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Center for Animal Health and Productivity, School of Veterinary Medicine, University of Pennsylvania Kennett Square 19348
Corresponding author: Z. Dou; e-mail: douzheng{at}vet.upenn.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: dairy farm • phosphorus • dietary management
Abbreviation key: TP = phosphorus determined in acid digests, Pi = inorganic phosphorus, Po = organic phosphorus, Pt = total phosphorus (Pi and Po) in extracts.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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While it is indisputable that dietary management should be taken as the first defense against P buildup on farms, conclusions derived from research feeding trials need to be verified under actual farm conditions. Research trials are often performed with deliberately controlled experimental settings to isolate and highlight treatment effects while minimizing potential impacts of other factors. In the real world of commercial dairy farms, however, the situation is diverse and many factors interplay simultaneously to affect the dependent variables. Farms vary in resources (e.g., soils, crops, imported feeds, etc.) and management preferences (feeding programs, dietary P levels, etc.). Cows differ in genetic traits and physical or physiological characteristics such as BW or milk yield. These and other intrinsic or managerial differences inevitably add variability to the independent and dependent parameters and may dilute or diminish the numerical relationships between fecal P concentrations and dietary P levels.
The primary objective of the present work was to assess the variability of P concentrations in dairy feces across and within farms and identify factors affecting concentrations of fecal TP as well as water-soluble P, using samples from a diverse group of commercial dairies. A secondary objective was to determine if concentrations of water-soluble P extracted from dried-ground fecal samples differ from extractions using wet samples.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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From each farm, feed and fecal samples were collected on the same day from the lactating cow group only. Feed samples were obtained at the feed bunk at feed delivery, mixed by hand and randomly subsampled, resulting in one composite sample per farm and a total of 33 TMR samples. Fecal samples were rectal grabs from randomly selected healthy lactating cows and were kept individually. Number of cows sampled per farm ranged from 7 to 17 depending on the herd size. The total number of cow fecal samples was 381 in this study.
3-farm set.
Separate from the 33-farm set, an intensive fecal sampling scheme was conducted on 3 commercial dairies located in southeast Pennsylvania. The herds were Holstein breed and fed TMR with dietary P concentrations measuring 3.51, 4.75, and 5.09 g/kg of P in feed DM at the time of sampling. Besides collecting TMR samples (one per farm), rectal grab fecal samples were obtained from all healthy lactating cows in the feeding group, resulting in a total of 29, 27, and 30 fecal samples for the 3 farms, respectively. Analyses from the 3-farm set were used to verify the magnitude of within-farm variability and to further illustrate its independence from sample numbers.
Dried-ground vs. wet samples.
Fecal samples from 10 out of the 33-farm set were selected with 4 random samples per farm, totaling 40 samples. In the laboratory, each of the 40 samples was divided: one-half for wet-based extraction (2 g + 98 mL deionized water) and the other one-half was dried at 65°C and ground to pass a 2-mm screen prior to extraction (0.3 g + 100 mL water). The DM of fecal samples averaged 15%, thus the sample:water ratio was essentially the same for the wet vs. dried extractions. Concentrations of Pt, Pi, Ca, and Mg in water extracts were determined following the procedures described below.
Sample Handling and Laboratory Procedures
Feed samples were analyzed at a commercial laboratory for standard feed quality parameters (Table 1
) using established procedures. Fecal samples were kept frozen until laboratory analysis, upon which samples were thawed at room temperature, homogenized by hand, and then split into subsamples. The subsamples were used for: (1) DM determination in a convection oven at 65°C, (2) acid digestion with a microwave-assisted digester (Walter et al., 1997); the digests were used for determination of TP, and total Ca and Mg by inductively coupled plasma-atomic emission spectroscopy (ICP-AES), and (3) pH and Pt, Pi, Ca, and Mg in water extracts. The extraction was performed with 2 g of wet fecal sample in 98 mL of deionized water, shaken on a reciprocal shaker for 1 h, and filtered through Whatman 42 paper. After measuring the pH, an aliquot of the water extract was analyzed for Pi by the phosphomolybdate blue method of Murphy and Riley (1962). Another aliquot (0.5 mL) was digested in concentrated nitric acid (1 mL) at 90°C overnight, then determined for water-soluble Pt, Ca, and Mg on ICP-MS (mass spectroscopy). Water-soluble organic P (Po) was calculated by subtracting Pi from Pt (Dou et al., 2000).
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Data Analysis
Concentrations of P, Ca, and Mg in feed and fecal samples were reported on a DM basis. Least squares ANOVA and descriptive statistics were performed in SAS (SAS Institute, 1999). A mixed, stepwise regression was performed to identify factors affecting fecal P parameters. Stepwise variable entry and removal in the model examined the variables in the block at each step for entry or removal, taking intercorrelation between variables into consideration. All variables that had a significant impact at the 0.05 level were retained in the model.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). The relative variability, calculated as CV across the 33 farms, was lowest for CP (8.4%) and highest for total Ca (19.6%). The large variability is not surprising because farms differ in a variety of ways, such as selection of feed ingredients, sources and processing of feeds, and soil and water conditions under which forages are grown. The magnitudes of the CV in the present study are comparable to those in previous reports. For example, Sniffen et al. (1993) found a 25% CV for Ca concentrations in 25 commonly used ruminant feedstuffs. Also, based on forage samples analyzed over a 12-mo period, Kertz (1998) reported CV of 14 to 36% for total P, 15 to 30% for CP, and 7.5 to 17.5% for NDF and ADF, respectively. Phosphorus concentrations in the 33 TMR samples ranged from 3.45 to 5.78 g/kg feed DM; all but one exceeded the relevant recommendations for diet P published by the National Research Council (NRC, 2001). The apparent overfeeding of P on these farms is consistent with recent survey findings that many farms feed excess P to lactating cows (Sink et al., 2000; Powell et al., 2002; Dou et al., 2003). The TMR samples also had a wide range of Ca, from 4.58 to 11.81 g/kg feed DM, comparing to the NRC recommendation of 6.6 g/kg (NRC, 2001). The Ca:P ratio in the feed samples ranged from 1:1 to 2.65:1. Ruminants tolerate a wide range of dietary Ca:P ratio (from 1:1 to 7:1) as long as P supply is adequate (McDowell, 1992).
Concentration and Variability of Fecal P
Fecal TP concentrations differed by more than a factor of 2 among the farms (Table 2; n = 33). Individual fecal samples had an even wider range, 3.92 to 16.73 g/kg fecal DM (n = 381). From feed to feces, P concentrations became higher and the range wider (3.45 to 5.78 g/kg DM in feed as compared with 5.84 to 12.84 g/kg DM of TP in feces). This concentrating effect is due to a greater disappearance of feed DM than P between the front end (feed intake) and the rear end (fecal excretion). With a digestibility of 65 to 75% for feed DM (NRC, 2001), only 25 to 35% is excreted as undigested feed residues. However, P use efficiency (disappearance) is much lower and fecal excretion higher. For example, the amounts of TP excreted in feces accounted for 55, 67, and 76% of feed P intake when cows were fed diets containing 0.31, 0.39, and 0.47% P (Wu et al., 2001). A concentrating effect is also apparent for Ca and Mg for the same reason (Tables 1 and 2).
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) and accounted for roughly 50% of the fecal TP. On average, 83% of the Pt in water extracts was Pi. Increases in dietary P led to higher TP as well as water-soluble Pi of feces whereas water-soluble Po remained low and unchanged (Figure 1). Water-soluble Po is primarily phytate-like P (He et al., 2004), which probably derives from undigested feed residues. Water-soluble Pi may originate from both inorganic P contained in the TMR feeds and P that is released through digestion/hydrolysis within the animal’s digestive tract. We do not have detailed information on feed ingredients and compositions for these farms. Nevertheless, the trends displayed in Figure 1 suggest that increasing dietary P, no matter whether the increase is brought about by addition of inorganic P supplements or inclusion of organic feed ingredients that are intrinsically high in P content, is unlikely to change the bioavailability of P. Instead, a higher amount and proportion of P is excreted as water-soluble Pi in feces. It is also interesting to note that only 40% of the total Ca in feces was water-soluble, whereas water-soluble Mg was nearly 73% of the total Mg (Table 2).
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). As represented by the calculated CV, pH, and DM had the lowest variability (CV <10%), mainly attributed to the regulating and buffering functions of the animals’ digestive physiology (Kohn and Dunlap, 1998; Allen, 2000). The higher variability exhibited by the other parameters, 10 to 20% for acid digest total P, Ca, Mg, and 20 to 30% for water-soluble Pi, Pt, and Ca, perhaps reflects differences in feeds, animals, and management factors. Chemical characteristics of samples might be an additional factor contributing to the highest variability of water-soluble Pi, Pt, and Ca. Considering fecal TP, the magnitude of across-farm variability is equivalent to that reported from another farm-based study (Powell et al., 2002), in which farm means of samples from 98 Wisconsin dairies had an estimated CV of 25%. Variability of fecal P within farms, calculated as CV based on individual cow feces samples for each farm, exhibited a rather wide range. Fecal TP had CVs as low as 7.6% on one farm but as high as 32% on another; water-soluble Pi had CV as low as 11.6% but as high as 37%. The overall ranking order of the fecal parameter variability within-farm is similar to that across-farms, i.e., pH and DM < acid digest total P, Ca, Mg < water-soluble Pi, Pt, Ca.
The within-farm variability appears to be unrelated to number of samples tested per farm, as illustrated in Figure 2 for selected parameters pH and TP. This is further corroborated by data from the 3-farm set with the intensive sampling scheme (n = 27, 29, or 30). On these farms, within-farm variability had CV ranging from 16.0 to 23.2% for fecal TP and 21.6 to 31.4% for water-soluble Pi, similar to the magnitude for the 33-farm set. Also, when the 33 individual farms were ranked based on the CV for selected parameters (pH, fecal TP, and water-soluble Pi), there was a lack of consistency in the ranking order. In other words, a farm may rank relatively low in variability for one parameter but high in others. This seems to suggest that within-farm variability of fecal P and other parameters is likely to be intrinsic and random in nature. The practical implication is that on-farm sampling and testing must be based on multiple samples, or a composite of multiple samples; yet, large numbers of samples (n > 10) are not necessary, as it may not lower the variability.
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Factors Affecting Fecal P
Factors affecting fecal P concentrations as identified by mixed stepwise regression are shown in Table 3. Clearly, dietary P concentration remains the dominating factor affecting all fecal P parameters (TP, water-soluble Pt and Pi). This is in line with expectations based on existing knowledge. It also confirms the overall trend of higher dietary P leading to higher fecal P observed in P-feeding trials (e.g., Wu et al., 2000; Knowlton and Herbein, 2002; Powell et al., 2002; Dou et al., 2003). However, the relative magnitude of the coefficient of determination (r2) between dietary P and fecal P was substantially lower in farm-based studies than P-feeding trials. For instance, Wu et al. (2001) reported a coefficient of determination of 0.843 when regressing fecal TP (%) against P intake (g/d), and Dou et al. (2002) obtained a coefficient of determination of 0.95 regressing fecal TP (g/kg) against dietary P (g/kg) for research trials. For farm-based studies, Powell et al. (2002) observed a coefficient of determination of 0.55 for 47 Wisconsin dairies with single group feeding, and Dou et al. (2003) reported a coefficient of determination of 0.43 for 72 samples from dairy farms in the Northeast and Mid-Atlantic regions. The current study had an r2 of 0.38 when regressing fecal TP against dietary P alone. Usually, research feeding trials deliberately control major factors to be the same except the treatment, whereas field-based studies like ours, with little intervention except sample collection, typically reflect the impact of all factors potentially affecting the dependent variables.
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). The DIM effect on fecal P, however minor it appears to be, has not been explicitly noted previously. In an attempt to explore the theoretical basis for DIM effect, we estimated fecal TP concentrations at different DIM stages for a cow fed the same diet throughout a lactation with fixed digestibility, growth rate, and standard milk production curve. The results (detailed data not presented) indicated that fecal TP concentration would increase with increasing DIM because of decreasing milk yield (a sink for P). Perhaps, the lack of notice of a DIM effect in previous studies can be attributed to limited data points coupled with considerable variability, whereas the large data set in the current study enabled its detection.
The role of pH in affecting water-soluble P is comprehensible from the view of chemistry. Water-soluble P, mostly inorganic P or orthophosphate, exists in dairy feces mainly as Ca-P complexes. As pH decreases, P in these complexes becomes more soluble. In the current study, fecal pH ranged from 5.81 to 8.61; and a linear correlation between water-soluble Pi and fecal pH was statistically significant with a negative slope (r = –0.37, P < 0.0001). On the other hand, the role of Ca in affecting water-soluble P is not straightforward. Fecal Ca was not a significant factor in the water-soluble Pi model (Table 3), but a separate analysis revealed a strong correlation between fecal pH and acid digest total Ca (Figure 3; P < 0.0001). Also, fecal Ca has a major impact on the proportion of water-soluble Pi in TP (Figure 4; P < 0.0001). This provides strong evidence of Ca-P complex formation in dairy feces. Apparently, pH and Ca interact and together affect fecal P solubility.
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) remains a challenge. Because TP is a measure of total P in feces, it should be independent of fecal pH. Yet, the pH effect on TP may not be a mere artifact because regressing TP against pH alone also revealed a trend of increasing TP with pH, i.e., the slope taking a positive value (data not presented). In reference to the Ca effect, it may be argued that dietary P supplementation is usually achieved by adding mono-calcium or dicalcium phosphate minerals to diets. The supplementation is unlikely to enhance animal performance. Instead, it leads to increased fecal excretion of P and Ca. This may explain at least partially the contribution of Ca in the TP model. Considering the strong relationship between fecal pH and Ca (Figure 3) plus the common practice of limestone (CaCO3) being used as an inexpensive Ca supplement, we reason that increasing limestone in diets led to higher Ca and pH in feces, which consequently led to decreases in water-soluble Pi as well as the proportion of Pi:TP in feces. We further hypothesize that increasing limestone in feed may also lead to elevated pH along the digestive tract, which in turn may favor Ca-P complex formation, making P less soluble for absorption and thus leading to a higher excretion of fecal TP. In other words, increases in limestone in diets may produce a "P pulling effect" inside the digestive tract. Of course, this hypothesis, serving as a starting point for further discussion, remains to be thoroughly investigated. A supporting argument is that in monogastric species undergoing chronic kidney failure, calcium carbonate is often used as a phosphate binder to help reduce the absorption of P from the diets (Polzin et al., 2000).
Dried-Ground vs. Wet Samples
The drying-grinding process reduced water-soluble Pi (with little change in Po) in most cases and the reduction is not linear (Figure 5). The amount of water-soluble Ca also changed nonproportionally, whereas changes in water-soluble Mg were less dramatic and in most cases close to linear (data not presented). Moreover, the relationship between water-soluble Pi and fecal TP was less correlated with data from the dried-ground samples as compared with the wet-based results (Figure 6). At present, we do not know if the change in the amount of Pi associated with the drying-grinding process is chemical or physical in nature, or both. In general, wet-based extraction features minimal alteration of manure properties and thus the results would be more reflective of natural conditions. In addition, wet-based extraction saves time and labor in terms of sample processing and thus can be easily adopted in laboratories handling large numbers of samples. On the other hand, dried-ground samples offer the convenience of easy storage for repeated use and thus are often preferred for research purposes. Some previously published studies used dried-ground samples (e.g., Dou et al., 2000; 2002), while others used wet samples (e.g., Kleinman et al., 2002b; Dou et al., 2003). The outcome of the direct comparison in the current study emphasizes the need for caution when one compares results from different studies.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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FOOTNOTES |
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Received for publication May 11, 2004. Accepted for publication August 4, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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