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* Agriculture and Agri-Food Canada, Soils and Crops Research and Development Centre, Québec, Canada G1V 2J3
Department of Plant Science, Macdonald Campus, McGill University, Sainte-Anne-de-Bellevue, Canada H9X 3V9
Agriculture and Agri-Food Canada, Soils and Crops Research and Development Centre, Normandin, Canada G1M 4K3
Département de phytologie, Faculté des sciences de l’agriculture et de l’alimentation, Université Laval, Québec, Canada G1K 7P4
1 Corresponding author: pelletiers{at}agr.gc.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Key Words: digestibility • Phleum pratense • chloride fertilization • liquid swine manure
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Decreases in forage DCAD following Cl or LSM fertilization are the result of an increase in forage Cl concentration (Pelletier et al., 2007a,b, 2008). With an increased application from 0 to 240 kg of Cl/ha, Cl concentration of timothy forage increased by 8.5 and 15.1 mg/g of DM for forages grown on Canadian and Australian soils, respectively. In both cases, the greatest forage Cl concentration observed was close to 20 mg/g of DM. Nonwoody species, such as timothy, can generally tolerate plant Cl concentrations as high as 15 to 50 mg/g of DM (Xu et al., 2000). The NRC (2001) reports that the average Cl concentration of forage is 6.6 mg/g of DM. The effect of high forage Cl concentrations, as observed by Pelletier et al. (2007a,b, 2008), on the nutritive value of timothy has never been investigated.
Although the effects of Cl fertilization are site-specific and vary during the growing season, Cl fertilization may have an impact on grass development (Fixen, 1993). Chloride plays a role in several processes associated with DM production, including enzyme activation, phloem loading and unloading of sugars, interactions with N metabolism, and the increases in cell hydration and turgor pressure that support cell expansion and elongation (Fixen, 1993; Taiz and Zeiger, 1998; Xu et al., 2000; Britto et al., 2004).
Although Cl fertilization up to 240 kg of Cl/ha did not affect timothy DM yield (Pelletier et al., 2007a), mechanisms affected by Cl (e.g., N metabolism, cell expansion and extension) may have, in turn, affected forage digestibility and concentrations of CP and NDF. To our knowledge, there are no reports that document the effects of Cl fertilization on forage digestibility and concentrations of CP and NDF. The objective of our study was to document the effects of applying organic and inorganic Cl fertilizers on forage CP and NDF concentrations, in vitro true digestibility of DM (IVTD), and in vitro digestibility of NDF (dNDF) of timothy grown on different soils and harvested twice during the growing season.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Average DM yields for spring growth and summer regrowth were, respectively, 3.70 and 1.45 Mg/ha at Sainte-Anne-de-Bellevue (hereafter called Sainte-Anne), 3.76 and 2.58 Mg/ha at Normandin, 4.22 and 3.34 Mg/ha at Saint-Augustin-de-Desmaures (hereafter called Saint-Augustin), and 1.96 and 1.49 Mg/ha at Sainte-Perpétue. Details of DM yields are provided in Pelletier et al. (2007a).
LSM Fertilization Experiment.
The study sites and experimental setup were part of a larger experiment described in detail by Chantigny et al. (2007). Timothy was sown in 2000 at Saint-David-de-Lévis (46°48' N, 71°23' W) and at Saint-Lambert-de-Lévis, Quebec, Canada (46°05' N, 71°02' W). In 2001 and 2002, 7 fertilization treatments were applied: 4 pretreated LSM (decanted, filtered, digested, and flocculated), 1 raw LSM, 1 inorganic fertilizer, and 1 unfertilized control.
Raw LSM was obtained during the winters of 2001 and 2002 from a commercial swine finishing operation. Part of the collected LSM was transferred into a batch anaerobic digester. After standing for 1 mo in the batch digester, anaerobically digested LSM was transferred into a 1-m3 plastic container and labeled "digested LSM". The rest of the collected LSM was stored for 6 wk in four 1-m3 plastic containers. After this period, the upper half of the raw LSM was pumped out of 2 plastic containers and transferred to an empty 1-m3 container. This liquid material was labeled "decanted LSM" and represented the clarified fraction of raw LSM after the natural settling of solids. Manure from a third plastic container was strained through a bed of wood shavings and sawdust. The filtrate was collected in a plastic container and labeled "filtered LSM". A fifth LSM type was obtained from another commercial swine finishing operation. This manure was chemically treated with a CaCl2-based coagulant to remove solids, and 1 m3 of the liquid fraction was collected in a plastic container and labeled "flocculated LSM". Selected characteristics of the LSM are presented in Table 1
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Plots were harvested twice to a 5-cm height when timothy reached the early-heading stage of development with an REM flail forage harvester (Swift Machine & Welding) at Saint-Lambert-de-Lévis and with a self-propelled flail forage harvester (Carter MGF Co. Inc.) at Saint-David-de-Lévis. The second harvest was taken approximately 6 wk after the first one. Average DM yields for spring growth and summer regrowth were, respectively, 3.66 and 2.77 Mg/ha at Saint-Lambert-de-Lévis (hereafter called Saint-Lambert), and 5.66 and 2.05 Mg/ha at Saint-David-de-Lévis (hereafter called Saint-David).
Chemical Analyses
Manure.
For each field application of manure, a 2-L composite sample was collected from each LSM type for analysis. Samples were homogenized with a Polytron homogenizer (model PT 3100, Kinematica AG, Littau-Lucerne, Switzerland); pH was then measured directly by reading with a glass electrode. Dry matter content was determined as the weight of materials remaining after drying 100 mL of homogenized LSM at 55°C for 96 h. Total C concentrations were measured in the homogenized LSM samples by direct injection in an automated combustion C analyzer (model Formacs, Skalar Analytical, De Breda, the Netherlands).
Nitrogen and K concentrations of the various LSM types were determined by acid digestion modified from the method of Isaac and Johnson (1976). Nitrogen was measured with an automated continuous-flow injection analyzer (model QuikChem 8000 FIA+, Zellweger Analytics Inc., Lachat Instruments, Milwaukee, WI) as described in Chantigny et al. (2007), and K was determined by flame emission with a Perkin Elmer 3300 atomic absorption spectrometer (Perkin Elmer, Überlingen, Germany). Finally, Cl was extracted by mixing 5 mL of the LSM sample with 20 mL of distilled water for 30 min; extracts were centrifuged at 32,570 x g for 10 min and filtered, and Cl concentration was determined in the supernatant by chromatography as previously described by Pelletier et al. (2007a).
Plants.
For both experiments, sample preparation and N analyses are described in Pelletier et al. (2007a). Briefly, a fresh forage sample of approximately 500 g was taken from each plot, weighed, and dried at 55°C in a forced-draft oven for 2 d (LSM fertilization experiment) or 3 d (inorganic fertilization experiment) to determine the DM concentration. Samples were then ground using a Wiley mill (standard model 3, Arthur H. Thomas Co., Philadelphia, PA) to pass through a 1-mm screen. Nitrogen was extracted using a method adapted from Isaac and Johnson (1976) and measured as described for manure extracts. Crude protein was determined by multiplying the forage N concentration by 6.25.
For the LSM fertilization experiment, all forage samples were analyzed chemically. The NDF concentration was determined using the Ankom Fiber Analyzer (Ankom Technology Corp., Fairport, NY). The IVTD was measured using the method of Goering and Van Soest (1970) based on a 48-h incubation with buffered rumen fluid followed by an NDF determination of the postdigestion residues. The rumen fluid incubation was performed with Ankom F57 filter bags and an Ankom Daisy II incubator, using the batch incubation procedures outlined by Ankom Technology Corp. Rumen fluid was obtained from a lactating, ruminally fistulated dairy cow that was offered a diet of good quality silages [40% timothy, 60% alfalfa (Medicago sativa L.)], corn (Zea mays L.) grain, and a concentrate mix. The diet was formulated to meet the nutritional requirements of a 636-kg lactating cow producing 9,580 kg of milk per year.
The IVTD (g/kg of DM) and dNDF (g/kg of NDF) were calculated as follows:
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For the inorganic fertilization experiment, milled samples were scanned using a near-infrared spectrophotometer (Foss NIRsystems 6500, Foss, Silver Spring, MD). Reflectance from 400 to 2,500 nm at 2-nm intervals was collected from all samples. The near infrared spectroscopy calibration equations were developed using a modified least squares regression method of the WinISI III software (Infrasoft International LLC, State College, PA). The 160 spectra with the greatest relative significance were selected from all samples of the 2 production years and the 4 locations. Of these samples, 140 were selected for the calibration set, and the other 20 samples were selected for the validation set. The 160 selected samples were analyzed in duplicate for NDF concentration and IVTD as previously described. The calibration equations were selected based on standard errors of prediction bias corrected, and R2 for the validation set; these statistics were, respectively, 0.949, 0.103, and 0.97 for the NDF concentration, and 2.029, –1.02, and 0.92 for the IVTD. The NDF concentration and IVTD were predicted in all samples using the selected calibration equations. The dNDF (g/kg of NDF) was then calculated as follows:
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Statistical Analyses
Data of the inorganic fertilization experiment were analyzed by ANOVA as a split-split-plot design with 4 replicates. Locations were assigned as main plots, fertilizer treatments as subplots, and harvests as sub-subplots; sources of variation are presented in Table 2. The experimental design at each location of the LSM experiment was a randomized complete block with 4 replicates; sources of variation are presented in Table 3. For both experiments, production years and replicates within locations were considered to be random effects, and harvests within years were considered to be repeated measurements. Data were analyzed using the MIXED procedure (Littell et al., 1996) with the Repeated option of SAS (SAS Institute Inc., 1999). In both experiments, plots were not rerandomized each year. Years were not treated as repeated measurements because there was no residual effect of Cl and N fertilizations in the spring of the second year. Indeed, soil Cl, NO3, and NH4 contents from the inorganic fertilization experiment and soil NO3 content from the LSM fertilization experiment at the beginning of the second year were not significantly different among fertilization treatments. Statistical significance was postulated at P 
0.05. Least squares means and standard error of the means (SEM) were calculated. Averaged SEM allowing comparison of values within fertilization treatments (Tables 2 and 3) and within the interaction location x harvest (Figure 1) were calculated from SEM given for these sources of variations in the difference of least squares means output of SAS. Contrasts defined a priori were performed on treatments of the inorganic fertilization experiment. Comparisons of least squares means from the LSM fertilization experiment were carried out using the predicted difference (PDIFF) option of SAS.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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). Chloride fertilization significantly increased NDF concentration (P = 0.040) and decreased IVTD (P = 0.026) and dNDF (P = 0.028) but only at the greatest application of N fertilization (Table 2). These variations, as a proportion of values when no Cl was applied, remained small (from 1.2 to 1.9%) for all variables. Moreover, when analyzed for each location, the linear effect of Cl fertilization on NDF concentration was not significant at any of the locations, and the quadratic effect of Cl fertilization on IVTD and dNDF was significant only at Sainte-Perpétue (IVTD, P = 0.035; dNDF, P = 0.026; data not shown). The CP concentration was not affected by Cl fertilization. Both types of Cl fertilizer (CaCl2 vs. NH4Cl) had the same effect on the nutritive value of timothy (Table 2).
Increasing N fertilization generally increased CP and NDF concentrations (P < 0.001) and decreased IVTD and dNDF (P < 0.001) of timothy forage (Table 2). The significant interaction between fertilization treatments and locations for NDF concentration and IVTD (Table 2) was due to N fertilization. Variations in forage NDF concentration and IVTD with increasing N fertilization were greater at Sainte-Anne (+14.5 g/kg of DM for NDF; –12.2 g/kg of DM for IVTD) and Saint-Augustin (+15.4 g/kg of DM for NDF; –24.5 g/kg of DM for IVTD) than at Normandin (–0.5 g/kg of DM for NDF; –2.6 g/kg of DM for IVTD) and Sainte-Perpétue (+6.0 g/kg of DM for NDF; –3.1 g/kg of DM for IVTD). The effect of N fertilization on forage NDF concentration also varied with harvests (Table 2); the increase in NDF concentration with increasing N application rates was generally more important in summer regrowth than in spring growth (data not shown).
Forage CP and NDF concentrations, IVTD, and dNDF were affected by an interaction between locations and harvests (Table 2; Figure 1). The CP concentration was greatest at Normandin in spring growth and at Sainte-Perpétue in summer regrowth (Figure 1A). Neutral detergent fiber concentration was greatest at Saint-Augustin in spring growth and at Normandin and Saint-Augustin in summer regrowth (Figure 1B). The IVTD (Figure 1C) and dDNF (Figure 1D) were greatest at Saint-Anne and Normandin in spring growth and greatest at Sainte-Anne only in summer regrowth. Variations with harvests and locations were, on average, 45 g/kg of DM for CP, 73 g/kg of DM for NDF concentration, 75 g/kg of DM for IVTD, and 109 g/kg of NDF for dNDF (Figure 1). Proportionally, these variations remained small for NDF concentration, IVTD, and dNDF (9 to 14%), but were larger for CP (28%).
LSM Fertilization
The flocculated LSM, which had the greatest Cl content of all LSM types (Table 1), did not affect forage NDF concentration, IVTD, and dNDF compared with the other LSM types (Table 3). Timothy fertilized with LSM had greater CP and NDF concentrations and generally lower IVTD and dNDF than the unfertilized control forage. On the other hand, timothy fertilized with LSM had lower CP concentration, especially with the raw LSM, and generally greater IVTD and dNDF than forage fertilized with the inorganic fertilizer (Table 3).
The effect of fertilization treatments on forage CP and NDF concentrations varied with harvests (Table 3). The CP concentration was generally similar or lower in summer regrowth than in spring growth, except for the unfertilized control timothy, which had greater CP concentration in summer regrowth (data not shown). On the other hand, NDF concentration was smaller in summer regrowth than in spring growth for all treatments, but the difference between harvests was greater for the unfertilized control timothy.
Concentrations of CP and NDF, and IVTD and dNDF of timothy, were affected by an interaction between locations and harvests (Table 3; Figure 1). From spring growth to summer regrowth, CP concentration decreased in timothy grown at Saint-Lambert and increased in forage grown at Saint-David; on average for both harvests, CP concentration remained greater in forage grown at Saint-Lambert (Figure 1A). The NDF concentration was greater in forage grown at Saint-David, especially in spring growth (Figure 1B). Finally, forages grown in spring at Saint-David had lower IVTD (Figure 1C) and dNDF (Figure 1D) than those grown at Saint-Lambert. In summer regrowth, IVTD and dNDF of forages grown at Saint-David were greater than those of forages grown at Saint-Lambert. These variations over harvests and locations were of 47 g/kg of DM for NDF concentration, 16 g/kg of DM for IVTD, 2.5 g/kg of NDF for dNDF, and 31 g/kg of DM for CP. Similar to the inorganic fertilization experiment, these variations remained small for NDF, IVTD, and dNDF (3 to 8%) but were greater for CP concentration (20%).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Chloride Fertilization
The effect of Cl fertilization on forage NDF concentration and digestibility of DM and NDF (Figure 2), although small, was surprising. Plants contain several compounds with covalently bound Cl (White and Broadley, 2001), but this element is biochemically inert (Fixen, 1993). Forage digestibility (Claessens et al., 2004) and Cl concentration (Xu et al., 2000) are both related to plant growth. Plant Cl concentration may improve plant growth because its accumulation increases cell hydration and turgor pressure needed for cell expansion and elongation (Xu et al., 2000), whereas the production of cellulose, hemicellulose, and lignin, which are NDF components, is needed to maintain constant wall thickness during cell expansion (Carpita and McCann, 2000). Thus, cell expansion and continued deposition of NDF polymers into the cell wall must be tightly integrated events. Therefore, high Cl concentration could be associated with increased cell expansion and plant growth which could, in turn, be associated with increased deposition of NDF components.
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The effect of Cl in the inorganic fertilization experiment at the greatest N application rate could be due to interactions between Cl and N in their biochemical functions. Indeed, it is known that Cl plays a role in N metabolism and that a number of proteins are involved in modulating the requirement for Cl in the evolution of O2 (Britto et al., 2004).
Nitrogen Fertilization
Conflicting results have been reported for the effect of N fertilization on the DM digestibility of timothy (Bélanger et al., 2001). In studies conducted in the Netherlands (Deinum et al., 1968), Sweden (Thorvaldsson and Andersson, 1986), and Canada (Kunelius et al., 1976), increasing application rates of N fertilization decreased timothy DM digestibility. However, no effect of applied N on forage DM digestibility was reported by Calder and MacLeod (1968) and St-Pierre and Pelletier (1977) in eastern Canada. In a more recent study with inorganic N fertilization, Bélanger and McQueen (1998) report that N fertilization decreased DM digestibility of timothy, an effect that was attributed mainly to a decreased proportion of leaves. Bélanger and McQueen (1998) conclude that one possible explanation for the conflicting reports concerning the effect of N fertilization on the digestibility of DM is the varying degree of crop N deficiency in the studies. A relative yield (DM yield obtained for a given treatment divided by the maximal observed DM yield) of less than 60% usually resulted in increased DM digestibility.
In the LSM experiment, the DM yield with no N applied was 66% of the maximal DM yield obtained with the inorganic fertilizer (Chantigny et al., 2007). The intermediate values for DM yield and IVTD of forages fertilized with LSM are probably due to lower availability and greater losses of N from LSM compared with the inorganic fertilizer (Chantigny et al., 2007). Our results confirm that increasing N fertilization with LSM or inorganic fertilizer can have a negative effect on the digestibility of DM, at least for situations in which the relative yield is 66% or less in the absence of N fertilization. In the inorganic fertilization experiment, the average DM yield over Cl fertilization treatments, years, and locations of forages fertilized with 70 kg of N/ha was 90% of the maximum DM yield obtained with forage fertilized with 140 kg of N/ha (Pelletier et al., 2007a), suggesting that timothy growth was only slightly limited by N. The IVTD of forage fertilized with 140 kg of N/ha was only 10 g/kg of DM lower than that of forage fertilized with 70 kg of N/ha. This small difference supports the hypothesis that when N limitation is negligible, the digestibility of DM and NDF is not affected by N fertilization.
In the LSM experiment, N fertilization increased NDF concentration and decreased dNDF. Similar results were reported by Bélanger and McQueen (1998). Increasing N fertilization with LSM or inorganic fertilizer also resulted in an increased forage CP concentration. This was reported by MacLeod and Macleod (1974) and Guertin et al. (1979). Although statistically significant, the effect of N fertilization on the 4 attributes of nutritive value was relatively small (Table 3). When no N was applied, CP concentration was 14% lower, NDF concentration was 4% lower, and both IVTD and dNDF were 2% greater than when N was applied. Our results confirm that when timothy is grown under nonlimiting N conditions, the increase in DM yield caused by N fertilization does not result in a biologically significant decrease in nutritive value.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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Received for publication June 8, 2007. Accepted for publication October 2, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSACKNOWLEDGEMENTSREFERENCES |
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, •) and NDF concentrations (
, 
), in vitro true digestibility of DM (IVTD; 
, 
), and in vitro digestibility of NDF (dNDF; 
, 
). Values are averages of 17 fertilization treatments (inorganic fertilization experiment; seasonal applications of 0, 80, 160, and 240 kg of Cl/ha as CaCl2, 160 kg of Cl/ha as NH4Cl; all combined with 70 or 140 kg of N/ha as NH4NO3 or a mix of NH4NO3 and NH4Cl; liquid swine manure (LSM) fertilization experiment: unfertilized control, inorganic fertilizer, raw LSM, and decanted, filtered, digested, and flocculated LSM), 6 locations (Sainte-Anne, Normandin, Saint-Augustin, Sainte-Perpétue, Saint-Lambert, and Saint-David), 2 harvests (spring growth and summer regrowth), and 2 yr (inorganic fertilization experiment: 2003 and 2004; LSM fertilization experiment: 2001 and 2002).