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ENVIRONMENT, WELL-BEING, AND BEHAVIOR |
USDA-ARS, Animal Waste Management Research Unit, Bowling Green, KY 42104
1 Corresponding author: mrothrock{at}ars.usda.gov
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: ammonia • poultry litter • quantitative real-time polymerase chain reaction • urease
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Uric acid and urea constitute approximately 70% of the total N found in poultry litter (Shuler et al., 1979; Koerkamp, 1994). Microbial decomposition of these organic N sources leads to the production and subsequent volatilization of NH4-N. One of the major limiting factors in the final stages of this decomposition reaction is the activity of the bacterial urease enzyme (Nahm, 2003), a nickel-containing trimeric enzyme that catalyzes the hydrolysis of urea to NH4-N and CO2. Most research dealing with bacterial ureases has focused on its role in the pathogenesis of urogential and gastroduodenal disease, including those caused by Helicobacter pylori, Ureaplasma urealyticum, and Proteus mirabilis (Mobley and Hausinger, 1989; Mobley et al., 1995; Burne and Chen, 2000). Microbial populations from poultry litter have been characterized by both culture-dependent (Nodar et al., 1990; Martin et al., 1998) and independent means (Lu et al., 2003; Enticknap et al., 2006). Although urease activity has been measured in poultry litter (Tejada et al., 2006, 2007), no data are available on the organisms responsible for urease production in poultry litter.
The goal of this study was to better characterize urease-producing microbes in poultry litter. The first objective was to identify the ureolytic microbial community in poultry litter by specifically targeting the urease gene using general ureC (largest structural subunit of the urease enzyme) PCR primers (Koper et al., 2004) and cloning and sequencing the resultant PCR products. A clone library was constructed to identify the dominant group(s) of urease producers in poultry litter. The second objective was to develop methods to specifically detect these dominant ureolytic group(s) from poultry litter using quantitative real-time PCR (QRT-PCR). Quantitative real-time PCR is a molecular microbiological technique that permits rapid, sensitive detection of target organisms in environmental samples. The abundance of a target sequence is determined by measuring PCR products as they are accumulating and quantifying the amount of target while the PCR reaction is still in the exponential range (Brunk et al., 2002; Ginzinger, 2002). Products are detected and quantified as they are generated during each PCR cycle through the use of fluorescently labeled probes and standards run along with samples. The advantage of the fluorogenic probes is that specific hybridization between probe and target is required to generate fluorescent signal. The polymerase will cleave the probe only while it remains hybridized to its complementary strand. It has been found that QRT-PCR has been used to target human pathogens in waste materials (Ibekwe et al., 2002, Inglis and Kalischuk, 2004) and to characterize bacteria in the rumen (Tajima et al., 2001) and in the human and swine intestinal tract (Hill et al., 2005). The final objective of this study was to use the developed assay to compare the concentrations of this novel group of ureolytic microbes from diverse poultry litter types, varying in location, bedding material, and physiochemical parameters, and determine which biological and physiochemical parameter directly correlated to or influenced the concentrations of this novel group in those poultry litters.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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–20°C) until sample processing. The Kentucky and Oklahoma poultry litter samples were bulk samples representative of their poultry houses, whereas the Mississippi litter samples were obtained as part of a spatial variability study within a single house (Lovanh et al., 2007). Ten subsamples from sampling sites throughout the Mississippi house were pooled to obtain a representative sample for the entire house.
Molecular Analysis of ureC Genes in Poultry Litter
Deoxyribonucleic acid was extracted from poultry litter samples (0.3 g) using the Q-Biogene FastDNA Spin Kit for soil (Q-Biogene, Irvine, CA) according to the specifications of the manufacturer. Urease genes (ureC) were specifically amplified from 2 µL of the 1:500 dilution of the community DNA extract using the ureC1F/ureC2R primer set (Table 1
) using thermocycling conditions as previously described (Koper et al., 2004) using a PTC-200 DNA thermal cycler (MJ Research, Las Vegas, NV). Sequences were amplified using Qiagen HotStarTaq Master Mix (Qiagen Inc., Valencia, CA), with 800 nM of each primer and 1 to 10 ng of template DNA from the genomic extracts of poultry litter. Annealing temperatures of 58 and 60°C were used in 2 separate cloning reactions to determine if annealing temperature had an effect on the resultant ureC clone libraries. The PCR products were cloned into the pCR2.1-TOPO plasmid using a TA TOPO Cloning Kit (Invitrogen, Carlsbad, CA) according to the specifications of the manufacturer and were sent to the USDA-ARS MSA Genomics Laboratory (Stoneville, MS) for sequencing. The new ureC sequences, combined with appropriate known ureC sequences from the GenBank database, were aligned using MEGA version 3.1 (Kumar et al., 2004). A 338-bp region of the alignment containing data from all sequences was selected for further phylogenetic studies. The alignment files were used to create bootstrapped (n = 1,000) neighbor-joining trees, using the Kimura 2-parameter model in the MEGA version 3.1 software package.
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). The primers were obtained from Sigma Genosys (St. Louis, MO), and the dual-labeled Black Hole Quencher probe was prepared by Biosearch Technologies Inc. (Novato, CA). The annealing temperature (58 to 70°C), primer (300, 600, 900 nM) and probe (50, 100, 200 nM) concentrations, and MgCl2 (3 to 5 µM) concentrations were optimized for this assay. The QRT-PCR assays were run on the DNA Engine Opticon 2 (MJ Research Inc., Waltham, MA) and were carried out using the Qiagen HotStar-Taq Master Mix (Qiagen) in a total volume of 25 µL. The amplification mixture contained 3.0 mM of MgCl2, 300 nM of each primer and 200 nM of probe, and sample DNA or standard (from 101 to 108 copies). The QRT-PCR program was 15 min at 95°C followed by 35 cycles at 95°C for 20 s and 63°C for 20 s. Baseline values were set as the lowest fluorescence signal measured in the well over all cycles. The baseline was subtracted from all values, and the cycle threshold (CT) was set to 1 standard deviation of the mean. A positive result was determined to be amplification of product at a CT value 33, which represented 1 cycle beyond the average CT (31.99) for the 101 ureC standard. Template DNA consisted of DNA extract (5 µL of a 1:500 dilution) run in triplicate, with all PCR runs including duplicates of standards and control reactions without template (repeatability). All reactions were run on at least 2 different plates (reproducibility). Standard DNA consisted of plasmid PCR 2.1 vector (Invitrogen) carrying a ureC insert from a larger fragment of a representative ureC poultry litter clone, contained within our novel group (338 bp), and amplified using the ureC1F/ureC2R primer set (Koper et al., 2004).
QRT-PCR Efficiency and Sensitivity
The DNA concentration of a representative poultry litter extraction was determined using the Hoechst 33258 nucleic acid stain (Invitrogen) and measured with a Hoefer DyNA Quant 200 fluorometer (Amersham Biosciences, San Francisco, CA) according to the instructions of the manufacturer. A series of dilutions of the poultry litter extract (1,000 ng to 0.1 ng of template DNA per PCR reaction) were amplified using the designed assay, as were plasmid standards containing 108 to 101 copies of the ureC insert. No inhibition was observed using DNA template concentrations between 0.1 and 100 ng per 25 µL of PCR reaction. To determine the amplification efficiency of this assay, the original number of ureC copies per PCR reaction was compared with the CT value for that sample, and a regression analysis was performed. Amplification efficiency was calculated using the following formula: E = 10(–1/x) – 1 x 100, where x = the slope of the regression line. Assay efficiency of the poultry litter extracts and the plasmid standards were compared.
To determine the specificity of this assay for ureC sequences found in poultry litter, DNA was extracted (using the above procedure) from environmental samples, including cow manure, soil, swine lagoon slurry, and water samples. Fifty to 150 pg of genomic DNA extract was analyzed using the ureC QRT-PCR assay described above. Five pure cultures were analyzed to further determine the specificity of the assay. Bacillus subtilis ATCC 6633, Lactobacillus fermentum ATCC 9338, and Staphylococcus aureus ATCC 25923 were obtained by growing cultures from Kwik-Stik (Microbiologics Inc., St. Cloud, MN) on Bacto-Tryptic Soy Broth (TSB) agar (BD, Franklin Lakes, NJ) with 5% sheep’s blood (Hemostat Laboratories, Dixon, CA). Pseudomonas aeruginosa ATCC 9027 from a BioBall (TCS Water Sciences, Buckingham, UK) was rehydrated in 100 µL of 1x phosphate-buffered solution and grown on TSB-Blood agar. Pure cultures of Bacteroides thetaiotaomicron ATCC 29148 were obtained from ATCC and grown on anaerobic brain heart infusion agar (BD). Isolated colonies from each organism were grown up in 5 mL of TSB or anaerobic brain heart infusion broth (B. thetaiotaomicron) overnight at 37°C, spun down (10,000 x g for 10 min), and genomic DNA was extracted using the FastDNA Kit (Q-Biogene) according to the specifications of the manufacturer, and 1:10 extract dilutions were analyzed using the ureC QRT-PCR assay.
Evaluation of QRT-PCR Assay
The concentration of poultry litter urease producers (PLUP) cells per gram of poultry litter was calculated by dividing the copy number per gram of litter (calculated from the regression analysis of the plasmid standard DNA) by average copy number of ureC genes per cell (1.5; Koper et al., 2004). To compare our PLUP cell concentrations to total cell numbers in the poultry litter, QRT-PCR analysis of 16S rDNA was performed. The QRT-PCR analysis of 16S rDNA copies was carried out as previously described (Harms et al., 2003) using the 1055f and 1392r primers at 600 nM each and the 16STaq1115-BHQ at a concentration of 200 nM (Table 1). The amplification mixture contained 3.0 mM of MgCl2, 600 nM of each primer, 200 nM of probe, and sample DNA (1:500 dilution) or standard (from 102 to 108 copies). The QRT-PCR program was 15 min at 95°C, 39 cycles at 95°C for 15 s, and 58°C for 45 s. Standard DNA consisted of plasmid PCR 2.1 vector (Invitrogen) carrying a 16S rDNA insert. All PCR runs included duplicates of standards and control reactions without template, and the amplification efficiency was calculated as shown above. Total concentration was calculated by dividing the copy number per gram of litter (calculated from the regression analysis of the plasmid standard DNA) by average copy number of 16S genes per cell (4.0; Klappenbach et al., 2001). The percentage of the novel PLUP cells in each poultry litter sample was determined as follows: (PLUP cell concentration/16S cell concentration) x 100.
Poultry Litter Analyses
The percentage of moisture of the poultry litter was determined by drying the litter at 65°C overnight and comparing the weight before and after drying the litter. Litter pH was determined using a combination electrode (Fisher Scientific, Hampton, NH) at a 5:1 deionized H2O:litter ratio. Total N and C were determined by combustion of the litter, followed by gas chromatography using a Vario Max CN analyzer (Elementar Americas Inc., Mt. Laurel, NJ). The NH4-N and NO3-N content of the litter was determined by a 60:1 litter:2 M of KCl extraction followed by flow injection analysis using a Quickchem FIA+ (Lachat Instruments, Milwaukee, WI). Organic N was estimated by subtracting the NH4-N and NO3-N values from the total N value.
Principal Components Analysis
Factor analysis as a multivariate statistical method was used to find a small number of factors from a data set of many correlated variables. Factor analysis is a useful tool for extracting latent information or variables (principal component), such as underlying but not directly observable relationships between variables. The physiochemical parameters included litter composition, percentage of moisture, pH, NH4-N, organic N, total N, and total C. The litter composition parameter was reduced to a single number to indicate bedding material (sawdust = 0, rice hulls = 1, no bedding = 2, and wood shavings = 3). STATIS-TICA 7.0 (Statsoft, Tulsa, OK) was used for the principal components analysis (PCA), which was performed using the Varimax raw method. The original data matrix was decomposed into the product of a matrix of factor loadings and a matrix of factor scores plus a residual matrix. The residual matrix identifies the part of variance of the data set that cannot be explained by common factors (e.g., analytical uncertainties or feature-own variances).
Nucleotide Sequence Accession Numbers
A total of 194 sequences were submitted to the GenBank database and were assigned the accession numbers of EF587504
[GenBank]
-EF587697.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). All of the 145 clone sequences within this novel cluster were 
97% similar to each other. This novel PLUP group dominated both individual clone library groups equally, representing 87 and 85% of the 58 and 60°C libraries, respectively. The translated ureC protein sequences were only 76% similar to Campylobacter lari (BAD89502
[GenBank]
), which corroborated the novel nature of this group. The remaining 23 clones formed 6 smaller groups (n 7 clones), all of which ranged from 66 to 88% similar to their nearest known ureC sequences in the GenBank database.
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). One of the ureC clones was selected as the standard and exhibited a strong linear fluorescent response over 8 orders of magnitude (101 to 108 ureC copies per QRT-PCR reaction; r2 = 0.994). There was a strong linear relationship between the CT and the logarithm of the number of ureC copies per PCR reaction using both poultry litter DNA template (Figure 2A; r2 = 0.999, efficiency = 102%) and ureC standard DNA template (Figure 2B; r2 = 0.994, efficiency = 104%). High amplification efficiencies, calculated from the slope of the regression analyses, using both poultry litter and standard ureC DNA templates, indicate that most of the target sequences were replicated at each amplification cycle. The similarity of these values also confirms the use of the standard ureC DNA as an accurate measure of the amplification and quantification of the target ureC sequence from poultry litter. Based on regression analysis, the lowest genomic poultry litter DNA concentration at which amplification was observed was 0.1 ng per PCR reaction, which is equivalent to approximately 102 copies of the ureC gene matching the PLUP group per PCR reaction. This would be equivalent to approximately 1 x 104 PLUP cells per gram of poultry litter.
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). All environmental and pure culture DNA extracts were positive for general ureC amplification except the B. thetaiotaomicron. The only sample that yielded a positive result when the new ureC QRT-PCR assay was performed was the poultry litter sample (Table 2), suggesting that the PLUP group sequences are unique to poultry litter. Attempts at cloning the QRT-PCR product failed to produce clones for any samples except for the poultry litter samples.
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). All QRT-PCR ureC sequences (n = 28) were 94 to 97% similar to the cluster of ureC clone sequences used to design the QRT-PCR primers and probe. This indicates that the QRT-PCR ureC assay is amplifying the correct target sequence and supports the overall specificity of this assay to this novel PLUP group
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). Assuming an average ureC gene copy number of 1.5 per cell (Koper et al., 2004), the litters contained between 6.0 x 106 to 2.4 x 108 PLUP cells per gram of litter. Total cell numbers ranged from 1.3 x 1011 to 2.8 x 109 cells per gram of litter, which were based on 16S copy numbers assuming 4 copies of the 16S rDNA gene per cell (Klappenbach et al., 2001). Based on these data, the novel PLUP group represented between 0.1 to 3.1% of the total microbial populations in these poultry litters. Although all the litter parameters evaluated were positively correlated to PLUP cell concentrations, the only significant correlation was to total cell numbers (r = 0.76). None of the physiochemical parameters were found to be strongly correlated to PLUP cell concentrations, but the physical parameters (litter composition, moisture content, pH) were more positively correlated (r = 0.32 to 0.40) than were the chemical parameters (r = 0.05 to 0.13). Although none of the physiochemical parameters were directly correlated, it is possible that they may indirectly influence PLUP cell concentrations in the poultry litters. The PCA analysis of the PLUP cell concentrations and both physical and chemical litter parameters showed that more than 75% of the total variance in the data set from the various poultry litter samples was accounted for by 2 factors, with factor 1 and 2 accounting for 42.3 and 33.2% of this variance, respectively (Figure 4). The log-transformed PLUP cell concentrations clustered near the physical (litter composition, pH, moisture content), as well as NH4-N, litter parameters, whereas they were distinct from remaining chemical litter parameters (total N, total C, and organic N).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although limited work has been performed to reduce or eliminate urease activity by adding chemical inhibitors (Singh et al., 2005), no work has concentrated on identifying the microbial communities responsible for production of the NH4-N in the poultry litter. Poultry litter is an excellent environment for the survival and persistence of urease-producing microorganisms, given the abundance of urea and its precursors (uric acid, allatoin, allatoic acid; Nahm, 2003). Uric acid and urea represent 70% of the total N in poultry litter (Shuler et al., 1979), and the limiting step in the conversion of organic N to NH4-N is the activity of microbial ureases. Although decomposition of organic N sources can be achieved abiotically, it is kinetically very slow, and therefore microbial ureases are essential to mediate this NH4-N production (Nahm, 2003). In our study, the urease-producing community clone library from poultry litter was dominated by a single group of urease producers (145/168 clones, Figure 1), and this represented a novel group of urease producers (73% similarity to known urease producers in the GenBank database) found only in poultry litter. Given its dominance, the goal was to target our PLUP group from diverse poultry litters.
Quantitative real-time PCR assays have been developed targeting the ureC gene of specific urease producers such as Ureaplasma urealyticum and Ureaplasma parvum (Mallard et al., 2005) and Helicobacter pylori (He et al., 2002), but no assays were available for the amplification of ureC genes from an environmental microbial community. In this study, QRT-PCR primers and a probe were designed to target on a dominant group of ureolytic microbes in poultry litter. The designed assay was found to be highly efficient at amplifying both standard and environmental templates (Figure 2) and was able to amplify the target ureC DNA from 0.1 ng of genomic poultry litter DNA per PCR reaction. Considering that the PLUP cells represented approximately 1% of the total cells in the poultry litter, it is possible that as little as 10 pg of the target ureC DNA per PCR reaction could be detected. This assay was specific for this novel PLUP group, with no amplification found for DNA templates from other environmental samples or pure cultures shown to contain the urease enzyme (Table 2). The results from the pure cultures were not surprising, given the fact that the target sequences in the ureC gene for each of the known bacteria were only approximately 75% similar to the sequences of the designed QRT-PCR primers and probe (data not shown).
Because the copy number of the ureC gene is low (1 to 2 copies per genome; Koper et al., 2004), the concentration of ureC genes per gram of litter is a faithful estimate of the concentration of PLUP cells in the litter (Bach et al., 2002). Initially, the designed primers and probe were developed from a single litter type, with this novel group found in relatively high quantities (5.4 x 107 PLUP cells per gram of litter), but similar concentrations were found in poultry litter samples from various locations in other states (ranging from 6.0 x 106 to 2.4 x 108 PLUP cells per gram of litter) and from litters with different physiochemical properties (Table 3). The prevalence of dominant but specific microbial groups (i.e., Lactobacillales, Actinomycetes, Clostridia) in diverse poultry litters (varying locations, bedding materials) has been previously reported (Martin et al., 1998; Lu et al., 2003; Fries et al., 2005), so it was not unexpected that this group of ureolytic microbes was present in similar concentrations in our various poultry litter types. This novel group represented anywhere from 0.1 to 3.1% of the total number of cells (based on 16S QRT-PCR data) in the litter. These ratios are consistent with the other microbial enzymes vital for N cycling (narG, nirK, nosZ) that were shown to represent similar portions of the total bacterial populations from a variety of soils, including agricultural soils (Henry et al., 2004, 2006; Lopez-Gutierrez et al., 2004). These results indicate that this novel PLUP group is a significant component of the total bacterial population of poultry litter and could account for a significant amount of the NH4-N produced from those houses.
In this study, we found the concentration of our PLUP group to be between 6.0 x 106 and 2.4 x 108 PLUP cells per gram of litter. Given published data on average flock concentration and litter production (Perkins et al., 1964) and the specific activity of the urease enzyme for a mixed microbial community from animal agriculture under optimal conditions (Mahadevan et al., 1977), we have determined that between 1.5 x 106 and 5.9 x 107 mg of NH3 could be produced per house per day given our data. Assuming an average of 2 x 104 birds per house, NH4-N production from larger poultry production facilities in the United States can range from 9.4 x 106 to 2.4 x 107 mg of NH3 per house per day (Groot Koerkamp et al., 1998; Burns et al., 2003; Lacey et al., 2003; Wheeler et al., 2006), which indicated that our novel PLUP group could be responsible for more than 10% of the NH4-N produced from a poultry house.
To assess the applicability of this assay, diverse litter types were tested microbiologically, physically, and chemically, and the variance in the data from the different litters was compared using PCA (Figure 4). Although only total cell concentrations were directly correlated to PLUP cell concentrations, other physiochemical parameters were found to influence their concentrations in the different litters. Log-transformed PLUP cells clustered most closely to physical litter parameters (pH, moisture content, bedding materials) and NH4-N (Figure 4). This result was not surprising, considering that parameters such as pH (Elliot and Collins, 1982; Derikx et al., 1994) and moisture content (Nahm, 2003; Lovanh et al., 2007) have been previously reported to be the dominant environmental factors in NH4-N volatilization within poultry houses. Because our PLUP group could potentially produce greater than 10% of that NH4-N, it is logical that these physical parameters also affect the concentration of these PLUP cells. Chemical litter parameters (organic N, total N, and total C) did not cluster near the physical parameters or PLUP cell concentrations, while also exhibiting the lowest direct correlation to PLUP cell concentrations (r 0.13). Previous studies have shown that there is either a very weak (Barbarika. et al., 1985; Serna and Fomares, 1991) or no correlation (Castellanos and Pratt, 1981) between total C/N ratio and N mineralization. Because the microbial urease activity is a major limiting step in the mineralization of organic (urea) to inorganic (NH4-N) N, the total C and N numbers for each litter type were not expected to influence the concentrations of the PLUP cells.
In conclusion, primers and a probe specific to the ureC gene from a novel group of dominant ureolytic microbes found in poultry litter were designed and used to develop a new QRT-PCR assay. This assay was highly specific to poultry litter samples, and calculated concentrations of cells containing this target ureC gene were found in high numbers (
107 to 108 PLUP cells per gram of litter) in diverse litter types from 3 different states and using 4 different bedding materials, and these concentrations were found to be affected by the physiochemical parameters of the litter. Initial attempts at isolating the PLUP bacteria have been unsuccessful, although additional isolation experiments are currently ongoing. The characterization of our PLUP group will allow for a greater understanding of how they function within poultry litter and potentially lead to the development of remediation strategies to eliminate this group from the litter, which would aid in reducing NH4-N volatilization. The use of appropriate NH4-N-reducing strategies will lead to increased bird performance and health, a reduction in energy costs associated with ventilation, and an increase in the fertilizer value of the poultry litter due to higher organic N content.
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ACKNOWLEDGMENTS |
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Received for publication August 21, 2007. Accepted for publication February 9, 2008.
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