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Delaware Agricultural Experiment Station, Department of Animal and Food Science College of Agriculture and Natural Resources, University of Delaware, Newark 19717-1303
Corresponding author:
L. Kung, Jr.; e-mail:
lkung{at}udel.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Abbreviation key: BPA = barley silage treated with a buffered propionic-acid-based product (0.2% of fresh weight), IN = barley silage treated with an inoculant containing Lactobacillus plantarum (1 x 104 cfu/g), Pediococcus pentosaceus (1 x 104 cfu/g), and Propionibacterium freudenreichii (1 x 104 cfu/g), LB1 = barley silage treated with Lactobacillus buchneri 40788 (final application rate of 1 x 105 cfu/g of fresh forage), LB2 = barley silage treated with Lactobacillus buchneri 40788 (5 x 105 cfu/g of fresh forage), LB3 = barley silage treated with Lactobacillus buchneri 40788 (1 x 104 cfu/g of fresh forage), WSC = water-soluble carbohydrates
Key Words: Lactobacillus buchneri • silage • aerobic stability
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Recently, the aerobic stability of a variety of silage crops has been markedly improved by inoculation with a heterolactic acid bacterium, Lactobacillus buchneri. For example, improvements in aerobic stability brought about by this organism have been reported in corn silage (Muck, 1996; Driehuis et al., 1999a; Ranjit and Kung, 2000) and wheat and sorghum silages (Weinberg et al., 1999). Lactobacillus buchneri has been shown to inhibit the proliferation of yeasts in silage via the production of acetic acid. Opponents of using heterolactic acid bacteria to improve silage quality cite the fact that in some past studies, high concentrations of acetic acid in silages have had depressing effects on DMI (Jones et al., 1980; Buchanan-Smith, 1990). However, this tenet is controversial, as few strong relations between silage acids and intake have been observed in recent summaries (Steen et al., 1998; Wright et al., 2000). When the current study was undertaken, only one other study had been conducted where silage treated with L. buchneri was fed to lactating cows (Driehuis et al., 1999b). In that study, DMI was not affected when cows were fed treated silage.
The first objective of this study was to establish a minimum rate of application of L. buchneri 40788 that could improve the aerobic stability of barley silage ensiled in laboratory silos. Secondly, we compared the ability of L. buchneri 40788 to improve the aerobic stability of barley silage in laboratory silos with several commercial silage additives. We also determined whether L. buchneri 40788 could improve the aerobic stability of barley silage ensiled in a farm silo and whether feeding silage treated with L. buchneri 40788 would have negative effects on intake or production of cows because of its higher concentration of acetic acid.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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-amylase (21,000 IU/tonne), xylanase (22,800 IU/tonne) and galactomannase (3840 IU/tonne) (Biotal, Eden Prairie, MN [LB1]); 3) L. buchneri (5 x 105 cfu/g) and enzymes as in treatment 2 (LB2), 4) L. buchneri (1 x 106 cfu/g) and enzymes as in treatment 2 (LB3), 5) an inoculant (IN) containing L. plantarum (1 x 104 cfu/g), Pediococcus pentosaceus (9 x 104 cfu/g), Propionibacterium freudenreichii (1 x 104 cfu/g), ß-glucanase (14,000 IU/tonne of fresh forage), -amylase (7000 IU/tonne of forage), xylanase (7680 IU/tonne of forage), and galactomannase (1280 IU/tonne of forage); and 6) a buffered propionic-acid-based product ([BPA] 56% active ingredients containing buffered propionic acid, acetic acid, benzoic acid, and citric acid; Cargill, Inc., Minneapolis, MN) at the rate of 0.2% of the fresh forage weight. Treatments 1 through 5 were diluted in deionized water and applied at the rate of 0.5 ml/kg of forage with a sprayer. The buffered propionic acid-based product was a granular formulation and was sprinkled onto the forage by hand followed by the same amount of water as applied in the other treatments. All additives were applied to the forages in a uniform manner with constant mixing.
Forage from each treatment was packed into macro silos (
27 cm, diameter x 36 cm, height) in triplicate to achieve a packing density of 200 kg of DM/m3 and sealed immediately. Weights of these silos were recorded, and silos were then stored at ambient temperature (20 to 27°C) in an enclosed barn. Forage samples were obtained after application of the appropriate additive but before ensiling from each treatment. Samples were stored on ice (about 45 min) until returned to the laboratory for processing. Dry matter of the samples was determined in a forced-draft oven set at 60°C for 48 h. After drying, forage samples were ground through a Wiley Mill (1-mm screen; Arthur H. Thomas, Philadelphia, PA) and analyzed for laboratory DM (100°C oven for 24 h), NDF using sulfite and amlyase (Van Soest et al., 1991), and ADF (Robertson and Van Soest, 1981). Crude protein was calculated by multiplying total N by 6.25 after total combustion (LECO CNS 528 Analyzer; LECO Corporation, St. Joseph, MI). Starch was analyzed using the method described by Poore et al. (1993). Twenty-five grams of fresh forage from each replicate was also homogenized for 1 min with 225 ml of sterile quarter-strength Ringer’s solution (Oxoid BR52; Unipath, Basingstoke, UK). Yeasts and molds were enumerated by pour plating in malt extract agar (Oxoid CM59; Unipath, Basingstoke, UK) that had been acidified by the addition of 85% lactic acid at the rate of 0.5%, vol/vol. Plates were incubated aerobically at 32°C for 48 h. Colonies were counted from the plates of appropriate dilutions containing a minimum of 30 colonies. A portion of the water extract was filtered through Whatman 54 filter paper (Whatman, Clifton, NJ), acidified with 50% H2SO4, and frozen before further analysis. One milliliter of each filtered and acidified water extract was combined with 200 µl of 25% meta-phosphoric acid containing isocaproic acid as an internal standard. Samples were centrifuged for 15 min at 10,000 x g and analyzed for acetic, propionic, and butyric acids by gas chromatography using a Hewlett Packard 5890 GC (Hewlett Packard, Avondale, PA) with a 530-µm Carbowax 20 M column (Supelco, Bellefonte, PA). The chromatograph oven was programmed as follows: 70°C for 1 min, 5°C increase/min to 100°C, 45°C increase/min to 170°C, and a final holding time of 5 min. Ammonia-N was analyzed by the phenol-hypochlorite procedure described by Weatherburn (1967). Water-soluble carbohydrates (WSC) were determined as described by Nelson (1944).
After 120 d of ensiling, the final weights of the silos were recorded and each silo was opened and the silage mixed thoroughly. The weights of empty silos were recorded for the calculation of DM recovery. Silage samples were processed and analyzed as described for fresh forages. In addition, the filtered and acidified water extracts of silages were analyzed for lactic acid by an enzymatic procedure (kit 826-UV; Sigma, St. Louis, MO). For the analysis of D-lactic acid, L-lactic dehydrogenase was replaced with a similar amount of D-lactic dehydrogenase (Sigma L-9636). L-Lactic acid (Sigma L-2250) and D-lactic acid (Sigma L-1000) were used for standards for their respective assays. The sum of the L- and D-lactic acids was reported as the total lactate concentration. Ethanol concentration of the water extracts of silages was determined as per the alcohol procedure for markedly turbid samples with multi-assay vials (Sigma Procedure No. 332-UV). Aerobic stability was determined on all silages after silo opening. Samples (2 ± 0.005 kg) of each replicate from each treatment was placed loosely into clean, macro silos. Silages were exposed to air at room temperature (22°C), and thermocouple probes were placed in the geometric center of the silage masses. A double layer of cheesecloth was placed over each container to prevent drying and contamination, but allowing for the penetration of air. After 7 d of exposure to air, silages (30%, DM basis) were combined with alfalfa silage (30%) and a dairy concentrate (40%) from the University of Delaware Dairy to form a TMR. The TMR were returned to their original silage bucket and allowed to incubate aerobically for an additional 6 d. Ambient temperature, as well as the temperature from each silage and TMR, was recorded every min and averaged every 2 h by a data logger (model number CR10X; Campbell Scientific, Inc., Logan, UT). Aerobic stability was defined as the number of hours the silage or TMR mass remained stable before rising more than 2°C above the ambient temperature (Moran et al., 1996).
Lactation Study
Whole-plant barley from one field was harvested in the early heading stage of maturity and wilted to approximately 40% DM before chopping and packing (Kelly Ryan Equipment Co., Blair, NE) into a bag silo (Klerk’s Plastic Products Manufacturing, Inc., Richburg, SC). The bag silo was oriented in a north-south direction to minimize effects of afternoon sun on the silo faces during feed out. One half (about 30 tonne) of the barley forage was untreated and packed in a bag silo, and the remaining half was treated with L. buchneri 40788 (a final application rate of 4 x 105 cfu/g of fresh forage), ß-glucanase (42,000 IU/tonne of fresh forage), -amylase (21,000 IU/tonne), xylanase (22,800 IU/tonne) and galactomannase (3840 IU/tonne) applied via a mounted sprayer (Nevtro Sales, Ltd., London, Ontario, Canada) in water (2 L/tonne). Untreated and treated silages were sampled during wk 3 to 16 of ensiling at random locations in the bag. Silage was analyzed for chemical end products and yeasts and molds as described for laboratory silage.
After 10 mo of storage, both ends of the bag silo were opened simultaneously for feeding in a lactation study (ambient temperatures of 19 to 27°C). Fourteen multiparous and six primiparous Holstein cows, producing an average of 25 kg of milk/d and 184 ± 28 DIM, were offered a complete diet as a TMR once daily for a 10-d pretreatment period. The first 3 d were used to accustom cows to feeding (once daily at 1400 h) via Calan gates (American Calan, Northwood, NH) and the data from the next 7 d were used for adjustment to the diet. The TMR during the pretreatment period was 18% (DM basis) untreated barley silage, 17% barley silage that had been inoculated with L. buchneri 40788, 15% corn silage, and 50% of a pelleted concentrate. Diets met all requirements for lactating cows (NRC, 1989). After the pretreatment period, cows were blocked on DIM, parity, and milk yield and randomly allocated to one of two treatments. The TMR during the treatment period was 35% (DM basis) untreated or treated barley silage, 15% corn silage, and 50% of a pelleted concentrate. The study during the treatment period was a crossover design that consisted of two 4-wk periods. The last 2 wk in each period were used for collection of data. Cows were fed for ad libitum intake, and feed refusals were measured daily throughout the study. Fresh water was available at all times, and the care of animals was via accepted protocols (Federation of Animal Science Societies, 1999). Daily low and high temperatures were recorded.
Throughout the study, a computer recorded milk production twice daily at 0700 and 1900 h. Once weekly, milk was sampled proportionately from consecutive p.m. and a.m. milkings and analyzed for fat, protein, and somatic cells (Milk-O-Scan; Foss Technology, Hillerød, Denmark). Body weights were recorded at the start and end of the study. Samples of barley silages, alfalfa haylage, concentrate, and TMR were collected three times weekly and composited prior to analyses as previously described.
Statistical Analyses
All microbial data were transformed to log10. Chemical data are presented on a DM basis. Data from the laboratory silo study were analyzed as a completely randomized design and subjected to ANOVA by the general linear models procedure of SAS (1998). Data from the lactation study were analyzed using the general linear models procedure of SAS (1998) for a crossover design. In both studies, differences among means were tested using Tukey’s Test (Snedecor and Cochran, 1980), and an level of P < 0.05 was deemed significant unless noted otherwise.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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). Only BPA had detectable concentrations of propionic acid (0.28%). Yeasts ranged from 4.46 to 5.83 log10 cfu/g, and molds ranged from 3.85 to 4.50 log10 cfu/g.
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). The pH of treated silages were not different when compared to the pH of untreated silage. Concentrations of lactic acid were lower in silages treated with L. buchneri 40788 when compared to other silages. In contrast, the concentrations of acetic acid were greater in all silages treated with L. buchneri 40788. In addition, the concentrations of propionic acid were greater in silages treated with LB2 (1.29%) and LB3 (1.44%) than in untreated silage (0.28%). Treatment with IN and BPA had no effects on the concentrations of these acids compared with untreated silage. Butyric acid was 0.84% in untreated silage and was lower in silage treated with LB2, LB3, IN, and BPA, but treatment with L. buchneri 40788 caused an increase in the accumulation of ethanol in silages (0.43% in untreated silage and an average of 0.91% in silages treated with LB 40788). Treatments had no effects on the concentration of residual NH3-N, with the exception of a higher level of NH3-N for silage treated with LB1. Treatments also had no effects on the recovery of DM when compared to untreated silage. The numbers of yeasts and molds were very low (less than 2.5 log10 cfu/g) and not different among treatments.
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]). The alfalfa silage had been ensiled without additives and contained 5.17 and 3.00 log10 cfu of yeasts and molds, respectively. Alfalfa silage alone spoiled after 124 h of exposure to air. The aerobic stabilities of the experimental TMR are shown in Figure 1. All TMR containing barley silages inoculated with L. buchneri 40788 were more stable than the TMR containing untreated silage (95 h). Treatment with IN had no effect on aerobic stability, but silage treated with BPA was as stable (105 h) as silages treated with LB1 and LB2 (average 106 h).
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). Treated silage also had a tendency (P < 0.08) for a lower concentration of lactic acid but higher concentration of acetic acid than did untreated silage. Treated silage also had a higher concentration of ethanol than did untreated silage. Untreated silage contained 4.89 log10 cfu of yeasts per gram of silage, but treated silage had only 2.63 log10 cfu of yeasts per gram of silage. The aerobic stability of untreated silage was 66 h, whereas treated silage was stable for 190 h. The composition of corn silage fed in the TMR is shown in Table 5. Specifically, the number of yeasts averaged 5.22 log10 cfu/g, and the aerobic stability of corn silage alone was 60 h.
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. The aerobic stability of the TMR was greater when it contained barley treated with L. buchneri 40788. Dry matter intake, milk production, and milk composition were not different between treatments (Table 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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Treatment with L. buchneri 40788 has also increased the concentrations of propionic acid in some (Driehuis et al., 1999a; Kung and Ranjit, 2001) but not all silages (Ranjit and Kung, 2000), and the reason for this finding is unclear at this time. In the current study, barley silages that had been treated with L. buchneri 40788 and stored in laboratory silos had greater concentrations of propionic acid than did untreated silage. However, propionic acid was not detected in any of our silages from the farm silo. As in past studies, treatment with L. buchneri 40788 and enzymes had no effects on the ADF, NDF, CP, or starch content of silage (Ranjit and Kung, 2000; Kung and Ranjit, 2001). An exception in this study was a lower concentration of starch and CP in silage treated with LB2, and we have no explanation for this finding.
Because the numbers of yeasts and molds were low in all silages stored in the laboratory silos, these silages remained stable even after 7 d of exposure to air. However, when these silages were mixed with untreated alfalfa silage and a dairy concentrate to make an experimental TMR, the aerobic stabilities of the TMR were better when they contained silages that had been treated with L. buchneri 40788. In addition, silage treated with the buffered propionic acid preservative improved the aerobic stability of the TMR to an extent that was similar to that found from TMR made with barley silage treated with 1 and 5 x 105 cfu/g of L. buchneri 40788. In previous studies, treatment with low levels of buffered propionic acid additives have consistently improved the aerobic stability of silages ensiled in laboratory silos (Kung et al., 1998; Kung et al., 2000). In our farm silages, treatment with L. buchneri 40788 decreased the numbers of yeasts and molds and increased aerobic stability when compared to untreated silage. When compared to untreated silage, inoculation with the commercial inoculant had few effects on the fermentation of barley silage in laboratory silos, with the exception of a lower concentration of butyric acid. The commercial inoculant also had no effect on the aerobic stability of the TMR, and although it contained propionibacteria, it did not contain higher concentrations of propionic acid. Higginbotham et al. (1998) also added propionibacteria to forage but found no increases in the concentration of propionic acid in the resulting silage. As found in the laboratory study, mixing silage from a farm-scale silo that had been treated with L. buchneri 40788 to make a TMR improved its aerobic stability. Imparting improved aerobic stability to a TMR may be extremely useful because silages are commonly incorporated into TMR for feeding.
Critics of using heterolactic acid bacteria as silage inoculants suggest that poor palatability from high concentrations of acetic acid may be one reason to dismiss these organisms from use. Thus, our lactation study was designed to evaluate potential negative effects on animal performance from feeding silages treated with L. buchneri 40788 during moderate weather (22 to 28°C). Our study was not designed to show potential positive effects of animals fed silages treated with L. buchneri 40788 when compared to untreated silage under conditions of severely hot weather and/or poor management. Our results showed that feeding silage treated with L. buchneri 40788, which contained a high concentration of acetic acid, had no effect on the DMI or production of lactating cows. Driehuis et al. (1999b) also reported that cows fed silage treated with L. buchneri 40788 consumed similar amounts of DM, as did cows fed a diet with untreated silage. In past studies, high concentrations of acetic acid in silages have sometimes been associated with lower DMI (Wilkins et al., 1971; Rook and Gill, 1990). However, after evaluating 136 grass silages, Steen et al. (1998) concluded that silage intake was not closely related to products of fermentation such as lactic acid or VFA. In that study, the average DM was 21.9% and ranged from 15.5 to 41.3%, and concentrations of acetic acid averaged 2.93% and ranged from 0.45 to 6.28%. Similar results have also been reported by Wright et al. (2000). In circumstances where DMI is depressed and associated with feeding silages with high concentrations of acetic acid (but not from treatment with L. buchneri 40788), other unidentified factors related to the undesirable fermentation may be contributing to the depression in intake.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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FOOTNOTES |
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Received for publication November 26, 2001. Accepted for publication January 23, 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
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