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Published online 24 January 2006
Published in Crop Sci 46:256-263 (2006)
© 2006 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
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FORAGE & GRAZINGLANDS

Influence of Grass Species and Sample Preparation on Ensiling Characteristics

D. J. R. Cherney*,a, M. A. Alessib and J. H. Cherneya

a Cornell University, Ithaca, NY, USA 14853
b Universitá Degli Studi Di Palermo, Italy

* Corresponding author (djc6{at}cornell.edu)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Laboratory silos are considered a practical method of comparing a number of treatments and are necessary when evaluating numerous experimental variables and their interactions involving different grass silages. Objectives of this study were to evaluate the suitability of grasses ensiled in vacuum-sealed polyethylene bags to assess treatment differences. Four field replicates of three grass species, orchardgrass (Dactylis glomerata L., OG), reed canarygrass (Phalaris arundinacea L., RG), and tall fescue (Festuca arundinacea Schreb., TF), harvested at two dates were ensiled whole or chopped. Bacterially inoculated grass samples (500 g) were ensiled for 0, 2, 4, 8, 16, 24, or 30 d. At 30-d post ensiling, lactic acid was the predominant acid, suggesting good fermentation. Differences were noted among species, harvest date, and chop, suggesting that the polyethylene bag is sensitive to treatment differences. There was little or no butyric or propionic acids in the silages, indicating that the silages did not undergo clostridial fermentation. Ensiled grasses dropped rapidly in pH and were stable beyond 4 d for all treatments. Despite species and processing differences, pH of all silages tended to be under 4.7, considered acceptable for grass silages. We conclude that it is possible to use vacuum-sealed plastic bags to ensile temperate grasses to assess treatment differences.

Abbreviations: ADF, acid detergent fiber • CP, crude protein • DM, dry matter • NDF, neutral detergent fiber • NDFD, neutral detergent fiber digestibility • NSC, non-structural carbohydrates • OG, orchardgrass • RG, reed canarygrass • TF, tall fescue • VFA, volatile fatty acid • IVTD, in vitro true digestibility


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
LABORATORY SILOS are considered a practical method of comparing a number of treatments (O'Kiely, 1993) and are necessary when evaluating numerous experimental variables and their interactions involving different silages. A critical assumption is that the fermentation process is reasonably similar to that taking place in field-scale silos (Cherney and Cherney, 2003). A survey of the limited number of studies directly comparing fermentation in field-scale and small-scale silos resulted in the conclusion that forage in both silo types did undergo a similar fermentation (Meiske et al., 1975). May et al. (2001) described a vacuum bag system that they used to evaluate fungal communities associated with whole plant corn (Zea mays L.) silage. That system was successfully used to study the influence of two different bacterial inoculants compared to a noninoculated control. There are inherent problems in all small scale silo systems, and caution should always be used when extrapolating mini-silo data to field scale ensiling (Cherney et al., 2004). Laboratory silos, however, are a reliable experimentation unit, as long as oxygen can be excluded to allow for consistent ensiling (Cherney et al., 2004).

Cherney et al. (2004) reported that vacuum-sealed polyethylene bags effectively ensiled corn silage samples in the laboratory. Perennial grasses, with their inherently lower sugar levels, however, are generally more difficult to ensile (Pitt, 1990). Objectives were to evaluate the influence of forage species, harvest date, and chopping (whole vs. chopped) on pH and volatile fatty acid profile of grasses ensiled in vacuum-sealed polyethylene bags and to assess the suitability of this method as a technique for ensiling perennial cool-season grasses on a laboratory scale.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Samples were obtained from an established site on a Niagara silt loam (fine-silty, mixed, active, mesic Aeric Epiqualfs) soil with 0 to 2% slope at Ithaca, NY. Four field replicates of each species were established. Nitrogen was supplied at 277 kg ha–1 after initial spring green up on 9 April, 2004. The site was fertilized with P and K according to soil test recommendations. Four replicates of three grass species, orchardgrass, reed canarygrass, and tall fescue were harvested at two dates. Harvest dates were 14 May 2004 and 25 May 2004 for orchardgrass, 17 May 2004 and 1 June 2004 for reed canarygrass, and 19 May 2004 and 28 May 2004 for tall fescue, for early and late harvests, respectively. Both harvests were of first-cutting material. These grasses do not develop another seedhead once the growth point is removed.

Samples (about 10 kg) of each species x harvest date x chop x field replicate combination were collected with a hand-clipper and a 10-cm high metal frame in the early afternoon. Samples were all in the early-boot stage of development at the early harvest date and were early-inflorescence at the later harvest date. Samples were mixed and one half of each sample was chopped (about 2 cm in length) with a chipper-shredder (Mighty Mac LSC506, MacKissic, Inc., Parker Ford, PA). A small amount of forage material was passed through the chipper-shredder each time a new sample was chopped to remove any residual plant material that may still have been in the shredder. Chopped samples were bagged separately in a tightly closed plastic transport bag to be used for ensiling in vacuum sealer bags. Bags were transported to the lab and stored in a cooler until processed. These samples were used for all studies. Samples were randomly processed within field replicate. All samples were processed within 8 h of chopping. Individual species x field replicate x harvest date x chop for all studies were processed within 1 h of removal from the cooler.

An initial study indicated that wilting to a uniform DM across species, harvest dates, and field replicates was very difficult. To reduce this variation, samples were not wilted before ensiling. It is recognized that high-moisture silages are at increased risk of undersirable fermentation, including clostridial fermentation (Collins and Owens, 2003). We also was felt that high-moisture samples would stress the ensiling system more than wilted samples. Good fermentation of these samples would be suggestive of the ability of the system to lower pH rapidly enough to prevent improper fermentation.

Chopped material from each species x cutting x field replicate was thoroughly mixed, resulting in a homogeneous mixture. All samples were treated with a lactic acid bacterial inoculant (Pioneer inoculant #1132; Pioneer Hi-Bred International, Inc., Johnston, IA) applied at 0.9 g Mg–1 (the recommended rate), unless otherwise noted. Samples were vacuum-sealed into polyethylene bags (Nylon poly EVOH high barrier 3 mil 22 x 33 cm vacuum pouches), as previously described (Cherney et al., 2004) with a single chamber vacuum packaging machine [Model KVP-420T with 68.261 kPa (512 mm Hg) vacuum level; Process Plus Food Processing Equipment Inc., Parkers Prairie, MN] and stored in the laboratory (21°C) in black plastic bags. The ensiling process was very quick, once packed in the polyethylene bags. Samples were vacuum packed within 1 min, and two bags could be vacuum packed simultaneously.

In the primary study (Study 1), duplicate samples (500 g) from each species x harvest date x chop x field replicate combination were ensiled for 0, 2, 4, 8, 16, 24, and 30 d to evaluate the pH, lactic acid, and volatile fatty acid (VFA) profiles of the mini-silos; only two field replicates were analyzed for VFA and lactic acid. Samples were frozen after ensiling until processed. Samples were removed from the freezer and allowed to thaw for 2 h. Vacuum-sealed bags were opened and an aliquot of 50-g wet sample was homogenized with 500 mL of deionized water in a household blender (2 min) and filtered through two layers of cheesecloth, and then pH was determined (Cherney et al., 2004). For lactic acid and VFA analysis 8.35 mL of 1 M m-phosphoric acid was added to 25 mL of the filtered extract, which was frozen before analysis.

Fermentation analysis of grass silage samples was performed by Dairy One (DHI Forage Testing Lab, Ithaca, NY). Samples were filtered again through a disposable syringe filter [Millipore Millex 5.0 µm PVDF (Durapore) membrane, Millipore Corp., Billerica, MA] and analyzed for acetic, propionic, butyric, and iso-butyric acids by gas chromatography (Anonymous, 1990). Lactic acid was determined with an YSI 2700 SELECT Biochemistry Analyzer equipped with an L-lactate membrane. Total lactic acid is determined by multiplying L-lactate by 2.0. It is recognized that multiplying by L-lactate by 2.0 may not always be an accurate assumption depending on which strains of lactic acid bacteria dominated fermentation. If different inoculants are a treatment of concern, care in the method of lactic acid determination is cautioned.

Grass samples were oven dried at 60°C for dry matter (DM) determination and ground to pass a 1-mm screen. Samples (Day 0 and 24) were analyzed by near infrared reflectance spectroscopy for neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin, nonstructural carbohydrates (NSC), crude protein (CP), and in vitro true digestibility (IVTD) by Dairy One (AOAC-989.03., 1996). The NDF digestibility was calculated as the amount of fiber digested after 48-h digestion, expressed as a percentage of the original NDF.

In a second study (Study 2), the effect of bacterial inoculation, propionic acid addition (1 g propionic acid/kg wet forage), or no additive (control) on final pH was determined. Duplicate samples (500 g) were prepared as above for each species x chop x field replicate combination, except that one third received propionic acid (Storage-mate II, a buffered propionic acid-based product, Cargill, Inc., Wayzata, MN), and one third received no inoculant or acid. Only the first harvest date was used. Fermentation was stopped at 30 d by freezing samples until pH could be determined.

In another study (Study 3), rate of inoculation on final pH was evaluated. Duplicate samples (500 g) were prepared as in Study 1, except that samples were inoculated with the recommended rate or three times this rate. Only TF was used. Samples from these studies were frozen at 30 d until pH was determined.

Effect of sample size (250 or 500 g) on final pH was evaluated (Study 4). In this study, only TF was used. Samples from these studies were frozen at 30 d until pH was determined.

A split-plot in a randomized complete block design was used for the studies, except for the sample size study, with four field replicates. Grass species were the main plots and harvest dates and chop were subplots. A split-plot analysis of variance with repeated measures was used to test for statistical significance of treatment effects and interactions using PROC MIXED (Littell et al., 1996) in SAS, version 7.0 software (SAS Inst., 1998). The model assumed that species, cutting date, chop, and length of ensiling were fixed variables, while replication was considered a random variable. Separation of means was accomplished using the Tukey-Kramer procedure for multiple comparisons (P ≤ 0.05). Study 2 was analyzed as described for Study 1, with the exception silage additive was a fixed variable rather than length of ensiling, and harvest date was removed as a variable. Study 3 was analyzed a randomized block design, with harvest date as the main block. Study 4 was analyzed as a randomized block design, with harvest date being the main plot. Statistical analysis was the same as for the other studies, with the exception that species was removed from the model and sample size was added as a fixed variable. Rates in pH decline were calculated as the change in pH from Day 0 to 2, divided by the number of days. Significance is declared at P ≤ 0.05 unless otherwise stated.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Interactions among main effects for nutritive value generally were not significant (Table 1). Therefore main effects generally will be discussed, with interactions discussed where appropriate. There was a species x harvest date interaction for DM, CP, and NDFD. Crude protein and NDFD decreased from first harvest date to second harvest date for all species, but the magnitude of the change varied among species and was greatest for RC. Dry matter increased slightly at the second harvest date for OG and RC but was unchanged for TF. Nutritive value, with the exception of IVTD and NDFD did not differ (P > 0.05) among species. Orchardgrass had highest IVTD and NDFD. Tall fescue was lower in IVTD and NDFD than orchardgrass and lower in NDFD than reed canarygrass. As expected, the later harvest date resulted in lower CP, IVTD, NDFD, and sugar, and higher NDF, ADF, and lignin.


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  		Table 1. Nutritive value of chopped silage as influenced by species, harvest date, and length of time ensiled (g kg–1 DM).

 
Neutral detergent fiber decreased as ensiling proceeded from 0 to 24 d (Table 1). There is a slow acid hydrolysis of structural carbohydrates during the stable phase of fermentation (Pahlow et al., 2003), which could account for the slightly lower NDF at Day 24. Loss of NDF, with no loss in lignin would result in a lower NDFD, which was observed in this study. Increases in ADF and CP, which are small, are most likely due to DM loss from carbon dioxide produced during fermentation. It was not likely that DM loss was caused by microbial respiration following diffusion of oxygen through the silage bags, because the bags were Nylon poly EVOH high barrier 3-mil 22- x 33-cm vacuum pouches. There was no significant (P > 0.05) loss of DM in this study. As expected, sugar decreased from 94 to 40 g kg–1 DM as fermentation proceeded from 0 to 24 d. Nutritive value changes observed between 0 and 24 d were within expected ranges, suggesting normal fermentation. We did not observe mold growth in any of ensiled samples, which agrees with a previous study with corn silage (Cherney et al., 2004) suggesting that the vacuum pouches, once sealed, are very resistant to oxygen movement into the bags. As with the corn silage samples (Cherney et al., 2004), we observed essentially no heating of the mini-silos. The vacuum removal of air from the bags minimizes any aerobic reactions which would generate heat.

Rapidly lowering pH to prevent undesirable bacteria and yeasts is one of the most important management aspects of the ensiling process (Jones et al., 2004). There was a species x chop interaction (P = 0.01), with the chopped RC being much lower in pH than whole material (Table 2; Fig. 1 ). Chopped OG and TF had pH's which were only slightly lower than whole OG and TF, respectively (Fig. 1). Orchardgrass was lower in pH than RC and TF. Chopping tends to increase microbial numbers (Bolsen, 1995), and the finer chop could liberate more nutrients, resulting in a lower pH. Ensiled grasses dropped rapidly in pH, with most of the decline occurring in the first 2 d. The pH of ensiled grasses was stable after 4 d of fermentation (Table 2). The pH of OG declined faster during the first 2 d (P ≤ 0.03; 0.83 ± .039 d–1) than RC (0.51 ± 0.039 d–1). Tall fescue was intermediate (0.68 ± 0.039 d–1) between OG and RC, and did not differ from either (P > 0.05). Orchardgrass had higher NDFD (Table 1) than the other grasses. This may have contributed to a faster release of nonstructural carbohydrates to the bacteria, which would then contribute to a higher rate of pH decline. Chopped silages had a faster (P < 0.05) rate of decline (0.78 ± 0.032 d–1) than whole silages (0.57 ± .032 d–1) during the first 2 d, as might be expected because of smaller particle size. Harvest date did not affect rate of pH decline during the first 2 d (P > 0.05).


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  		Table 2. Fermentation acid profile (g kg–1) of silage as influenced by species, chop, and harvest date (length of time ensiled).

 

Figure 1
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  		Fig. 1. Influence of chop and species on pH. Vertical bars indicate standard error of the mean.

 
Chopped silages had lower pH during the entire ensiling period than whole silages (Table 2). Laboratory ensiling experiments have consistently shown that shorter chop length or greater degree of shredding results in a more rapid drop in pH and a lower final pH (Muck et al., 1989; Seale et al., 1982; Marsh, 1978). Silages harvested at the more mature harvest date had lower pH than those harvested earlier (Table 2). Buffering capacity was most likely lower in the more mature grasses given their lower quality (Table 1). Muck et al. (1991) reported decreased buffering capacity with increasing maturity of permanent grassland swards. Higher buffering capacity results in higher pH (Wilkinson et al., 2003). Most of the pH values were below 4.7. Kung and Shaver (2001) indicated that grass silages typically range in pH from 4.3 to 4.7. On the basis of this criterion, the polyethylene vacuum bag technique resulted in well ensiled forages.

The pH of silage alone does not indicate the type of fermentation that occurred (Jones et al., 2004). In addition, relative concentrations of acids are more likely to influence silage intake, an important consideration in ruminant nutrition, than pH alone (Jones et al., 2004). Jones et al. (2004) indicated that lactic acid in well-fermented silages should account for 30 to 80 g kg–1 of silage DM and 600 g kg–1 of total acids. Lactic acid accounted for the majority of the acids in the ensiled forages in this study (Table 2) and was within the range suggested by Jones et al. (2004) for well-fermented silage. There were species x chop (P = 0.06) and species x harvest date (P = 0.06) interactions for lactic acid (Fig. 2a and 2b ). Lactic acid tended to be lower in whole silages than chopped silages (Fig. 2a) and was significantly lower for whole RC than chopped RC. Reed canarygrass has a wide leaf compared to OG and TF. This may have exaggerated the difference between chopped and whole material in RC. Reed canarygrass tended to be lower in lactic acid than OG and TF, with the difference between species being larger in whole than chopped material. Reed canarygrass was lower in lactic acid in the later harvest date, but lactic acid concentration did not differ among harvest dates for OG and TF (Fig. 2b).


Figure 2
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  		Fig. 2. Influence of chop and species (a) and harvest date and species (b) on lactic acid, g kg–1 of DM. Vertical bars indicate standard error of the mean. Harvest dates were 14 May 2004 and 25 May 2004 for orchardgrass, 17 May 2004 and 1 June 2004 for reed canarygrass, and 19 May 2004 and 28 May 2004 for tall fescue.

 
Jones et al. (2004) indicated that acetic acid should be between 10 and 40 g kg–1 of DM. All silages, with the exception of those sampled at Day 2, were within this range, suggestive of adequate fermentation (Table 2). There were species x chop, species x harvest date, harvest date x day and species x harvest date x chop interactions (Table 2). Acetic acid tended to be higher in chopped silages than whole silages in all species, although magnitude of the difference varied (Fig. 3 ). There were no consistent differences between harvest dates across species (Fig. 3). Acetic acid from silages from the earlier harvest date, with the exception of the 16 d silages, was higher than the later harvest date (Fig. 4 ).


Figure 3
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  		Fig. 3. Influence of chop, harvest date and species on acetic acid, g kg–1 of DM. Vertical bars indicate standard error of the mean. Harvest dates were 14 May 2004 and 25 May 2004 for orchardgrass, 17 May 2004 and 1 June 2004 for reed canarygrass, and 19 May 2004 and 28 May 2004 for tall fescue.

 

Figure 4
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  		Fig. 4. Influence of harvest date and day on acetic acid, g kg–1 of DM. Vertical bars indicate standard error of the mean. Harvest dates were 14 May 2004 and 25 May 2004 for orchardgrass, 17 May 2004 and 1 June 2004 for reed canarygrass, and 19 May 2004 and 28 May 2004 for tall fescue.

 
There was little or no propionic or butyric acids in the silages, indicating that the silages did not undergo clostridial fermentation (Weiss et al., 2003; Jones et al., 2004). Propionic acid did not vary among species or between chops and harvest dates (P > 0.05; Table 2). Propionic acid was higher at Day 30 than at Days 2 through 8 (Table 2). The value at 30 d (0.094 ± 0.034, percentage of DM) would not be considered a problem. There were species x harvest date and species x day interactions in butyric acid. Differences were small, however, and unlikely to suggest problems with clostridial fermentations.

Total acids, with the exception of RC, were unaffected by harvest date. Total acids were lower in whole silages than chopped silages (Fig. 5a ), but the magnitude of difference between whole and chopped was greater for RC than OG and TF (Fig. 5b). Muck et al. (2003) suggested that shredding resulting in greater plant cell rupture will lead to a greater proportion of sugars available, which permits a more rapid and extensive bacterial fermentation. This should result in greater total acids for the chopped silages. Total acids in RC silages harvested at the later date were lower than those harvested at the earlier date (Fig. 5b). This was due to both lower lactic and acetic acid in late cut RC (Fig. 2b and 3).


Figure 5
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  		Fig. 5. Influence of chop and species (a) and harvest date and species (b) on total acids, g kg–1 of DM. Vertical bars indicate standard error of the mean. Harvest dates were 14 May 2004 and 25 May 2004 for orchardgrass, 17 May 2004 and 1 June 2004 for reed canarygrass, and 19 May 2004 and 28 May 2004 for tall fescue.

 
Lactic:acetic acid ratios were above 2:1 (Table 2), which is considered minimal for quality silage (Jones et al., 2004). Orchardgrass had higher lactic:acetic acid ratios than RC or TF (Table 2). This was due to lactic acid being higher in OG than RC silages and acetic acid being lower in OG than RC and TF. Chopped silages had lowered lactic:acetic acid ratios than whole silages. Lactic:acetic acid ratios were relatively constant throughout the ensiling process.

In the study looking at source of silage additive (Study 2; microbial inoculant, acid or control), we observed no differences in pH due to treatment (Table 3). There were no interactions between treatment and species or chop. Chop had no effect on TF (4.39 ± 0.022). In Study 3, there was no difference in pH because of inoculation with the recommended silage rate or three times the recommended rate (Table 3). No interactions were significant. Silage additives are used to improve fermentation and prevent butyric acid production (Bolsen et al., 1995). Kung et al. (2003) indicated that additives do not always improve fermentation. They noted that this is particularly true when the control silages undergo excellent fermentations. In Study 2, control silages did not differ in pH from treated silages, and pH of all silages were below 4.7, suggesting that fermentations were good and that the addition of inoculant at three times the recommended rate would not be expected to have an effect on fermentations.


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  		Table 3. pH of silage as influenced by silage additive (Study 2), silage inoculation rate (Study 3) and sample size (Study 4).

 
In the sample size study (Study 4), there was no difference in pH between silages of 250-g sample size versus those of 500-g sample size (Table 3). There was no difference in pH between harvest dates and whole or chopped silages. Interactions were not significant. Cherney et al. (2004) vacuum ensiled corn silage in plastic bags with sample sizes of 50, 100, 200, 400, and 600 g. The pH did not change for sample size above 100 g in that study. They concluded that it is possible to use vacuum-sealed plastic bags to ensile corn, with samples as small as 200 g. On the basis of results with grass, vacuum-sealed plastic bags can be used with samples as small as 250 g. It should be remembered, however, that sampling is often the largest source of error in the analysis process (Youden and Steiner, 1975) and that this error is likely to increase with smaller sample size (Cherney and Cherney, 2003).

In all studies, we observed essentially no heating of the mini-silos, in agreement with results previously reported (Cherney et al., 2004). If not properly sealed, bags will expand and fog up within 5 to 10 min of packaging. This is easily observable and silages were rebagged immediately. In all four studies, only three samples (of 976) were lost because of improper fermentation. This was readily observed, visually, olfactorily, and by high pH (>5.8). Poor fermentation most likely resulted from improper sealing as duplicate sample bags properly fermented in all cases. Because the three samples were obviously poorly fermented and were more than two standard deviations from the mean of duplicate and field replicate samples, these samples were discarded and not included in the averages. Silage effluent production was not a problem in any of the studies.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Despite inherent problems in all small scale silo systems (Cherney et al., 2004), laboratory silos are an accurate and reliable experimentation unit. They are essential when evaluating numerous experimental variables and interactions involving different forages. The pH and VFA fermentation characteristics suggested that all samples in this study were well ensiled within 4 d of ensiling. Sample size (250 or 500 g) did not influence final pH. Addition of bacterial inoculant or propionic acid in relation to an untreated control did not affect final pH in this study, suggesting that conditions for proper fermentation existed. We conclude that it is possible to use vacuum-sealed plastic bags to ensile temperate grasses to assess treatment differences.


   ACKNOWLEDGMENTS
 
This research was supported in part by the Cornell University Agricultural Experiment Station federal formula funds, Project No. NYC-1277455 received from Cooperative State Research, Education, and Extension Service, U.S. Department of Agriculture. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the U.S. Department of Agriculture. The assistance of Samuel Beer and Molly Leibowitz is especially appreciated.

Received for publication April 25, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 





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