fed tallgrass-prairie hay
 
Ruminal ammonia concentration also demonstrated (P < 0.001) a treatment x time interaction (Figure 1Go). Provision of supplemental DIP linearly (P < 0.001) increased ruminal ammonia concentration. Additionally, 61/d led to decreased average ammonia concentrations than did 183/3d. Treatment 61/3d produced small peaks (averaging 1.4 mM) in ammonia concentration 3 h after supplementation that quickly returned to baseline and remained there for the remainder of the sampling time. Treatment 61/d also produced small peaks (averaging 1.4 mM) at 3 h after supplementation. Treatment 183/3d produced a larger peak (5.2 mM) at 6 h after supplementation and then returned to baseline near 39 h after supplementation. Treatment 183/d produced peaks in ammonia concentration at 6 h after supplementation averaging 6.7 mM. Treatment 549/3d produced the largest ammonia peak (13.5 mM) at 9 h after supplementation and then returned to baseline near 45 h after supplementation.


Figure 1
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  		Figure 1. Effect of frequency and level of protein supplementation on ruminal ammonia concentration in steers fed tallgrass-prairie hay. 61/3d = 61 mg of N/kg of BW every third day; 61/d = 61 mg of N/kg of BW daily; 183/3d = 183 mg of N/kg of BW every third day; 183/d = 183 mg of N/kg of BW daily; 549/3d = 549 mg of N/kg of BW every third day; all N was provided as casein dosed ruminally as a source of degradable intake protein (DIP). Treatment x time interaction (P < 0.001). Linear effect (P < 0.001) of amount of supplemental DIP provided per 3-d period. Quadratic effect (P = 0.64) of amount of supplemental DIP provided per 3-d period. Effect of 61/d vs. 183/3d (P = 0.01). Effect of 183/d vs. 549/3d (P = 0.22). SEM = 0.32.

 
Plasma urea concentration exhibited a treatment x day interaction (P < 0.001; Figure 2Go). Plasma urea concentration increased linearly (P < 0.001) with increasing supplemental DIP. Plasma urea concentration for 183/3d on the day of supplementation was greater than the next 2 d when no supplement was provided (P < 0.05). Similarly, on the day of supplementation, 549/3d led to greater plasma urea concentrations than the day after supplementation, which was greater than the second day after supplementation (P < 0.05). For steers supplemented daily, there were no differences among days.


Figure 2
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  		Figure 2. Effect of frequency and level of protein supplementation on plasma urea-N concentration in steers fed tallgrass-prairie hay. 61/3d = 61 mg of N/ kg of BW every third day; 61/d = 61 mg of N/kg of BW daily; 183/3d = 183 mg of N/kg of BW every third day; 183/d = 183 mg of N/kg of BW daily; 549/3d = 549 mg of N/kg of BW every third day; all N was provided as casein dosed ruminally as a source of degradable intake protein (DIP). For 61/3d, 183/3d, and 549/3d, steers received supplemental casein on d 1. Linear effect (P < 0.001) of amount of supplemental DIP provided per 3-d period. Quadratic effect (P = 0.28) of amount of supplemental DIP provided per 3-d period. Effect of 61/d vs. 183/3d (P = 0.15). Effect of 183/d vs. 549/3d (P = 0.57). Error bars are SEM.

 

   DISCUSSION
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Amount of Supplemental DIP

Provision of supplemental DIP resulted in increases in forage OM intake, total OM intake, and total digestible OM intake similar to those previously observed using similar forage (Wickersham et al., 2008Go). The intake response has previously been observed to plateau with increasing DIP (Köster et al., 1996Go; Klevesahl et al., 2003Go; Wickersham et al., 2004Go), but our greatest level of supplemental DIP was designed to be near the requirement for maximal forage utilization. The intake response is the result of increased supply of RAN directly from supplemental DIP and from recycled urea. The increased supply of RAN is demonstrated by the increases in ruminal ammonia concentrations and in microbial N flow with increasing DIP supplementation. Similar increases in ruminal ammonia and microbial N flow with supplementation of DIP as casein were observed by Köster et al. (1996)Go and Wickersham et al. (2008)Go. Although microbial activity was increased by DIP provision, there were no improvements in ruminal NDF digestibility. In contrast, Wickersham et al. (2008)Go demonstrated that increasing DIP increased total tract digestibility of OM and NDF. Additionally, Guthrie and Wagner (1988)Go and Köster et al. (1996)Go reported increased digestion of OM and NDF when DIP was supplemented. It is possible that improvements in digestion with increased DIP supplementation were not observed because passage rate was increased in response to increased RAN supply (Guthrie and Wagner, 1988Go; Köster et al., 1996Go; Olson et al., 1999Go) reducing the length of time ruminal microbes had access to the substrate.

Provision of casein as a source of supplemental DIP increased the supply of a highly degradable source of N, which improved the N status of the steers. The end result was increases in apparent N absorption and N retention similar to Wickersham et al. (2008)Go. An increase in N retention from 3.6 to 15.6 g of N/d for un-supplemented and supplemented steers was observed by Hennessy and Nolan (1988)Go when supplemental protein was provided at 100 mg of N/kg of BW daily. By supplying DIP, the supply of MP to the steer is not directly increased by the supplement but rather by the resulting increase in microbial N flowing to the duodenum, which increases the supply of AA to the steer for tissue synthesis. Additionally, total digestible OM intake was increased by DIP, which increased the amount of energy available for tissue synthesis and maintenance. Increases in AA and energy supplies explain the improvements in N retention with supplemental DIP.

Ammonia-N absorbed across the ruminal wall is a key contributor to N available for urea production, and plasma urea concentrations increase in response to protein supplementation (Cocimano and Leng, 1967Go; Thornton, 1970Go; Neutze et al., 1986Go). Supplemental DIP also increased the supply of AA (i.e., microbial N flow increased), the catabolism of which can also contribute to urea production. Increases in urea production with protein supplementation to cattle consuming low-quality forage are an expected result (Hennessy and Nolan, 1988Go; Wickersham, 2006Go; Wickersham et al., 2008Go). Additionally, increased N intake in dairy heifers increased urea production (Marini and Van Amburgh, 2003Go). Urea that is produced and subsequently enters the gut can serve a productive function if it is incorporated into microbial N. Urea production, plasma urea concentration, and ammonia concentration in the rumen typically are positively related to each other, whereas gut entry of urea is negatively related to ruminal ammonia concentration and positively related to plasma urea concentration (Harrop and Phillipson, 1974Go; Kennedy and Milligan, 1978Go; Rémond et al., 1993Go). Despite the greater ruminal ammonia concentrations with DIP supplementation, the amount of urea recycled into the gut was increased with increasing DIP supplementation. The increased gut entry in response to increasing DIP resulted from the following: 1) increases in plasma urea concentrations, which favored the transfer of plasma urea from the blood to the rumen (Weston and Hogan, 1967Go; Ford and Milligan, 1970Go; Kennedy, 1980Go); 2) plasma urea-N concentrations, despite increasing with DIP, generally remained below 8.6 mM, the point at which Vercoe (1969)Go observed no further increases in gut entry of urea as plasma urea increased; 3) ruminal ammonia concentrations generally less than 6 mM, the point above which Kennedy (1980)Go observed that degradation of urea in the rumen was inhibited; and 4) increases in fermentable OM intake (Houpt, 1959Go; Kennedy, 1980Go; Norton et al., 1982Go). The high percentage of urea directed toward gut entry rather than urinary excretion demonstrates the remarkable ability of cattle to conserve N by recycling urea in the face of a RAN deficiency.

With increasing provision of supplemental DIP, the percentage of gut entry of urea-N that was returned to the ornithine cycle increased, and, concomitantly, the anabolic utilization as a percentage of gut entry of urea-N linearly decreased; fecal excretion of recycled urea-N was not altered by DIP supplementation. Anabolic utilization as a percentage of recycled urea-N was greater with the decreased levels of supplementation, because microbial N incorporation of recycled urea as a percentage of gut entry was greater. In other words, at the low levels of supplemental DIP (61/3d, 61/d, and 183/3d), a greater proportion of the urea entering the gut was subsequently incorporated into microbial N, likely because there was less RAN provided directly as DIP that would compete with the recycled urea-N for incorporation by ruminal microbes. Recycled urea-N incorporated into microbial N would be less likely to be returned to the ornithine cycle than that not captured by microbes, explaining why the percentage of urea returned to the ornithine cycle was less when low levels of DIP were supplemented. Similar observations were made with DIP supplementation to steers fed similar forage (Wickersham et al., 2008Go). Marini and Van Amburgh (2003)Go also observed that as N intake in dairy heifers increased, the percentage of N returning to the ornithine cycle increased from 17 to 32%, whereas anabolic utilization remained unchanged. In contrast to the current study and that of Wickersham et al. (2008)Go, microbial incorporation of N (g of N/d) did not increase with increasing provision of N in the study by Marini and Van Amburgh (2003)Go, likely because all their diets met RAN requirements. At all amounts of supplemental DIP, anabolic utilization as a percentage of gut entry was less than microbial incorporation of recycled urea-N. This was expected, because microbial incorporation of recycled N into AA would generally serve as the primary precursor to anabolic utilization of recycled urea.

Frequency of Supplementation at Low Levels of DIP

Forage OM intake, total OM intake, and total digestible OM intake were not different between frequencies of supplementation for the low levels of supplemental DIP (61/d vs. 183/3d). Bohnert et al. (2002b)Go observed quadratic increases in forage and total OM intake when supplementation frequency decreased from daily, to every third day, to every sixth day. In contrast, Farmer et al. (2001)Go observed linear reductions in OM intake as supplementation frequency decreased from daily to twice weekly. Beaty et al. (1994)Go also observed decreased intake when steers were supplemented thrice weekly rather than daily. Our observations may differ from both Beaty et al. (1994)Go and Farmer et al. (2001)Go because the CP content of our supplement was much greater than the supplements fed in their studies (95 vs. 12 to 43% CP). This allowed us to provide less supplement per supplementation event, thereby preventing the substitution effect reported by both studies to be, in part, responsible for the reductions in forage intake. Similar urinary N excretions and urinary urea-N excretions between 61/d and 183/3d indicate that there was no measurable inefficiency in N metabolism with more infrequent supplementation. Urea kinetics and microbial incorporation of recycled N were similar between 61/d and 183/3d with similar urea production, gut entry as a percentage of urea production, anabolic utilization as a percentage of urea production, and microbial N incorporation of recycled urea, indicating that there was not a substantial difference between the treatments in the way N was metabolized. A reason for the lack of differences between 61/d and 183/3d is that, on the days of supplementation, ammonia was sufficiently captured by ruminal microbes, which prevented large increases in ruminal ammonia concentration and the associated absorption of ammonia from the rumen. As a result, urea production was similar between 61/d and 183/3d. Because ruminal microbes directly assimilated most of the N provided by the 183/3d treatment, the 183/3d and 61/d treatments were equally dependent on N recycling as a N source. At this decreased level of supplementation, the larger dose of DIP for 183/3d on the day of supplementation was handled very similar to when 61 mg of N/kg of BW was provided daily.

Frequency of Supplementation at High Levels of DIP

When supplements were provided at the high level at different frequencies (183/d and 549/3d), intake was almost identical between the treatments. Urea metabolism, however, was different, with greater urea production for 549/3d than for 183/d. Urea production in our study is an average over 3 d, which for 549/3d would include the day of supplementation and the following 2 d without supplementation. In light of the high plasma urea concentration on the day of supplementation for 549/3d and the decreasing plasma urea concentration on the days after supplementation, urea synthesis was likely greatest on the day of supplementation and then decreased over time. This conclusion is supported by the positive relationship that exists between urea production and plasma urea concentrations (Cocimano and Leng, 1967Go; Kennedy, 1980Go). Urinary urea excretion as a percentage of urea production was greater for 183/d than for 549/3d, which was the result of similar amounts of urinary urea-N excretion between the treatments but greater urea production for 549/3d. Although ruminal ammonia concentration was greater for 549/3d than 183/d throughout the day of supplementation, ammonia concentrations were less for 549/3d throughout the 2 d when steers were not supplemented. The decreased ruminal ammonia concentrations would favor the transfer of urea to the gut, which may explain why gut entry of urea was greater for 549/3d than 183/d. In contrast to the low level of supplementation, different frequencies at the high level of supplementation resulted in differences in ruminal ammonia utilization. Ammonia concentration for 549/3d was high on the day of supplementation, because ammonia was produced from the DIP faster than ruminal microbes could incorporate the N, leading to inefficient capture of N from the DIP. As ammonia concentration increases, absorption of ammonia from the rumen and the subsequent production of urea is increased. Because ammonia concentrations increased to a greater extent for 549/3d than for 183/d, more urea was produced, and when ammonia concentrations fell at times distant from supplementation, ruminal microbes from cattle supplemented every third day were more dependent on N recycling than those from cattle with daily supplementation. Anabolic utilization of recycled urea made up a much greater percentage of gut entry for 549/3d than for 183/d (42 vs. 34%). Even more striking, 45.7 g of recycled urea-N/d was used for anabolic purposes for 549/3d vs. only 23.9 g of urea-N/d for 183/d. The amount of recycled N incorporated into microbial N was 47.4 g of N/d for 549/3d vs. 30.8 g of N/d for 183/d. Similarly, 42.1 vs. 22.8% of microbial N came from recycling for 549/3d vs. 183/d. In light of the high rate of gut entry of urea-N for 549/3d and the low ammonia concentrations for 48 h of the 72-h supplementation period, the high dependency of microbial populations on N recycling would be expected. Additionally, these results indicate that urea recycling was used as a N-conserving mechanism in infrequently supplemented steers consuming low-quality forage at the high rate of supplementation. Despite the value of recycling in providing RAN to steers fed 549/3d, the decreased microbial N flows for 549/3d than for 183/d suggest that microbial activity may have been impaired slightly by the less frequent supplementation. In contrast, whole animal utilization of N (N retention) was numerically greater for 549/3d than 183/d.

Increasing supplemental DIP produced increases in urea production, urea recycling to the gut, microbial incorporation of recycled N, and low-quality forage utilization that have been observed previously. At the decreased level of supplementation, daily versus every third day supplementation led to urea kinetics that were similar. In contrast, at the high level of supplementation, supplementation every third day led to greater dependency on urea recycling for meeting ruminal N requirements than daily supplementation. From these data, it is apparent that urea recycling plays a significant role in enabling producers to utilize infrequent supplementation as a management option to control supplementation cost without having a severely negative effect on animal performance.


   Footnotes
 
1 Contribution No. 07-289J from the Kansas Agricultural Experiment Station, Manhattan. Back

2 Present address: Texas A&M University, College Station, TX 77843. Back

3 Corresponding author: etitgeme{at}ksu.edu

Received for publication June 4, 2007. Accepted for publication June 5, 2008.


   LITERATURE CITED
Top
 Abstract
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 


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