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a Dep. of Plant Science, McGill University, Sainte-Anne-de-Bellevue, QC, Canada H9X 3V9
b Agriculture and Agri-Food Canada, Soils and Crops Research and Development Centre, 2560 Hochelaga Blvd., Sainte-Foy, QC, Canada G1V 2J3
* Corresponding author (michaudr{at}agr.gc.ca).
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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Abbreviations: ADF, acid detergent fiber • ADL, acid detergent lignin • CEL, cellulose • DM, dry matter • HEM, hemicellulose • IVNDFD, in vitro neutral detergent fiber digestibility • IVTD, in vitro true digestibility • NDF, neutral detergent fiber
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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Forage digestibility, which is commonly used to estimate forage nutritive value, can be improved by selecting for reduced fiber concentration (Shenk and Elliot, 1970; Carpenter and Casler, 1990; Casler 1999). While the reduction of fiber concentration might improve digestibility, it could also have the undesirable correlated response of reducing forage yield. Surprenant et al. (1988) in reed canarygrass (Phalaris arundinacea L.) and Casler (1999) in smooth bromegrass (Bromus inermis Leyss.) reported a reduction in forage yield in populations selected for reduced fiber concentration.
Another approach to improve forage digestibility would be to increase the digestibility of the cell wall, which is composed primarily of lignin, cellulose, and hemicellulose. Lignin is indigestible and reduces cell wall digestibility directly (Casler, 2001). It also cross-links with cell wall polysaccharides, particularly hemicellulose, limiting the access of rumen microorganisms to hemicellulose and cellulose, thus reducing the digestibility of those fiber components (Jung and Deetz, 1993; Moore and Hatfield, 1994; Hatfield et al., 1999). Cellulose and hemicellulose also limit forage digestibility through their own composition and structure. They are both more digestible than lignin, with hemicellulose at least as digestible as cellulose (Casler, 1987; Buxton and Brasche, 1991). Because the three fiber components (lignin, cellulose, and hemicellulose) have different digestibilities, their proportion in the cell wall should affect forage digestibility. We hypothesized that selection based on the concentration of the fiber components and/or their ratios may be effective in increasing timothy digestibility while maintaining DM yield.
The objectives of this study were (i) to assess genetic variation for the concentrations of fiber components and for ratios between these concentrations in timothy germplasm, (ii) to identify and evaluate selection criteria which could have a meaningful effect on timothy digestibility without adversely affecting DM yield, and (iii) to study relationships among in vitro true digestibility (IVTD), in vitro NDF digestibility (IVNDFD), fiber component concentrations and their ratios, and DM yield of timothy.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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45 m) in late May 1994, with plants spaced at 90 cm between and within rows. The herbicide 4-chloro-2-methylphenoxyacetic acid was applied at 2 L ha–1 about 1 mo after transplantation for control of annual weeds. In late August, plants were clipped with a flail-type harvester to a 10-cm stubble height and fertilized with 40 kg N ha–1. In each of the two years following establishment (1995 and 1996), the nursery was fertilized with 55 kg N ha–1 in early May and 1.3 L ha–1 of a mixture of 295 g L–1 2,4-D (2, 4-dichlorophenoxyacetic acid), 110 g L–1 mecoprop [2(4-chloro-2-methylphenoxypropanoic acid)], and 80 g L–1 dicamba (3, 6-dichloro-2-methoxybenzoic acid) was applied for weed control after the second harvest. In June 1995, visual phenotypic selection was applied for general appearance and vigor in each population and 107, 124, 124, and 140 plants were kept from AC Alliance, AC Antoine, AC Apex, and SF8602, respectively. The 495 selected plants were sampled at a 10-cm stubble height with a hand sickle on 20 and 23 June in 1995 and on 17 and 26 June in 1996; plants had reached a growth stage ranging from 50 to 54 in 1999 and 52 to 58 in 1996 according to the scale of Simon and Park (1981). In late August 1995, plants were cut to a 10-cm stubble height with a flail-type harvester and 40 kg N ha–1 was applied; no samples were taken. Each hand-harvested plant sample from the first harvest in 1995 and 1996 was air-dried at 55°C for 2 d and DM yield was measured on a single-plant basis. Forage samples were ground to pass through a 1-mm screen in a Wiley mill (Thomas-Wiley Laboratory Mills, Philadelphia, PA).
All 495 samples from the first harvest in each year were scanned on a NIR spectrophotometer (Pacific Scientific Model 6150, Silver Spring, MD). Each year, 60 forage samples were chosen for calibration purposes by cluster analysis (Infrasoft International, 1993). The calibration samples were analyzed in duplicate for NDF, ADF, and ADL concentrations by the detergent fiber system of Goering and Van Soest (1970), except that neither sodium sulfite nor 
-amylase were used and the initial sample dry weight was 0.25 g instead of 0.50 g. Separate samples were used for NDF and ADF analyses. Determinations from the 60 calibration samples were used each year to develop NIR spectroscopy calibration equations for NDF, ADF, and ADL concentrations expressed in grams per kilogram DM. The R2 values of the calibration equations were 0.91 for NDF, 0.93 for ADF, and 0.76 for ADL in 1995 and 0.97 for NDF, 0.96 for ADF, and 0.79 for ADL in 1996. Standard errors of calibration for NDF, ADF, and ADL were respectively 6.0, 7.6, and 3.9 g kg–1 DM in 1995 and 11.4, 7.7, and 3.0 g kg–1 DM in 1996. The predicted values of NDF, ADF, and ADL concentrations were used to estimate hemicellulose concentration by subtracting ADF from NDF and to estimate cellulose concentration by subtracting ADL from ADF. No correction for ash was made.
Selection for DM yield was performed in each population, on the basis of mean data from 1995 and 1996. Plants that were superior to the mean yield of the population by at least one standard error were selected. This resulted in 78 plants with 16, 17, 21, and 24 plants coming from AC Alliance, AC Antoine, AC Apex, and SF8602, respectively. Following this selection, the 2-yr average concentrations of NDF, ADF, ADL, hemicellulose (HEM), and cellulose (CEL) and four ratios calculated from these values (ADL/HEM, ADL/CEL, ADL/(HEM + CEL), and HEM/CEL) were used as criteria for divergent selection. For each of the nine criteria, the two plants with the highest values and the two plants with the lowest values were selected from within each of the four populations. This resulted in the selection of 45 genotypes, 36 of which were selected for more than one criterion. Means and standard errors were calculated for the base population of 495 plants and for each group of eight selected genotypes. There was very limited difference in maturity among the selected genotypes, which ranged from growth stage 52 to 58 (Simon and Park, 1981) at harvest. No correlation was found between growth stage values and NDF concentration in either year (data not shown).
Evaluation of Selected Genotypes
The 45 selected genotypes were vegetatively propagated in the greenhouse in April 1998 and transplanted to the field in May 1998 at Lévis, QC, Canada. The genotypes were arranged in a randomized complete block design with four blocks. Each plot consisted of two plants per genotype. Plants were spaced at 90 cm between and within rows. Fertilizers and herbicides were applied as described for the selection nursery.
Average monthly temperatures in 1999 were 4.2, 14.7, and 18.2°C for April, May, and June respectively, and 3.7, 10.9, and 15.0°C for the same months in 2000. All eight plants of each genotype were harvested when at least four of them had reached growth stage 52. In 1999, harvest operations were interrupted by rain, with 35 genotypes harvested on 12 June at growth stages between 52 and 56 and 10 genotypes harvested at growth stages between 54 and 58 on 17 June. In 2000, plants were harvested from 19 to 23 June, at growth stages ranging from 52 to 56. In both years, each plant was cut to a 10-cm stubble height with a hand sickle. Plants were air-dried at 55°C for 2 d and DM yield was measured on a single-plant basis. Forage samples were ground to pass through a 1-mm screen in a Wiley mill. All samples were scanned on a NIR spectrophotometer (Pacific Scientific Model 6150, Silver Spring, MD). Each year, 60 forage samples were chosen for calibration purposes and were analyzed for NDF, ADF, and ADL concentrations as described for the samples from the selection nursery. New calibration equations were developed for NDF, ADF, and ADL concentrations. The R2 values of the calibration equations were 0.97 for NDF, 0.94 for ADF, and 0.82 for ADL in 1999 and 0.96 for NDF, 0.99 for ADF, and 0.90 for ADL in 2000. Standard errors of calibration for NDF, ADF, and ADL were respectively 9.8, 7.8, and 2.5 g kg–1 DM in 1999 and 11.8, 8.3, and 2.1 g kg–1 DM in 2000. Neutral detergent fiber, ADF, and ADL predicted values were used to estimate HEM and CEL concentrations.
The calibration samples were also analyzed for in vitro true digestibility (IVTD), which estimates digestibility of the forage DM, and in vitro NDF digestibility (IVNDFD), which estimates the digestibility of the NDF fraction. The IVNDFD was calculated from NDF values as follows: IVNDFD (g kg–1) = [1– (postdigestion NDF dry weight/predigestion NDF dry weight)] x 1000. No correction for ash was made. The NDF determination of the postdigestion samples was done with an ANKOM Fiber Analyzer (Ankom Technology, Fairport, NY), using sodium sulfite and -amylase. The rumen fluid digestion used to measure digestibility was done with an ANKOM Daisy II incubator (Ankom Technology, Fairport, NY). Rumen fluid was obtained from a lactating ruminally fistulated dairy cow. Determinations from the 60 calibration samples were used each year to develop NIR spectroscopy calibration equations for IVTD and IVNDFD concentrations. The R2 values of the calibration equations were 0.95 for IVTD and 0.93 for IVNDFD in 1999 and 0.91 for IVTD and 0.90 for IVNDFD in 2000. Standard errors of calibration for IVTD and IVNDFD were respectively 16.7 g kg–1 DM and 21.9 g kg–1 NDF in 1999 and 25.1 g kg–1 DM and 22.2 g kg–1 NDF in 2000.
Statistical Analysis
Analysis of variance (GLM Procedure, SAS Institute, 1996, Cary, NC) was conducted on the data from 1999 and 2000, with genotypes as fixed effects and years and replications as random effects. Data from each year were also analyzed separately. The data from the genotypes were then grouped by selection criterion and evaluated by performing contrasts between the divergent groups. Statistical significance was defined as P < 0.05.
Correlation analysis was conducted to study the relationships among IVTD, IVNDFD, fiber components and their ratios, and DM yield, by means of genotype mean values within years and the correlation matrix method (SAS Institute, 1996, Cary, NC).
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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Digestibility
ADL-Based Selection Criteria
Selections that involved ADL concentrations [ADL, ADL/HEM, ADL/CEL, and ADL/(HEM+CEL)] were generally the most stable across years for forage IVTD. Divergent ADL, ADL/HEM, ADL/CEL, and ADL/(HEM+CEL) groups differed for IVTD in 1999 and 2000 (Table 3). Differences in IVTD averaged over 2 yr for the ADL (22 g kg–1 DM), ADL/HEM (24 g kg–1 DM), and ADL/(HEM+CEL) (23 g kg–1 DM) selections were generally greater than that for the ADL/CEL (14 g kg–1 DM) selection. Our results highlight the importance of lignin in timothy digestibility and they are similar to observations by Carpenter and Casler (1990) in smooth bromegrass. They reported that one cycle of phenotypic selection for reduced ADL concentration was more effective in improving digestibility than selection for reduced NDF, ADF, HEM, or CEL concentrations or even direct selection for digestibility.
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The low-ADL/HEM group had lower ADF and ADL concentrations and higher HEM concentrations than the high-ADL/HEM group in 1999, but had lower NDF, ADF, ADL, and CEL concentrations than the high group in 2000 (Table 2). Despite these differences between the two years, the low-ADL/HEM group had higher IVTD and IVNDFD values than the high-ADL/HEM group in both years (Table 3). Selection based on ADL/HEM produced groups as divergent for IVTD and IVNDFD than selection based on ADL only (Table 3). The ADL/HEM groups had 10 genotypes in common with the ADL groups and, therefore, only six genotypes were specific to each selection. This helps explain the similarity of the effect of the ADL and ADL/HEM selections on digestibility.
The low-ADL/CEL group had lower ADL than the high-ADL/CEL group in both years. The two groups had similar NDF concentration in 2000 but not in 1999, whereas they had similar ADF, HEM, and CEL concentrations in both years (Table 2). Hence, the lower IVTD and IVNDFD of the low-ADL/CEL group were generally associated only with reduced ADL concentration (Table 3). Selection based on ADL/CEL produced groups as divergent for IVTD and IVNDFD as selection based on ADL only, without changing the total cell wall concentration (Table 3). The ADL/CEL groups had seven genotypes in common with the ADL groups indicating that the differences observed between the two selections were due to the nine genotypes not found in both selections.
The low-ADL/(HEM+CEL) group had lower ADL than the high-ADL/(HEM+CEL) group in both years, but the two groups had similar NDF, ADF, HEM, and CEL concentrations (Table 2). The selection based on ADL/(HEM+CEL) resulted in groups that were more divergent for IVNDFD in both years than those from selection based on ADL (Table 3). Reduction in lignin concentration within the cell walls apparently allowed for a more complete digestion of other cell-wall components. This selection also affected IVTD, for which the ADL/(HEM+CEL) groups were divergent, but usually no more so than the ADL/HEM or ADL groups (Table 3). The greater effect for IVNDFD than for IVTD is probably due to the selection for reduced ADL/(HEM+CEL) modifying cell wall composition (reducing lignin relative to the other fractions) without affecting the overall fiber concentration in the plant. The ADL/(HEM+CEL) groups had 10 genotypes in common with the ADL groups indicating that the differences observed between the two selections were due to the six genotypes not found in both selections.
Selecting for ratios of indigestible (ADL) to digestible (HEM and/or CEL) fiber components allowed the increase of cell wall digestibility with no consistent reductions in the total cell wall concentration, which is important for maintaining DM yield.
NDF, ADF, and CEL-Based Selection Criteria
In the analysis of variance combined over years, the divergent NDF and ADF groups differed for digestibility (data not shown). However, the analysis of variance for each year indicated that the IVTD and IVNDFD values of divergent NDF and ADF groups were similar in 1999 but different in 2000 (Table 3). In 1999, the divergent NDF and ADF groups differed less for their NDF and ADF concentrations than in 2000 (Table 2). Since the fiber concentration has an important effect on digestibility, the lack of difference in digestibility in 1999 could be due to the lower variability in NDF and ADF concentrations between divergent groups as opposed to 2000. These major differences between years could be due to the different climatic conditions prevailing in 1999 and 2000. Temperature has been reported to accelerate the rate of plant development (Buxton and Casler, 1993). The warmer temperatures in 1999 increased the rate of development, which might have reduced the difference among genotypes. Our results show that the NDF and ADF selection criteria were less stable across years than the selection criteria involving ADL.
The low-CEL group showed similar IVNDFD value in both years and higher IVTD value in 2000 compared to the high-CEL group (Table 3), indicating that selection for cellulose concentration had no effect on the cell wall digestibility and only a limited impact on forage digestibility. These results are similar to those of Carpenter and Casler (1990) who conducted divergent selection for cellulose concentration in smooth bromegrass and did not observe a significant difference in in vitro DM digestibility.
Dry Matter Yield
In the analysis of variance combined over years, the NDF, ADL, ADL/HEM, ADL/CEL, and ADL/(HEM+CEL) differed for DM yield (data not shown). The low-NDF and -ADL/HEM groups had lower DM yield than their corresponding high groups. Selection for low NDF concentration has been reported to reduce DM yield in reed canarygrass (Surprenant et al., 1988) and smooth bromegrass (Casler, 1999). However, when we analyzed each year separately, no significant difference for DM yield between divergent NDF groups was observed (Table 3). Therefore, in our study, the visual selection for vigor followed by modified independent culling selection for DM yield and fiber components may have prevented this expected negative association between DM yield and fiber concentration. Similarly, this lack of a forage yield response to selection for high nutritive value has also been observed in alfalfa and switchgrass (Panicum virgatum L.) populations that were simultaneously selected for both criteria (Coors et al., 1986; Hopkins et al., 1993). As for the divergent ADL and ADL/HEM selections, the DM yield response was not consistent across years.
The low-ADL/CEL and -ADL/(HEM+CEL) groups, characterized by higher IVTD and IVNDFD values, had higher DM yield than their corresponding high groups in both years (Table 3). Given that the divergent ADL/CEL and ADL/(HEM+CEL) groups had very similar NDF, ADF, HEM, and CEL concentrations, their differences in DM yield were not related to fiber concentration.
Relationships among Variables
Digestibility and Cell Wall Components
It is well accepted that digestibility decreases with increases in cell wall concentrations (Buxton and Casler, 1993; Jung and Allen, 1995; Casler, 2001). The selection responses observed here were consistent with this only in 2000 where IVTD was negatively correlated to NDF concentration (Table 4). The traits ADL, ADL/HEM, ADL/CEL, and ADL/(HEM+CEL) were all highly negatively associated with IVTD and IVNDFD in both years, indicating that plants with low values for these traits would tend to be more digestible than plants with high values (Table 4). These relationships are consistent with the divergence in IVTD and IVNDFD observed after selection based on ADL, ADL/HEM, ADL/CEL, and ADL/(HEM+CEL), and they indicate that there is potential for further genetic improvement. The ADF concentration was also negatively correlated to IVTD and IVNDFD in both years (Table 4). However, the divergent selection based on ADF concentration did not provide consistent improvements in IVTD or IVNDFD (Table 3).
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Digestibility and DM Yield
Many studies have reported a negative correlation between digestibility and DM yield (Durand and Surprenant, 1993; Bélanger and McQueen, 1998; Casler and Vogel, 1999). Here, IVTD and DM yield exhibited a moderate negative relationship in 1999 (r = –0.44) but were not significantly correlated in 2000 (Table 4). This lack of strong association between IVTD and DM yield may have resulted from selection pressure for DM yield applied to the genotypes before the selection for fiber traits. Therefore, our selection results indicate that it is possible to dissociate this negative relationship by selection for DM yield combined with the selection for an appropriate nutritive value criterion.
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SUMMARY AND CONCLUSIONS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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Received for publication January 22, 2003.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUMMARY AND CONCLUSIONSREFERENCES |
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