Effects of supplemental urea sources and feeding frequency on ruminal fermentation, fiber digestion, and nitrogen balance in beef steers

Animal Feed Science and Technology 171 (2012) 136–145

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Effects of supplemental urea sources and feeding frequency on ruminal fermentation, fiber digestion, and nitrogen balance in beef steers E.G. Alvarez Almora a , G.B. Huntington b,∗ , J.C. Burns b,c a b c

Universidad Autónoma de Baja California, Mexicali, Mexico North Carolina State University, Raleigh, NC 27695-7621, United States US Department of Agriculture, Agricultural Research Service, United States

a r t i c l e

i n f o

Article history: Received 24 June 2011 Received in revised form 27 October 2011 Accepted 28 October 2011

Keywords: Nitrogen Urea Fiber Digestion Steers

a b s t r a c t The objective of two experiments was to evaluate non-protein N supplementation with protected urea sources in terms of rumen fermentation products, nutrient digestibility, and N balance in ruminally fistulated beef steers (initial bodyweight 239 ± 18 kg) fed switchgrass hay. Experiment 1 compared urea with Optigen II® , and Experiment 2 compared urea with RumaPro® . In both experiments, supplements (400 g/kg of daily dietary dry matter) were fed once daily or every 2 h in a balanced design. Supplements contained soybean hulls, corn grain, vitamins, and minerals as well as non-protein N sources. Non-protein N provided 0.18 g/g of dietary N. Switchgrass hay was fed once daily, at the same time as the supplement in the once-daily treatments. Dry matter intake (4.1 kg/d in Experiment 1, 4.5 kg/d in Experiment 2), dry matter digestibility (P<0.25, 0.58 ± 0.014 g/g in Experiment 1, 0.58 ± 0.010 g/g in Experiment 2), N balance (P<0.83, 11.3 ± 1.9 g/d in Experiment 1, 11.8 ± 3.6 g/d in Experiment 2), ruminal ammonia concentrations (P<0.29, 15.2 ± 1.4 mM in Experiment 1, 11.8 ± 0.6 mM in Experiment 2), and ruminal short-chain fatty acid concentrations (P<0.13, 77.7 ± 3.0 mM in Experiment 1, 75.4 ± 3.0 mM in Experiment 2) were not affected by feeding protected urea sources. Providing a steady supply of ruminally degradable N by feeding supplement every 2 h vs once daily decreased ruminal ammonia concentrations by approximately one-half by 4 h after feeding hay (P<0.01 in both experiments) and increased (P<0.02 in Experiment 1, P<0.08) in Experiment 2) apparent digestibility of dry matter (0.58–0.62 in Experiment 1, 0.56–0.61 in Experiment 2) and dietary fiber components. © 2011 Elsevier B.V. All rights reserved.

1. Introduction The rate and extent of structural carbohydrate fermentation in the rumen is affected not only by the amount of dietary N supply, but also by rate and extent of degradation of dietary and endogenous N supplies to the rumen (McAllan and Cockburn, 1988). Degradation of N in the rumen is a function of degradability of N sources and size and frequency of meals. Ruminal ammonia concentrations can fluctuate by a factor of 5–8 mM in animals consuming meals (Gustafsson and Palmquist, 1993; Robinson and McQueen, 1994; Horney et al., 1996; Rodriguez et al., 1997), with less (Shabi et al., 1998) to essentially no variation (Lana and Russell, 1997) as frequency of meals increase. Ruminal ammonia concentrations are positively related to

Abbreviations: CELL, cellulose; CP, crude protein; DM, dry matter; NIRS, near infrared reflectance spectroscopy; NPN, non-protein N; RDP, ruminal degradable protein; SCFA, short-chain fatty acids; SMD, standard error of the difference between means. ∗ Corresponding author. Tel.: +1 919 513 1693; fax: +1 919 515 4463. E-mail address: gerald [email protected] (G.B. Huntington). 0377-8401/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2011.10.012

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Table 1 Diet composition (g/kg DM). Experiment 1 Urea Switchgrass hay Supplement Soybean hulls Corn, cracked Soybean meal Optigen II RumaPro TraceMineral Premix Limestone Vitamins A, D, Ea Urea Calcium sulfate

a

Experiment 2 Optigen

Urea

Optigen

600.0

600.0

600.0

600.0

98.3 240.0 39.0 0.0 0.0 0.8 1.7 3.0 16.0 1.2

96.4 240.0 39.0 17.9 0.0 0.8 1.7 3.0 0.0 1.2

90.0 226.0 39.0 0.0 0.0 0.9 0.0 3.0 32.0 1.0

91.0 235.0 39.0 0.0 16.0 0.9 7.0 3.0 0.0 1.0

1000.0

1000.0

1000.0

1000.0

Added to provide 5000 IU A, 1100 IU D, and 2.2 IU E per kg of diet.

degradable or fermentable crude protein (CP) intake (Poore et al., 1993; Nagaraja, 1995; Taniguchi et al., 1995; Elizalde et al., 1999; Philippeau et al., 1999). Supply of exogenous urea (and subsequent increase in rumen ammonia) increased organic matter digestibility in steers fed sugarcane tops (Ortiz-Rubio et al., 2007), sheep fed rice straw (Hettiarachchi et al., 1999), grass-like browse (Lee et al., 1987) or mature bromegrass hay (Ferrell et al., 1999). Supplementation with soybean meal or a mixture of feather meal:blood meal (ruminally undegradable protein) also increased organic matter digestibility in the latter study. In terms of dynamic factors, ruminal ammonia concentrations decrease in response to factors that diminish ammonia production, such as intake of less degradable N sources (Horney et al., 1996; Lana and Russell, 1997; Rodriguez et al., 1997), or factors that promote use of ammonia, such as synchronization of fermentable energy and N (Petit and Veira, 1994; Kolver et al., 1998; Olson et al., 1999). Forages typically have elevated concentrations of readily degradable non-protein N (NPN), and may contain degradable protein concentrations that result in ruminal ammonia release that exceeds capture as microbial protein (NRC, 2000; Magee et al., 2005; Archibeque et al., 2001, 2002; Huntington et al., 2007; Huntington and Burns, 2008). However, mature forages can contain low concentrations of crude protein (Hettiarachchi et al., 1999; Lee et al., 1987; Ferrell et al., 1999), which are not conducive to ammonia release and optimal ruminal organic matter fermentation. Therefore, the goal of theses studies was to evaluate the ability of protected urea to improve fiber and N use by ruminants by slowing urea degradation in the rumen. 2. Materials and methods 2.1. Experiment 1 2.1.1. Animal and experimental procedures The experimental design was a crossover with a 2 × 2 factorial arrangement of treatments. Supplements containing one of two sources of supplemental N, urea or coated urea (Optigen II® , Alltech Inc., Lexington, KY, USA) were fed either once daily or every 2 h (12 times daily). All steers received both supplements in a balanced design, and feeding frequency was balanced in time, but feeding frequency of supplement did not change for a given steer. Therefore, the experiment had two periods. Alamo switchgrass (Panicum virgatum L.) hay was the base feed composing 600 g/kg of diet, with the remaining part a supplement to provide energy, protein, vitamins, minerals, and either urea or Optigen (Table 1). Prior to feeding, hay was processed through a Van Dale Bale Processor, Model S600 (J-Star Industries, Ft. Atkinson, WI, USA) with knives spaced 12.5 cm apart. Hay was fed once daily between 08:00 and 09:00. When supplement was fed once daily, it was fed at the same time as the hay. When supplement was fed every 2 h, it was delivered by automatic feeders for each steer. Eight crossbred, ruminally fistulated steers (initial bodyweight 239 ± 18 kg) were acclimated to working closely with people, familiarized with the handling facilities, and trained to lead by halter. Handling, sampling, and care of the steers were approved by the North Carolina State University Animal Care and Use Committee (IACUC # 06-114-A). Steers were placed in indoor, individual tie-stalls (115 cm × 178 cm) for the entire experiment with individual feeders and automatic waterers. Steers were adapted to the facilities with a diurnal pattern of 12 h of light and 12 h of darkness. Except during collection of urine and feces, steers had at least 2 sessions of unrestrained, outdoor exercise each week. After determination of ad libitum intake for each steer in a preliminary period, daily feed offered was maintained at that level. On day 10 of each 21-d period, a sterile catheter was placed in the exterior jugular vein of each steer, filled with sterile saline containing 3.5 g/L of sodium citrate, and secured with elastic bandage wrapped around the steer’s neck. On day 11, blood and rumen fluid samples were collected 30 min before, and 30, 60, 90, 120, 240, 480, and 720 min after the morning feeding. Blood samples were collected into heparinized tubes, centrifuged at 2500 × g for 15 min, and plasma

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was collected and stored frozen. Jugular catheters were removed after the last blood sample was collected. Ruminal fluid samples (approximately 100 mL for each collection) were collected with a manual vacuum pump, and strained through four layers of cheesecloth. Ruminal fluid pH was determined immediately after collection, then samples were chilled on ice and subsequently stored frozen at −20 ◦ C. The sensitivity of the pH was 0.1 pH units. Starting on day 14, all urine and fecal excretions were collected for 5 d. Procedures for collection, aliquot, and storage of urine and feces during the balance trial were those described by Matthews et al. (2005), except 6 N acetic acid was used to maintain urine 4 ≤ pH ≤ 6. Rectal temperatures were recorded daily throughout the balance trial for each steer; the overall average ± SD among steers was 38.2 ± 0.3 ◦ C. On day 21, rumen contents were evacuated through the rumen cannula as described by Álvarez et al. (2008). The contents were weighed, pH was recorded, and an aliquot was retained and stored frozen. Remaining ruminal contents were returned to the steer through the rumen cannula. 2.2. Experiment 2 2.2.1. Animal and experimental procedures The protocol used in Experiment 1 was repeated with the same steers (BW = 284 ± 20 kg), after the conclusion of Experiment 1. Feeding frequency for supplement (once daily or every 2 h) was retained for all steers. Treatments were the same except in this experiment the supplemental N sources (Table 1) were either urea or an aqueous solution of CaCl2 and urea (RumaPro® , Unipro International, 5626 W. 19th Street, Greeley, CO, USA). The overall average ± SD rectal temperature among steers was 38.5 ± 0.2 ◦ C. 2.3. Chemical analysis of feed, orts, and fecal samples All hay samples, supplement samples, orts, and fecal samples were dried to a constant weight at 60 ◦ C. The rumen content samples were dried in a freeze drier (VirTis Model USM-15, Gardiner, NY, USA). Hay, supplement, ort, and fecal samples were ground in a Wiley Mill (Thomas Scientific, Swedesboro, NJ, USA) to pass through a 1 mm screen. All of these samples were thoroughly mixed, subsampled, and stored in plastic bags until analyzed. Freeze-dried samples were stored in a freezer (−20 to −30 ◦ C). All samples were scanned in a near-infrared spectrophotometer (NIRS Model 5000, FOSS NIRSystems Inc., Laurel, MD, USA) and a global H statistic of ≤3.0 was used to define the spectral boundaries of the population. A neighborhood H statistic of ≤0.6 was used to identify samples whose spectra were not within the boundaries of our data libraries for hays, supplements, feces and orts. Those samples were then analyzed in the laboratory as described below. Dry matter (DM) was determined by Method # 934.01 of AOAC (1990) procedures. Neutral detergent fiber with heat stable amylase (aNDF), ADF, cellulose (CELL), lignin (sa), and acid insoluble ash were determined in sequential assays according to Robertson and Van Soest (1981) in a batch processor (Ankom Technology Corp., Fairport, NY, USA). Sulfite was not added to the NDF solution, and both NDF and ADF include residual ash. Crude protein was calculated as 6.25 times the N concentration as determined with an auto analyzer (Method # 976.06, AOAC, 1990). Composition of selected samples was then added to data libraries of feeds, ort, or fecal samples previously analyzed by near infrared reflectance spectroscopy (NIRS) and by chemical procedures described above, and concentrations of crude protein and fiber fractions were predicted by NIRS (Westerhaus et al., 2004). Prediction equations were generated with NIRS for the total population and predicted compositions of all feed, ort, and fecal samples were used in subsequent calculations and statistical analyses. All supplement samples in Experiments 1 and 2 were analyzed chemically for CP content. Those values were added to the NIRS data library, and crude protein concentration of supplements was predicted for all supplement samples. Rumen fluid was treated with m-phosphoric acid and centrifuged before short-chain fatty acids (SCFA) were determined by gas–liquid chromatography (Varian model 3800 model, Chromatography Systems Business, Sugar Land, TX, USA). The SCFA were analyzed using a NUKOL Fuses Silica capillary column with a 30 m × 0.25 mm × 0.5 ␮m film thickness (Supelco, Supelco Park, Bellefonte, PA, USA). Rumen fluid ammonia was determined by the automated procedure used for Kjeldahl N (Method # 976.06, AOAC, 1990). Urine and plasma were analyzed for urea N using the diacetyl monoxime method of Marsh et al. (1965) on a Technicon Auto Analyzer (Technicon Instruments Corporation, Tarrytown, NY, USA). Urine was also analyzed for total N and ammonia N using an auto analyzer (Method # 976.06, AOAC, 1990). 2.4. Statistical analysis All intake, digestion, and balance data, and prefeeding concentrations of plasma urea and rumen fluid ammonia data from Experiment 1 and Experiment 2 were analyzed using the PROC MIXED procedure of SAS (1990). The model accounted for supplemental CP source, supplement feeding frequency, CP source by frequency interaction, period, period × frequency interaction, and residual sources of variation. Steer within supplement feeding frequency was a random effect. Sampling time was included as a repeated measure in statistical analyses of plasma urea N and for ruminal concentrations of ammonia and short-chain fatty acids. Feeding frequency was tested by the test term ‘steer within N source’ while the remaining variables were tested against the residual.

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Table 2 Body weight (BW), and rumen dry matter (DM), plasma urea N, rumen fluid pH and concentrations of ammonia, short-chain fatty acids (SCFA), acetate (C2), propionate (C3), isobutyrate (iC4), n-butyrate (nC4), isocaproate (iC5), and n-valerate (nC5) in steers fed supplements (Supp) containing urea or Optigen once daily or every 2 h (Freq). Item

BW (kg) Rumen DM (kg) Rumen DM (g/kg content) Plasma urea N (mM) Rumen fluid pH Ammonia (mM) SCFA (mM) C2 (mol/100 mol) C3 (mol/100 mol) C2:C3 iC4 (mol/100 mol) nC4 (mol/100 mol) iC5 (mol/100 mol) nC5 (mol/100 mol) a

Once daily

SMDa

Every 2 h

P=

Urea

Optigen

Urea

Optigen

Supp

Freq

S×F

255 4.62 136 9.2

257 4.23 144 9.6

257 4.83 134 8.7

260 4.45 136 8.4

12 0.56 11.0 0.7

0.42 0.38 0.91

0.47 0.66 0.36

0.96 0.66 0.57

6.85 21.2 73.5 73.85 15.72 4.71 0.91 8.35 1.29 0.87

6.72 19.8 78.4 71.35 17.54 4.11 0.90 8.01 1.34 0.86

6.75 9.7 79.2 71.96 16.43 4.39 0.92 9.24 1.76 0.81

6.78 10.0 79.6 70.41 19.78 4.23 0.90 9.58 1.47 0.86

0.07 2.0 4.2 0.68 1.19 0.34 0.07 0.74 0.16 0.05

0.08 0.46 0.13 0.01 0.03 0.04 0.73 0.99 0.27 0.58

0.15 0.01 0.42 0.09 0.94 0.77 0.92 0.14 0.06 0.52

0.37 0.25 0.18 0.04 0.12 0.15 0.83 0.14 0.15 0.43

Standard error of the difference between means.

3. Results 3.1. Experiment 1 All steers consumed their ration of hay and supplement with essentially no feed refusals; the largest feed refusal was 32 g/d during the digestion and balance trial. Average ± SD for dry matter concentrations of CP, NDF and ADF in the 7 hay samples collected during the experiment were 42.4 ± 1.4 g/kg, 797 ± 4 g/kg, and 456 ± 3 g/kg, respectively. Concentrations of CP in the 8 samples of supplement containing urea or Optigen were 247 ± 7 g/kg and 231 ± 11 g/kg, respectively. Optigen supplementation decreased the proportion of acetate and increased the proportion of propionate in rumen fluid, and thereby decreased the acetate:propionate ratio (P<0.04; Table 2). The effect of Optigen on decreasing acetate proportion was greater when steers were supplemented once vs every 2 h (−3.4% vs −2.2%, respectively, supplement × feeding frequency interaction P=0.04). Rumen fluid pH was lower (P=0.08) when steers were fed Optigen vs urea (6.72 vs 6.78). Supplement did not affect rumen fluid ammonia N concentration (P<0.46, Table 2), including the time pattern postfeeding (Fig. 1). Feeding supplement every 2 h decreased rumen ammonia N concentration 52% when compared with once daily feeding of supplement. Additionally, feeding supplement every 2 h vs once daily decreased fecal output of DM, aNDF, and ADF, increasing apparent digestibility of DM by 6.1% (P=0.07; Table 3), and apparent digestibility of aNDF by 9% (P=0.06; Table 4), and removed the postfeeding peak in ruminal ammonia N, SCFA, and plasma urea N (Fig. 1). Treatments did not affect excretion or digestion of CELL (P>0.15). There were sampling time and sampling time by feeding frequency responses (P<0.01) for ruminal ammonia N, SCFA, and plasma urea N (Fig. 1). Feeding supplement every 2 h vs once daily also decreased (P=0.09) the molar proportion of C2 by 1.9%, but increased (P=0.06) the molar proportion of iC5 in rumen fluid by 26% (Table 2). Treatments had no effect on ruminal DM fill, DM proportion of rumen content, plasma urea N, or total SCFA concentrations (P>0.35, Table 2). Treatments did not differ in any of the measures of N excretion, digestion, or retention (P>0.12, Table 3). Urinary excretion of ammonia N was minimal, indicating successful preservation of urine during collection. Within steers and periods, N balance ranged from 4.1 to 17.8 g/d. 3.2. Experiment 2 All steers consumed their ration of hay and supplement with essentially no feed refusals (average daily amounts of 10 g or less); therefore, feed refusals were not included in calculations of intake and digestibility. Average ± SD concentrations of CP, aNDF and ADF in the 7 hay samples collected during the experiment were 414 ± 2.7 g/kg, 800 ± 4 g/kg, and 463 ± 4 g/kg, respectively. Concentrations of CP in the 7 samples of supplement containing urea or RumaPro were 240 ± 3 g/kg and 276 ± 4 g/kg, respectively. Compared with urea, RumaPro supplementation increased (P=0.03) rumen fluid concentrations of ammonia by 16% but feeding supplement every 2 h vs once daily decreased (P=0.01) ammonia concentration by 43%. RumaPro decreased (P<0.07) molar percentages of i-butyrate and n-valerate (Table 4). Compared with urea, RumaPro decreased (P=0.03) total SCFA concentrations in rumen fluid after adjustment for prefeeding values from 2.09 to 1. 94 mM. Treatments did not affect rumen fluid pH (P>0.15, Table 4). Feeding supplement every 2 h vs once daily decreased (P=0.01) fecal excretion and thereby increased (P≤0.04) apparent digestibility of DM by 9.1%, aNDF by 12.2%, ADF by 5.5%, CELL by 12.6%, and CP by 5.5% (Table 5). Increased feeding frequency decreased (P=0.01) rumen fluid ammonia concentration, but increased (P=0.01) plasma urea N and rumen fluid VFA

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Fig. 1. The pattern of ruminal ammonia N concentrations (A), ruminal short-chain fatty acid (SCFA) concentrations (B), and plasma urea N concentrations post feeding (C) of steers fed supplement containing urea fed once daily (1×) or every 2 h (12×), or Optigen fed once daily (1×) or every 2 h (12×). Concentrations are adjusted within steer for pre-feeding values. There were feeding frequency × time postfeeding interactions for all variables (P<0.01).

concentrations (Table 4). The effect of decreased ruminal ammonia concentrations was greater (P=0.05) for urea (52%) than for RumaPro (35%, supplement × feeding frequency interaction, Table 4). Sampling time by feeding frequency responses (P<0.04) for ruminal ammonia N, SCFA, and plasma urea N (Fig. 2) were caused by no postfeeding increase in those variables when steers were fed supplement every 2 h. Interactions between supplement and feeding frequency for fecal output of ADF (P=0.04), and apparent digestibilities of ADF (P=0.02) and CELL (P=0.03, Table 5) are due to a negative response with once daily supplement feeding but a positive response with supplement feeding every 2 h for steers when fed RumaPro than when fed urea. The decrease in rumen DM content with RumaPro when supplement was fed once daily was not evident when supplements were fed every 2 h (supplement by feeding frequency interaction, P<0.01, Table 4). There was a trend (P<0.15) for a similar interaction on DM percentage of ruminal content (Table 4). Greater intake of CP for steers when fed RumaPro

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Table 3 Dry matter intake during the digestion trial, fecal dry matter, fecal excretion and digestion of dry matter, aNDF, ADF, cellulose, and crude protein, urine N excretion, urine concentration of (NH4 N) and urea N, and N balance in steers fed switchgrass hay supplemented with urea or Optigen (Supp) fed once daily or every 2 h (Freq). Item

Once daily Urea

Dry matter Intake (kg/d) Feces (kg/d) Digestibility aNDF Intake (kg/d) Feces (kg/d) Digestibility ADF Intake (kg/d) Feces (kg/d) Digestibility Cellulose Intake (kg/d) Feces (kg/d) Digestibility Crude protein Intake (kg/d) Feces (kg/d) Digestibility Urine N (g/d) Urine NH4 N (g/kg total N) Urine urea N (g/kg total N) N balance (g/d) N balance (g/kg intake) N balance (g/kg digested) a

SMDa

Every 2 h Optigen

Urea

Optigen

P= Supp

Freq

S×F

4.08 1.65 0.594

4.08 1.78 0.565

4.08 1.58 0.613

4.08 1.56 0.617

0.01 0.08 0.020

0.26 0.33

0.07 0.07

0.16 0.20

2.38 1.17 0.507

2.44 1.26 0.486

2.38 1.12 0.529

2.44 1.09 0.550

0.02 0.05 0.024

0.45 0.91

0.06 0.06

0.15 0.14

1.35 0.69 0.485

1.34 0.73 0.457

1.34 0.66 0.510

1.34 0.63 0.532

0.01 0.03 0.226

0.95 0.84

0.04 0.04

0.18 0.17

1.16 0.57 0.511

1.17 0.56 0.513

1.17 0.56 0.520

1.17 0.49 0.581

0.01 0.04 0.03

0.16 0.21

0.17 0.17

0.27 0.27

0.489 0.158 0.677 41.3 28 800 11.7 149 219

0.488 0.168 0.656 41.7 24 764 9.5 121 182

0.489 0.150 0.693 42.9 19 723 11.3 144 233

0.492 0.151 0.693 41.8 23 736 12.7 162 210

0.009 0.009 0.021 3.4 9.7 38 2.8 35 5.3

0.40 0.49 0.79 0.98 0.61 0.82 0.81 0.83

0.14 0.13 0.79 0.48 0.15 0.55 0.56 0.66

0.49 0.51 0.62 0.60 0.32 0.31 0.29 0.35

Standard error of the difference between means.

Table 4 Body weight (BW), and rumen dry matter (DM), plasma urea N, rumen fluid pH and concentrations of ammonia, short-chain fatty acids (SCFA), acetate (C2), propionate (C3), isobutyrate (iC4), n-butyrate (nC4), isocaproate (iC5), and n-valerate (nC5) in steers fed supplements (Supp) containing urea or RumaPro once daily or every 2 h (Freq). Item

BW (kg) Rumen DM (kg) Rumen DM (g/kg content) Plasma urea N (mM) Rumen fluid pH Ammonia (mM) VFA (mM) C2 (mol/100 mol) C3 (mol/100 mol) C2:C3 iC4 (mol/100 mol) nC4 (mol/100 mol) iC5 (mol/100 mol) nC5 (mol/100 mol) a

Once daily

SMDa

Every 2 h

Urea

RumaPro

Urea

RumaPro

278 5.39 139 9.5

279 4.96 130 9.2

287 4.75 130 11.8

291 4.83 126 11.3

6.93 19.0 68.6 69.7 17.1 4.1 1.1 9.6 1.5 1.0

6.94 19.3 72.2 71.5 16.6 4.3 1.0 8.7 1.5 0.9

6.77 9.2 78.2 70.0 16.0 4.4 1.0 10.5 1.6 1.0

6.82 12.5 82.8 70.1 15.9 4.4 1.0 10.7 1.4 0.9

P= Supp

Freq

S×F

12 0.54 8.0 0.92

0.50 0.55 0.52

0.06 0.40 0.01

0.01 0.15 0.92

0.09 0.91 4.6 0.94 0.68 0.22 0.04 0.66 0.11 0.07

0.29 0.03 0.21 0.14 0.54 0.41 0.05 0.41 0.16 0.07

0.16 0.01 0.01 0.46 0.12 0.29 0.23 0.03 0.84 0.65

0.48 0.05 0.87 0.19 0.69 0.48 0.88 0.18 0.45 0.50

Standard error of the difference between means.

apparently is due to mixing errors of the supplement, because chemical analysis and desired proportions of ingredients indicated similar CP concentration. Urinary total N excretion (P=0.01), and urinary proportions of ammonia N (P=0.02) and urea-N (P=0.06) were greater for steers when fed RumaPro than when fed urea, but N balance in grams per day, or as a percentage of N intake or N digested, did not differ (P>0.05) between feeding frequency or supplement (Table 5). Urinary excretion of ammonia N was minimal, indicating successful preservation of urine during collection. There was appreciable variation in N balance, which ranged within steer and period from 1 to 25 g/d.

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Table 5 Dry matter intake during the digestion trial, fecal DM, fecal excretion and digestion of DM, aNDF, ADF, cellulose, and crude protein, urine N excretion, urine concentration of ammonia N (NH4 N) and urea N, and N balance in steers fed switchgrass hay supplemented with urea or RumaPro (Supp) fed once daily or every 2 h (Freq). Item

Once daily Urea

Dry matter Intake (kg/d) Feces (kg/d) Digestibility aNDF Intake (kg/d) Feces (kg/d) Digestibility ADF Intake (kg/d) Feces (kg/d) Digestibility Cellulose Intake (kg/d) Feces (kg/d) Digestibility Crude protein Intake (kg/d) Feces (kg/d) Digestibility Urine N (g/d) Urine NH4 N (g/kg total N) Urine urea N (g/kg total N) N balance (g/d) N balance (g/kg intake) N balance (g/kg digested) a

SMDa

Every 2 h RumaPro

Urea

RumaPro

P= Supp

Freq

S×F

4.47 1.98 0.557

4.47 1.98 0.558

4.47 1.78 0.602

4.47 1.73 0.614

0.0 0.06 0.014

0.34 0.25

0.01 0.01

0.96 0.31

2.60 1.39 0.472

2.57 1.40 0.454

2.60 1.25 0.519

2.59 1.23 0.520

0.02 0.04 0.017

0.79 0.28

0.01 0.01

0.19 0.22

2.39 0.82 0.656

2.36 0.85 0.640

2.39 0.76 0.680

2.36 0.74 0.687

0.01 0.02 0.086

0.81 0.26

0.01 0.01

0.04 0.02

1.29 0.76 0.413

1.28 0.82 0.359

1.29 0.76 0.397

1.29 0.68 0.472

0.01 0.03 0.019

0.82 0.59

0.01 0.01

0.01 0.03

0.544 0.180 0.669 48.4 64 714 9.8 112 162

0.608 0.172 0.717 63.3 46 841 6.4 66 92

0.544 0.154 0.717 48.9 32 844 13.4 154 215

0.608 0.152 0.750 55.2 41 812 17.7 181 242

0.007 0.009 0.016 4.6 17 66 5.1 54 76

0.29 0.01 0.01 0.02 0.06 0.89 0.78 0.63

0.02 0.04 0.36 0.31 0.42 0.12 0.13 0.16

0.51 0.27 0.16 0.28 0.20 0.26 0.30 0.32

Standard error of the difference between means.

4. Discussion Results of both experiments support our hypothesis on the effects of synchronization of fermentable energy and nitrogen in the decreasing postfeeding concentrations of ruminal ammonia, with concomitant decreases in blood urea concentrations. Similarly, both experiments also show the effect of feeding frequency on increased digestion of fiber components, but no influence of N supplement type. Additionally in both experiments rumen ammonia concentration was more depleted by feeding supplement every 2 h (approximately 50%) than for N supplement type. Albeit minimal, Optigen or RumaPro consistently increased aNDF, ADF or CELL digestion when fed every 12 h. Thus, response to slow-release N is related to feeding frequency. The important influence of restricted DM intake level or continuous infusion of N supplements on organic matter digestion has been reported by Rihani et al. (1993). In both experiments, prefeeding concentrations of rumen fluid ammonia N (P>0.11) and plasma urea N (P>0.65) were similar for N supplements. For Experiments 1 and 2, experimental prefeeding rumen fluid ammonia N and plasma urea N concentrations were 8.5 and 9.0 mM and 8.2 and 8.5 mM, respectively. However, feeding supplement every 2 h vs once daily increased (P<0.01) prefeeding concentrations of rumen fluid ammonia N (10.5 vs 6.5 mM in Experiment 1, 10.5 vs 5.9 in Experiment 2) and plasma urea N (7.8 vs 6.4 mM (P<0.07) in Experiment 1 and 11.0 vs 6.4 mM (P<0.01) in Experiment 2). Therefore, feeding supplement every 2 h vs once daily increased ruminal ammonia N and plasma urea N concentrations between meals, but eliminated the post-feeding increase in those variables (Figs. 1 and 2). We expected to see decreased ruminal ammonia concentrations, increased fiber digestibility, and possibly increased N retention in response to feeding the protected urea sources compared with urea. This was based on published responses with the feeding of Optigen (Garcia-Gomez et al., 2007) or dosing cattle with RumaPro (Huntington et al., 2006). Approximately 0.46 of the supplemental protein or 0.18 of total CP consumed was provided by urea or protected urea. Switchgrass has high proportions of ruminally degradable protein (RDP), 0.50–0.55 of CP (Archibeque et al., 2001). Therefore, the calculated dietary proportions of RDP and ruminally undegradable protein were approximately 1:1. The putative RDP proportion in the diet is within the range of, or less than, proportions in which dietary changes in RDP affected ruminal ammonia concentrations in dairy cows (Davidson et al., 2003; Broderick and Renal, 2009) or other cattle (Coomer et al., 1993). Therefore, it is possible that our diets did not contain adequate proportions of RDP to allow detection of a difference between supplementation with the protected urea sources compared with urea. It is also possible that transfer of urea from blood or saliva into the rumen, and subsequent hydrolysis of urea to ammonia, masked differences in RDP of the supplements (Huntington and Archibeque, 1999). Data from dairy cows do not link greater milk yield in cows fed Optigen to decreasing RDP by addition of Optigen

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Fig. 2. The pattern of ruminal ammonia N concentrations (A), ruminal short-chain fatty acid (SCFA) concentrations (B), and plasma urea N concentrations post feeding (C) of steers fed supplement containing urea fed once daily (1×) or every 2 h (12×), or RumaPro fed once daily (1×) or every 2 h (12×). Concentrations are adjusted within steer for pre-feeding values. There were feeding frequency × time postfeeding interactions for all variables (P<0.04).

to the diet (Inostroza et al., 2010). Decreased rumen fluid concentrations of the isobutyrate and n-valerate in response to feeding RumaPro are consistent with decreased amino acid degradation in the rumen. 5. Conclusion We conclude that the hypothesis of improving fiber digestion by creating a constant release of nitrogen in the rumen is supported by the positive effects of feeding supplement every 2 h on ruminal concentration pattern of ammonia and apparent digestibilities of DM and fiber components. Interestingly, feed supplement every 2 h did not improve N balance, which is consistent with a nutrient other than protein, or energy, being the primary limiter of growth. The ruminal ammonia and plasma urea patterns do not support the concept that Optigen or RumaPro changed the pattern of degradation of dietary N in the rumen. However, both affected microbial fermentation in the rumen as evidenced by changes in concentrations of SCFA in ruminal fluid.

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Acknowledgements This research was supported in part by Alltech Inc., Lexington, KY, USA, and Unipro International, 5626 W. 19th Street, Greeley, CO, USA.

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