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a USDA-ARS, Dairy Forage Res. Cent., 1925 Linden Drive West, Madison, WI 53706
b Dep. of Dairy and Anim. Sci., Pennsylvania State Univ., 324 William L. Henning Bldg., University Park, PA 16802
c Dep. of Soil Sci., Univ. of Wisconsin–Madison, 1525 Observatory Drive, Madison, WI 53706
* Corresponding author (jmpowel2{at}wisc.edu).
Received for publication October 7, 2002.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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Abbreviations: NDF, neutral detergent fiber • NDIN, neutral detergent insoluble nitrogen
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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Manure nutrient availability to crops is estimated by indirect methods and varies widely. For example, using the difference method and the fertilizer equivalent approach, 12 to 63% of dairy manure N may be taken up by corn during the first growing season after application (Motavalli et al., 1989; Klausner et al., 1994). Nutrient availability in the second and subsequent years can be more difficult to predict. The difference method assumes that the difference in total nutrient uptake between manure-amended and nonamended plots are attributed to the addition of manure. The fertilizer equivalent approach compares crop N uptake in manure- and fertilizer-amended plots (Klausner and Guest, 1981; Harmsen and Moraghan, 1988; Motavalli et al., 1989; Muñoz et al., 2004). The fertilizer equivalent of manure is the amount of fertilizer N required to achieve the same yield and N uptake achieved with manure. Both the difference method and the fertilizer equivalent approach assume that crop N uptake in the manure-amended, fertilizer-amended, and control plots are accomplished with the same efficiencies. However, whereas approximately half (or more) of manure N is organically bound and must be mineralized by soil microbes before becoming available for crop uptake, fertilizer N is more water soluble and potentially more readily available for crop uptake.
The stable isotope 15N has been used extensively to evaluate the availability of fertilizer N to crops. The use of 15N to determine the availability of manure N to crops has been studied using two approaches: (i) postexcretion 15N enrichment of the NH4 pool or (2) 15N enrichment of feedstuffs, which are then fed to ruminant livestock. Whereas approximately 50 to 60% of the N in slurry is in the NH4 form (Dittert et al., 1998), semisolid dairy manure, the most important manure type on Wisconsin dairy farms (Jackson-Smith et al., 1997), typically contains much lower amounts of NH4 and higher amounts of organically bound N. The chemical composition and mineralization of organic N in dairy manure are not well understood. More accurate estimates of manure N availability to crops are needed if we are to expect farmers to improve manure management.
Only 20 to 30% of the N (protein) fed to a dairy cow is converted into milk, with the remaining excreted about equally in urine and feces (Castillo et al., 2000; Broderick, 2003). Fecal N can be divided into two pools: (i) endogenous N consisting of microbial products and microorganisms from the rumen, the intestine, and the hind gut, and the N originating from the digestive tract itself; and (ii) undigested feed N (Mason and Frederiksen, 1979). Rumen microbial products and other endogenous, organic N forms in feces may make a significant contribution to crop N requirements the year following manure application. Fecal undigested feed N mineralizes slowly in soil and is therefore unavailable to plants over the short term (Sørensen et al., 1994; Powell et al., 1999). Postexcretion labeling of the NH4 pool would not, therefore, be an appropriate technique for the study of N cycling in soils amended with semisolid manure.
The labeling of ruminant livestock manure by feeding 15N forage is expensive and laborious (Sørensen et al., 1994; Powell and Wu, 1999). However, this is the only current method for labeling the urinary and fecal N components of semisolid dairy manure. However, if urine and fecal endogenous N are the only major components that contribute to crop N requirements over the short term, then an alternative method may be available to selectively label these manure N pools in a more cost-effective and less laborious manner. This paper describes two techniques that can differentially enrich in 15N the urinary N, fecal endogenous N, and fecal undigested feed N excreted by dairy cows.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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Starter fertilizer (6, 5, and 110 kg ha–1 of N, P, and K, respectively) was applied annually to corn plots (approximately 32700 plants ha–1) before planting in early June. Ammonium sulfate—12.3 atom% 15N in 1997 and 10.0 atom% 15N in 1998, 1999, and 2000—was dissolved in water and applied using a watering can at an equivalent rate of 75 kg N ha–1 during the growth period to two adjoining corn rows [5 m in length (5 m2) in 1997, 6.65 m in 1998 and 1999, and 9.11 m in 2000] in each of three applications (total application of 225 kg N ha–1). We estimated that each gram of manure (urinary plus fecal) N would require approximately 2 g of forage N (an assumed forage N digestibility of 50%). Probable alfalfa and corn silage dry matter (DM) yields, their N contents, and a target diet comprised of approximately 50% of each forage type (DM basis) were used to estimate land area and fertilizer 15N requirements for each forage type. Corn plants were harvested at approximately one-third milkline (60–65% moisture), chopped to 2- to 3-cm lengths, and ensiled in PVC silos.
A 20-m2 area of a second-year alfalfa stand was used in 1997, a 28-m2 area in 1998 and 1999, and a 40-m2 area in 2000. Alfalfa was fertilized with 10.0 atom% 15N in the same manner as corn, at an equivalent rate of 100 kg N ha–1 in each of two applications (total application of 200 kg N ha–1). The first application was made in early to mid-June, the day after cutting alfalfa growth to an aboveground height of 2 cm. The first alfalfa 15N harvest (all 15N harvests involved cutting total aboveground biomass to a 2-cm residual height) occurred approximately one month thereafter. The second fertilizer application was made immediately after the first 15N harvest. The second 15N harvest occurred 5 to 6 wk after the first 15N harvest. No further fertilizer applications were made. A third alfalfa 15N harvest was taken in the fall, before first frost, in mid- to late October. All alfalfa was dried to make hay. Irrigation was applied as necessary to corn and alfalfa by central pivot.
Nitrogen-15 Labeling of Dairy Urine and Feces
Two methods were used to differentially enrich dairy urine and fecal N components in 15N (Fig. 1)
. The forage method involved labeling alfalfa hay and corn silage and then feeding these forages to dry dairy cows (Powell and Wu, 1999). This technique labeled urine N, fecal endogenous N, and fecal undigested feed N. The urea method involved directly feeding 15N-enriched urea to cows with unlabeled forage. This technique only labeled urine N and fecal endogenous N. No labeled undigested feed N in feces was expected using this technique since no 15N forage was fed.
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The cows were kept in adjoining stanchions and bedded with rubber mats. For both labeling methods, cows were first adapted to a diet consisting of approximately 55% alfalfa hay and 45% corn silage on a DM basis (atom% 15N at natural abundance) for 7 d. On the last day of the adaptation period, indwelling catheters were inserted into the bladders for urine collection.
For the forage method, 15N-enriched alfalfa hay from each harvest and corn silage (Table 1) were divided into 6 to 10 equal parts (to assure uniform 15N feeding) on a weight basis. Alfalfa and corn harvested from the 15N-treated plots as well from border areas (15 cm from treated alfalfa plot's edge and 30 cm from each corn row end) were fed. The hay–silage mixtures were each mixed carefully by hand. Mixtures were offered ad libitum to each of the two cows until all feed was consumed, or approximately 28 to 70 h after feeding was initiated.
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Total feces and urine were collected at 4-, 8-, or 12-h intervals after initial feeding of 15N-enriched forage or urea up to a total of 192 h (Fig. 2–5) . Feces were hand-scraped from metal catchment containers fitted into the gutters. Urine was collected from catheter tubes draining into plastic containers embedded in ice. Feces and urine from each collection were subsampled and frozen immediately. Feces were subsampled from the metal catchment containers in 1998 and directly from the rectum at the end of each sampling period in 1999, 2000, and 2001. No apparent differences in fecal 15N concentrations were noted between either technique (Fig. 3). Urine subsamples were taken from catheter tubes at the end of each sampling period.
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Cell wall components of feces were determined using the detergent system (Goering and Van Soest, 1970) as neutral detergent fiber (NDF). Total N and 15N contained in cell walls of feces, or the undigested feed N component in feces, were determined as neutral detergent insoluble N (NDIN). The NDF soluble N fraction in feces (endogenous N) was estimated as the difference between total N and NDIN (Mason and Frederiksen, 1979). The homogeneous 15N labeling of fecal N components (Sørensen et al., 1994) was evaluated by comparing 15N concentrations in total N and NDIN (Fig. 4).
The percentage recovery of 15N fertilizer in alfalfa and corn and the percentage recovery of 15N forage and urea in feces and urine were calculated as follows:
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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For both alfalfa and corn, 15N concentrations in plants harvested from the border areas of fertilized plots were four to five times greater than atom% 15N in unfertilized plots (data not shown). These border row 15N concentrations were approximately one-third to one-half the atom% 15N measured in plants harvested from the central 15N-fertilized plots. This border forage DM was, therefore, used as feed (Table 1). It provided approximately 15 to 20% more forage than if only plot forage was harvested and fed.
Nitrogen-15 Feeding and Excretion
Both cows fed 15N-enriched forage had similar patterns of 15N excretion in urine and feces during all four study years (Fig. 2 and 3). Nitrogen-15 appeared in urine before feces. Except for one cow's 15N excretion in feces in 2000, 15N concentrations in urine and feces increased to a single maximum point and decreased thereafter. Peak 15N concentrations in urine occurred between 25 and 70 h and coincided closely with the final offer of 15N-enriched forage (Fig. 2). Peak 15N concentrations in feces occurred between 32 and 108 h, or approximately 4 to 44 h after the final offer of 15N-enriched forage (Fig. 3). Peak 15N concentrations in urine and feces were attained the latest during those years (2000 and 2001) when the most 15N-enriched forage was fed (Table 1). Regression analyses showed no relationship between 15N concentrations in feed (Table 1) and peak 15N concentrations excrete in urine (Fig. 2) or feces (Fig. 3). Sørensen et al. (1994) found that it was necessary to feed sheep 15N-enriched forage for at least 7 d to achieve peak and uniform 15N concentrations in feces.
The pattern of 15N excretion in urine and feces after feeding 15N-enriched urea (Fig. 4) was very different from the observed pattern of 15N excretion after feeding 15N-enriched forage. The single 100-g dose of 5 atom% 15N urea fed in 1999 resulted in a single peak of 15N enrichment in urine (approximately 1.25 atom% 15N) 8 h after dosing and a single peak of 15N enrichment in feces (approximately 0.75 atom% 15N) 32 h after dosing. The eight doses of 15N-enriched urea fed in 2000 resulted in eight 15N peaks (from 1.25–2.15 atom% 15N) in urine. Each peak was recorded within 4 h after feeding 15N-enriched urea. A single 15N enrichment peak in feces occurred (approximately 1.25 atom% 15N) approximately 56 h after the initial offer of 15N-enriched urea. Peak 15N concentration in both urine and feces was attained only up to the sixth dosing. No increases in urinary or fecal 15N concentrations were observed after the seventh or eighth dosing.
Nitrogen-15 Labeling of Fecal Nitrogen Components
The undigested feed N in feces (NDIN) accounted for 21% of the total fecal N, or 9% of the total N (urine plus feces) excreted by the cows in this study (Table 2). The homogeneous 15N labeling of fecal N components was evaluated by comparing 15N concentrations in total fecal N to that in fecal NDIN. Fecal endogenous N was calculated as the difference between total fecal N and NDIN. Nitrogen-15 concentrations in NDIN were generally lower than in total fecal N during the period before maximum fecal 15N concentrations were attained and higher after maximum fecal 15N concentrations were attained (Fig. 5). This differential labeling of fecal N components requires that one of two strategies be used to obtain uniformly labeled fecal N components: (i) proportionately combining feces from excretion periods before and after peak fecal 15N concentrations (Powell and Wu, 1999) or (ii) feeding 15N-enriched forage for a longer period and using feces sampled after 15 to 20 d (Sørensen and Jensen, 1998). The latter observation was made in a trial involving feeding 15N-enriched forage to sheep. Feeding 15N-enriched forage to dairy cows for 2 to 4 d was very expensive (Table 3). Feeding for a longer period, such as 15 to 20 d, to obtain uniform 15N distribution in feces would be cost prohibitive.
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The 15N distribution in urine and feces using the urea method could have been influenced by feeding conditions, such as the amount of urea fed, protein content, and digestibility of the feed, which would influence how much urea is assimilated by microbes and how much is excreted in urine. Most studies of feeding urea to ruminants indicate that the utilization of urea N is inferior to that of conventional protein supplements. The limiting factor is rapid urea hydrolysis with much of the NH3 absorbed from the rumen before microorganisms can incorporate it into microbial protein (Helmer and Bartley, 1971). Excess NH3 is excreted in the urine of dairy cows (Castillo et al., 2000; Broderick, 2003). The dietary protein level of urea-fed cows was approximately 14.4 to 16.4%, of which 1.9 to 3.9% was derived from urea. These dietary protein levels and the amount of urea fed were not excessive (Helmer and Bartley, 1971), so a proportionate 15N labeling of urine N and fecal microbial N likely occurred.
A slight 15N enrichment of fecal NDIN derived from cows fed 15N-enriched urea was observed both in 1999 and 2000 (data not shown). This result was unexpected and, in the context of this study, has no apparent theoretical basis for which it could be explained. No 15N-enriched forage was fed, and therefore, no 15N enrichment of fecal NDIN should have been detected. This 15N contamination of NDIN was likely due to the inability of neutral detergent solution to remove 15N-enriched bacterial nucleic acids and cell walls and glycocalyces on undigested feed fiber. Mason (1969) has shown that NDF from ovine feces retained 5 to 38% of fecal diaminopimelic acid (a bacterial cell wall component). Approximately 80% of the microbial protein is true protein, and the remaining 20% is associated with nucleic acids. Of the true protein, 80% is considered digestible (Natl. Res. Counc., 2001). Thus, 36% of the microbial protein could be excreted in feces, and this fraction would be partitioned into NDIN. However, the proportion of the microbial residual N as 15N would be small as the contribution of labeled urea to microbial protein was small.
Further contamination of the NDF with glycocalyx (glycoprotein encrustations produced by bacteria to facilitate attachment of cells to fiber and other solid surfaces) is likely as these materials are often poorly soluble in detergents (Gibson et al., 1999; Landa et al., 1999; Merritt et al., 2000). We further washed urea-derived feces in acid detergent solution to remove heteropolysaccharides, the major constituents of glycocalyx structures, but even this treatment did not result in complete 15N removal. This is not surprising, in view of the tenacity with which gastrointestinal bacteria are known to attach to fiber particles (Costerton et al., 1987). The continued 15N enrichment of fecal acid detergent insoluble N in 2000 (data not shown) indicates that the detergent system may not be an adequate procedure for fractionating fecal N into microbial and undigested feed components.
Using Nitrogen-15 to Label Dairy Manure Components
The selection of a manure 15N labeling technique depends on the intended use of the 15N-labeled manure and associated costs and labor. Nitrogen cycling studies involving long-term (more than 2 yr) turnover of manure N in soils may require that urine N and both fecal endogenous and undigested feed N be labeled using the forage method. In a 3-yr field trial, recovery of applied dairy manure 15N (enriched using the forage method) in harvested corn silage averaged 18%, and approximately 46% of applied manure 15N remained in upper 90-cm soil profile (Muñoz et al., 2003). One of the conclusions of this study was that although costly and time-consuming to prepare, the use of 15N-labeled manure using the forage method provided a much better approach to study the fate of manure N within the soil–crop system compared with unlabeled manure.
On many dairy farms, the straw of a small-grain crop is used as bedding, and this, therefore, is a manure N component. Many other low-N bedding sources (e.g., sand) are used. Nitrogen-15-labeled straw bedding has been found not to contribute significantly to crop N uptake over the short term (Jensen et al., 1999). However, this manure N component, like the undigested feed N in feces, may make a significant contribution over the long term to crop N requirements and be an important component of organic matter in soils amended often with manure.
Of the total 15N-enriched ammonium sulfate used in this study, 22 to 28% was incorporated into feces and urine using the forage method. Of the total 15N-enriched urea fed to dairy cows, 64 to 78% was incorporated into feces and urine (Table 3). This difference in 15N use efficiency between the two 15N labeling techniques was due to the loss of 15N in soil when labeling forage. Of applied 15N, 36 to 44% was taken up by alfalfa and 26 to 65% by corn. The lowest 15N uptake by corn occurred in 1998 when high-rainfall events following the second and third fertilizer applications likely resulted in 15N leaching losses. Of the total 15N fed, 51 to 64% was recovered in feces and urine from cows fed 15N-enriched forage vs. 64 or 78% recovery from cows fed 15N urea. Higher 15N recovery using the urea method was likely due to less input to the cows and different uptakes by body tissue.
The forage method is much more laborious and costly than the urea method. The forage method requires 15N application to field plots and forage harvest, handling, storage, and feeding (Fig. 1). Nitrogen-15-labeled urea can be mixed directly with forage and fed. In this study, ruminally fistulated cows were available to allow 15N-enriched urea to be applied directly into the rumen via cannula. Our observations during the urea adaptation period were that urea could be just as well mixed with water and sprinkled over (unlabeled) forage and fed. However, the feeding of urea in this way may result in less recovery due to feed refusals than if urea was applied directly into the rumen.
The cost of the isotope to label urine and both fecal N components using the forage method ranged from $291 to $376 per gram of labeled manure N, and to label urine and fecal endogenous N using the urea method, the cost ranged from $269 to $277 (Table 3). These estimates were based on manure produced over the entire 8-d collection period (Fig. 2–4) using only feces and urine that contained a minimum atom% 15N level of 0.403, or approximately 10% over natural abundance. Feces and urine of the highest enrichment would be needed for long-term N cycling studies, and lower enrichments could be used for shorter-term studies. Dairy manure containing at least 0.566 atom% 15N would be needed to track manure N uptake by corn over a 3-yr period (Powell and Wu, 1999).
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SUMMARY AND CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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