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ANIMAL NUTRITION |
* Department of Animal Sciences, University of Florida-IFAS, Gainesville 32611; and
University of Florida-IFAS, Range Cattle Research and Education Center, Ona 33865
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: citrus pulp • fecal nitrogen • fermentation • protein • steer
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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). Dried citrus pulp (DCP) is typified (Arthington et al., 2002; Bampidis and Robinson, 2006) by high concentrations of pectin (22 to 40%) and a high Ca:P ratio (3.5 ± 1.7% Ca to 0.34 ± 0.07% P), and relatively low concentrations of CP (7.2 ± 0.2%), fat (3 ± 1.0%), NDF (19.3 ± 1.3%), and ADF (16.9 ± 2%; DM basis). Previous studies concluded that up to 40% of dietary corn grain could be replaced with DCP without compromising digestibility, ruminal function, or performance in ruminants (Vijchulata et al., 1980; Wing, 1982; Bueno et al., 2002).
Pectin is the predominant carbohydrate in DCP, and it is quickly and extensively degraded by ruminal bacteria (Dehority, 1969; Sniffen and Robinson, 1987; Sunvold et al., 1995). Pectin supplementation or abomasal infusion can reduce urinary N excretion in ruminants (Mason et al., 1981; Gressley and Armentano, 2005) and mitigate ammonia toxicity arising from intake of excess urea due to its high fermentability and acetate-stimulating properties (Wing, 1982). Citrus pulp supplementation may improve N utilization and performance of ruminants, but the response most likely depends on the form of N supplied in the diet. Effects of different forms of supplemental N on performance of cattle fed citrus pulp diets have not been documented.
Our objectives were to compare the effect of supplemental N source on the fermentation of DCP and performance of growing steers fed DCP and grass hay.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Exp. 1: In Vitro Fermentation
In the first of 3 experiments, the effect of supplemental N source on the fermentation of citrus pulp in rumen fluid was evaluated. Dried citrus pulp alone (control), and isonitrogenous (15% CP, DM basis) mixtures of DCP and solvent soybean meal (SBM), expeller soybean meal (SP; SoyPLUS, West Central Cooperative, Ralston, IA), or urea (UR) were ground through a 1-mm screen in a Wiley mill (Arthur H. Thomas, Philadelphia, PA). Substrates were analyzed for rate and extent of fermentation gas production using the method of Adesogan et al. (2005). Five replicates of each substrate were incubated in buffered rumen fluid for 24 h within 250-mL Pyrex bottles (No. 1395, Fisher Scientific, Pittsburgh, PA) fitted with radio-frequency pressure sensors (Point Six Wireless, Lexington, KY). The culture bottle-pressure sensor assembly was examined for leakage on the day before inoculation by injecting 103 kPa of air into the sensors and monitoring the pressure overnight.
The rumen fluid was collected by aspiration before the morning feeding from 2 nonlactating, nonpregnant Holstein cows fed Coastal Bermudagrass [Cynodon dactylon (L) Pers.] hay for ad libitum intake and 900 g/d of SBM. Rumen fluid was filtered through 2 layers of cheesecloth and immediately transported to the laboratory in a prewarmed thermos flask, where it was mixed (1:2 ratio) under a CO2 stream with warmed (39°C) artificial saliva (McDougall, 1948). Leak-tested culture bottles containing 0.5 g of sample were placed in a 39°C water bath, and 40 mL of the rumen fluid/culture medium solution was added to each bottle under a CO2 stream. The bottles were sealed and placed in an incubator at 39°C, and the pressure sensors were set to measure gas pressure hourly for 24 h. The pressure sensors contained built-in microprocessors and radio transmitters that sent digital pressure data via radio frequency signals to an integrated web server (Point Server, Point Six Wireless). Data were downloaded from the Web server into Excel using Pointware software (Point Six Wireless). Pressure data were converted to volume equivalents using a regression equation developed by injecting known volumes of CO2 into the culture bottles and measuring the corresponding pressures with a digital manometer (No. 840084, Sper Scientific, Scottsdale, AZ).
Chemical Analysis.
After fermentation, bottle contents were filtered through Whatman No. 541 filter paper (Fisher Scientific), and the residues were dried at 60°C for 48 h to measure DM digestibility. The pH of the filtrate was measured immediately (Corning Model 12, Corning Scientific Instruments, Medfield, MA), and 0.5 mL of 9 M H2SO4 was added to prevent further fermentation. The filtrate was centrifuged at 4°C at 10,400 x g for 15 min, and the supernatant was analyzed for ammonia-N and VFA. The VFA concentration was measured after filtering the supernatant through a 0.22-µm syringe filter (Fisher Scientific) with the method of Canale et al. (1984), and an HPLC (FL 7485, Hitachi, Tokyo, Japan) system. The HPLC system was coupled to an autosampler (L 7200, Hitachi) and a UV detector (Spectroflow 757, ABI Analytical Kratos Division, Ramsey, NJ) set at 210 nm. The supernatant (20 µL) was injected into a BioRad Aminex HPX-87H column (BioRad Laboratories, Hercules, CA) containing a 0.015 M H2SO4 mobile phase flowing at a rate of 0.7 mL/min at 45°C. The peaks were identified by comparison with known standards, and the peak areas were used to quantify VFA. Ammonia-N concentration was determined using an adaptation of the Noel and Hambleton (1976) procedure for the Alpkem Auto Analyzer (Alpkem Corp., Clackamas, OR).
Exp. 2: Feed Intake and Apparent Digestibility In Vivo
Animals, Diets, and Feeding Regimen.
Experiment 2 determined the effect of supplemental N source on feed intake, digestibility, and plasma metabolite concentration in beef steers fed basal diets of hay from a 9-wk regrowth of bahiagrass (Paspalum notatum), at the University of Florida-IFAS Range Cattle Research and Education Center (Ona, Florida). The hay contained 92.5% DM, 5.4% CP, 78.4% NDF, and 41.9% ADF (DM basis). Four yearling Braford steers with 254 ± 22 kg of BW were randomly assigned to 1 of 4 DCP-based supplements fortified with no N supplement (control) or supplements in which CP was predominantly supplied from UR, SBM, or SP. Protein-supplemented diets were formulated to be isonitrogenous (11.9% CP), and all diets were formulated to be isocaloric (66% TDN; DM basis) and to meet the nutrient requirements of the steers (NRC, 2000), except that protein requirements were not met by the control diet. Urea, SBM, and SP accounted for 44, 39, and 32% of total dietary CP and 82, 65, and 54% of total supplement CP in the respective diets (DM basis). Sulfur was not limiting in the urea diet.
The experiment had a 4 x 4 Latin square design with four 21-d periods, each consisting of 14 d of diet acclimation followed by 7 d of total feces collection. Steers were individually housed in covered (15 m2) pens and fed concentrates (3.3 kg/d) in 2 equal meals at 0900 and 1700 h during the adaptation period. During the 7-d trial period, steers were housed in metabolism crates, and daily fecal output was collected in a pan placed behind the crates. The bahiagrass hay was ground to pass a 2.5-cm screen using a hay grinder (Haybuster 2544, Haybuster Agricultural Products, Jamestown, ND) and offered separately for ad libitum consumption. Steers had free-choice access to water and trace mineral-containing salt blocks throughout the study.
Sampling and Analysis. The concentrates and hay were subsampled each week and frozen (–25°C) until analyzed. To determine daily DMI, the refused concentrates and hay were collected just before the morning feeding and analyzed for DM. The daily total fecal output of each steer was weighed, mixed, subsampled, composited for each period, and subsampled again for analysis of DM, CP, and NDF. On d 3 and 5 of each collection period, blood samples were collected by coccygeal venipuncture into evacuated tubes containing sodium heparin anticoagulant (Becton Dickinson, Franklin Lakes, NJ) and immediately placed on ice. The tubes were centrifuged at 2,000 x g for 30 min, and the plasma was frozen at –20°C until analyzed for plasma urea N and glucose.
Chemical Analysis.
The diet, orts, and feces samples were dried at 60°C for 48 h to determine DM, and then ground through a 1-mm screen in a Wiley mill. Crude protein concentration was calculated as N x 6.25, with N measured with a Vario Max CN Elemental N analyzer (Elementar Americas Inc., Mt. Laurel, NJ). Concentrations of NDF and ADF were measured with an Ankom200 Fiber Analyzer (Ankom Technology, Macedon, NY) using adaptations of the methods of Van Soest et al. (1991). Amylase and sodium sulfite were used for NDF determination, and the results were expressed on a DM basis. Plasma glucose was analyzed with kit B-7551-120 (liquid glucose oxidase reagent set, Pointe Scientific Inc., Lincoln Park, MI), and plasma urea was analyzed with kit G-7521-120 (Urea Nitrogen Berthelot method, Pointe Scientific Inc.).
Exp. 3: Growth Performance
Experiment 3 determined the effect of supplemental N source on ADG of steers fed the same hay and the same control, SBM, and UR supplements as used in Exp. 2. The SP supplement was not included in this trial because it produced results similar to those of the SBM supplement in Exp. 1 and 2. Twenty-four yearling Brahman x British crossbred steers, with an average BW of 261 ± 25 kg, were randomly assigned to 1 of the 3 treatments, housed in individual covered pens (15 m2), and fed the experimental diets for 56 d. Steers were adapted to the diets in the first 2 wk, and shrunk BW were measured after a 16-h fast on d 0 and 56. The hay and supplements were offered separately, at the same rates as in Exp. 2, at 0800 h daily.
Statistics
The gas production parameters in Exp. 1 were described with the model of McDonald (1981), which has the form Y = A + B (1 – e – c(t – L)), where Y = gas pressure at time t, A = the immediately fermentable fraction, B = the potentially fermentable fraction, A + B = the total fermentable fraction, c = the fractional fermentation rate of B, t = the time incubated, and L = the lag phase. The NLIN procedure (SAS Inst. Inc., Cary, NC) was used to fit the model to the data, and the parameters of the model were analyzed using the GLM procedure of SAS for a completely randomized design. Orthogonal contrasts were used to compare the effect of N supplementation (control vs. N supplements) and N source (SBM + SP vs. UR; SBM vs. SP).
Data from Exp. 2 were analyzed with the MIXED procedure of SAS for a Latin square design with 4 treatments, 4 steers, and 4 periods. The model statement included the effects of treatment and period and the random statement contained the effect of steer. The data were compared using the same orthogonal contrasts as used for Exp. 1.
Data from Exp. 3 were analyzed with the MIXED procedure of SAS for a completely randomized design. A repeated measures statement was used to analyze the DMI data. The model statement included the effects of treatment, week, and their interaction, and the random statement included the effect of steer nested within treatment. Steer was the experimental unit. The data were compared using orthogonal contrasts similar to those for Exp. 1 and 2.
A P-value < 0.05 was considered significant, and values of 0.06 to 0.10 were considered tendencies toward significance.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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The ingredient and chemical compositions of the Exp. 1 substrates are shown in Table 1. Nitrogen supplementation tended to increase (P < 0.10) IVDMD and the potentially and total fermentable fractions (Table 2). It also increased (P < 0.05) the immediately fermentable fraction, but decreased the lag phase (P < 0.01) and fermentation rate (P < 0.10). The increases in fermentable fractions and IVDMD probably reflect increased microbial protein synthesis (MPS), which typically improves digestibility (Olmos Colmenero and Broderick, 2006). Therefore, N supplementation is important for increasing ruminal fermentation and nutrient utilization from DCP. The decreased fermentation rate due to N supplementation reflects the slower fermentation rates for SP and UR compared with the control.
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). Nevertheless, gas production results from the urea substrate should be cautiously interpreted because the batch culture used did not permit the normal rapid outflow of urea from the rumen. Gas production kinetics were similar between soy-based substrates except that SBM fermented at a faster (P < 0.001) rate than SP. This may reflect the slower rate of carbohydrate fermentation with SP or processing of SP to reduce ruminal degradability (Maiga et al., 1996; Gonzalez et al., 2002).
Supplemental N source did not affect IVDMD. This finding supports results from in vivo (Fujimaki et al., 1989) and continuous culture (Griswold et al., 1996) studies in which cell-wall carbohydrate digestion was unaffected by replacing peptides or AA with urea or NH3. Such results are typically due to deamination of AA to release NH3, which is the primary N source for structural carbohydrate-fermenting bacteria (Russell et al., 1992). Similar IVDMD results across treatments may also reflect the ability of pectin-fermenting bacteria to use different forms of N (Dehority, 1969).
In Vitro Fermentation Characteristics
Concentrations of NH3-N (Table 3) exceeded the threshold at which ruminal digestion (8 mg/dL; Hoover, 1986) and MPS are maximized (5 mg/dL; Satter and Slyter, 1974). The molar proportions of VFA were similar to that typical for forage- or citrus pulp-based diets (Ariza et al., 2001). The absence of lactic acid accumulation and relatively high acetate to propionate ratio also reflect the fermentation of NDF and pectin (Marounek et al., 1985; Strobel and Russell, 1986; Hall et al., 1998), which collectively account for approximately half of the chemical composition (DM basis) of citrus pulp.
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). The pH increase largely reflects the corresponding increase in the NH3-N:total VFA ratio (0.08, 0.13, 0.12, and 0.14 mg/(dL·mM) for control, SBM, SP, and UR, respectively). Nevertheless, the pH values were normal and pH differences between treatments were likely biologically insignificant. Nitrogen supplementation also decreased (P < 0.05) the proportion of acetate and increased (P < 0.01) the proportion of propionate, thereby reducing (P < 0.001) the acetate:propionate ratio. Therefore, N supplementation improved the efficiency of ruminal fermentation probably because of increased MPS.
Urea supplementation produced more alkaline pH values (P < 0.05) and greater concentrations of NH3-N (P < 0.01) and total VFA (P < 0.10) than supplementation with SBM and SP. The difference in NH3-N concentration between substrates supplemented with UR vs. SP or SBM was small. However, it suggests that care is needed when supplementing with urea vs. natural protein sources due to increased risk of NH3 toxicity (Villar et al., 2003). Excess ruminal NH3 can also contribute to urinary urea excretion and thereby increase environmental N pollution (Kebreab et al., 2002).
For unknown reasons, supplementation with SBM produced a lower molar proportion of propionate (P < 0.01), a greater (P < 0.05) molar proportion of butyrate, and a greater (P < 0.10) acetate:propionate ratio than SP supplementation. Other than these differences and a greater fermentation rate for SBM, supplementation with SP vs. SBM did not affect in vitro fermentation or digestion.
Feed Intake, Apparent Digestibility, and Plasma Metabolites
The ingredient and chemical composition of the supplements fed in Exp. 2 and 3 are shown in Table 4. Nitrogen supplementation increased (P < 0.05) concentrate, hay, and total DMI, as well as intakes of N, NDF, and ADF (Table 5). Apparent digestibility of DM, N, and ADF as well as plasma urea N concentration increased (P < 0.05) with N supplementation, as did NDF digestibility and plasma glucose concentration (P < 0.10). The improved intake and digestibility associated with N supplementation likely reflects increased N supply for ruminal microbial activity and synthesis. Diets with high CP or rumen-degradable protein concentrations usually have greater apparent digestibility of OM and N due to increased intake of more digestible feeds and increased NH3-N synthesis for incorporation into microbial protein (Broderick, 2003; Olmos Colmenero and Broderick, 2006). Plasma urea N concentrations were increased by N supplementation because they are usually positively correlated to concentrations of dietary CP, rumen-degradable protein, and ruminal NH3- N concentration (Broderick and Clayton, 1997; Hristov et al., 2004; Olmos Colmenero and Broderick, 2006).
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) or to the lower level of molasses in the UR diet. Although the greater DCP level in the UR diet can also be implicated, a review of several feeding studies concluded that supplemental citrus byproducts do not affect feed intake in ruminants (Bampidis and Robinson, 2006). In contrast, other studies have found that feed intake is increased by molasses supplementation (Brown, 1993; Royes et al., 2001) or by replacing urea with SP or SBM in cattle diets (Ludden and Cecava, 1995; Ludden et al., 1995).
Steers fed UR had lower (P < 0.05) N and ADF digestibilities, and less (P < 0.01) plasma urea and glucose concentrations than steers fed soy-based supplemental N. These differences are consistent with previous studies (Lana et al., 1997; Robinson et al., 1998) and suggest that MPS was compromised in steers fed the UR supplement most likely due to the rapid hydrolysis and outflow of urea from the rumen. Less CP was apparently digested in steers fed UR vs. SP or SBM, although fecal N excretion was less for the UR diet because steers fed UR consumed less N. Although fecal N can be used as an organic fertilizer, increased production of fecal N is economically and environmentally undesirable. Future studies should evaluate the effect of supplemental N source for DCP and forage rations on urinary N excretion, which is a greater source of environmental pollution than fecal N.
Expeller soybean meal has outperformed SBM at increasing animal performance in some studies (Broderick, 1986; Dado et al., 1990) but not others (Ludden et al., 1995). As in Exp. 1, digestibility was similar for SBM and SP in Exp. 2, and feed intake or plasma metabolite concentrations were similar between SBM and SP. This may reflect the fact that that there was only a small difference in degradable intake protein supply from the SP and SBM supplements, or that both supplements exceeded the degradable intake protein requirement. Nevertheless, rather than a specific requirement for undegradable intake protein, cattle require specific AA that may be more efficiently utilized if they are supplied postruminally rather than ruminally. Titgemeyer and Loest (2001) noted that studies demonstrating a response to undegradable intake protein supplementation by forage-fed cattle typically reflect improved metabolizable AA supply. Methionine, Lys, His, Leu, and Val have been described as the AA most likely to limit performance of forage-fed growing ruminants (Titgemeyer and Loest, 2001). Modeling with the Cornell Net Carbohydrate and Protein System (CNCPS, Release 5.0; Dept. Anim. Sci., Cornell University, Ithaca, NY) suggests that Met- and Lys-allowable gain for control, UR, SBM, and SP diets averaged 0.0 ± 0.0, 0.58 ± 0.02, 0.96 ± 0.01, and 0.78 ± 0.01 kg/d, respectively. Therefore, the control diet was predicted to be limiting in Met and Lys, but the others were not. Soy-based diets supplied more Lys and Met than did the UR diet. Ludden and Cecava (1995) found that feeding SP instead of SBM did not improve the flow of metabolizable AA to the small intestine.
Growth Performance
In Exp. 3, N supplementation increased (P < 0.05) feed intake as well as final BW, ADG, and G:F (Table 6). Steers fed SBM had greater (P < 0.05) DMI (kg/d) and ADG than those fed the UR diet. These results support those from Exp. 1 and 2 by indicating that N supplementation improved the performance of the steers. This was likely due to improved N and AA flow to the small intestine (Titgemeyer et al., 1989; Coomer et al., 1993), which can improve the growth of steers (Stock et al., 1983; Goedeken et al., 1990). However, supplementation with SBM was more effective than supplementation with UR. Our results support those of Stock et al. (1983), Pate et al. (1990), and Ludden et al. (1995), who reported that natural or true protein sources are better sources of supplemental N than urea for cattle.
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1 Corresponding author: adesogan{at}ufl.edu
Received for publication January 23, 2007. Accepted for publication May 22, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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