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Effect of Lactobacillus hilgardii, Lactobacillus buchneri, or their combination on the fermentation and nutritive value of sorghum silage and corn silage
* Present address: National Institute of Animal Science, RDA, Cheonan 31000, South Korea † Present address: Agricultural Research Station, Fort Valley State University, Fort Valley, Georgia 31030 ‡ Present address: Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Madison 53706
The objective of this study was to determine the effect of inoculation with Lactobacillus hilgardii with or without Lactobacillus buchneri on the fermentation, chemical composition, and aerobic stability of sorghum and corn silage after 2 ensiling durations. Sorghum forage was harvested at 27% dry matter (DM; experiment 1), and different corn hybrids were harvested at late (43.8% DM; experiment 2) or normal maturity (34% DM; experiment 3). All harvested forages were chopped and ensiled in quadruplicate in vacuum-sealed nylon-polyethylene bags (40 × 61 cm) for 30 and 90 d after treatment with (1) deionized water (uninoculated) or (2) L. buchneri (1.5 × 105 cfu/g of fresh weight; LB); (3) L. hilgardii (1.5 × 105 cfu/g of fresh weight; LH); or (4) L. buchneri and L. hilgardii (1.5 × 105 cfu/g of fresh weight of each inoculant). Data for each experiment were analyzed separately accounting for the 2 × 2 × 2 factorial treatment arrangement. Inoculating sorghum forage with LB or LH separately increased acetate and 1,2 propanediol concentration, tended to increase DM loss, reduced lactate concentration and the lactate-to-acetate ratio, and increased aerobic stability after 90 but not after 30 d of ensiling. Inoculating late-harvested corn silage with LB or LH separately increased and decreased DM loss, respectively, increased 1,2 propanediol concentration, reduced lactate-to-acetate ratio and yeast counts but did not affect aerobic stability. Inoculating normal-harvested corn silage with LH reduced DM loss and increased 1,2 propanediol concentration and yeast counts; LB reduced lactate concentration, lactate-to-acetate ratio, and total acids. Either inoculant alone increased aerobic stability after 30 or 90 d. The main benefit of combining LB with LH was prevention of increases in DM losses by LH or LB separately. No improvement in aerobic stability resulted from applying LH instead of LB separately or from combining them. Application of LB or LH separately improved aerobic stability of sorghum silage after 90 d and normal-harvested corn silage after 30 or 90 d but did not affect that of late-harvested corn silage.
Provision of high quality and safe silage is a pivotal determinant of the profitability of dairy production. Adding bacterial inoculants to freshly harvested forages can increase the likelihood of making good quality silage. Bacterial inoculants containing homofermentative or facultative heterofermentative bacteria accelerate the decrease in silage pH by fermenting sugars into lactate (
). However, such inoculants have not increased aerobic stability in many studies (Muck and Kung, 2007). Aerobic stability has been improved by inoculating forages with heterolactic Lactobacillus buchneri (LB;
Meta-analysis of effects of inoculation with Lactobacillus buchneri with or without other bacteria, on silage fermentation, aerobic stability, and performance of dairy cows.
The effect of Propionibacterium acidipropionici, with or without Lactobacillus plantarum, on the fermentation and aerobic stability of wheat, sorghum, and maize silages.
; Kung, 2010). Consequently, LB has been widely used to improve the preservation of silage. However, the effects of LB inoculation on aerobic stability take 45 to 60 d because of the slow conversion of lactate to acetate (
Effects of conservation period and Lactobacillus hilgardii inoculum on the fermentation profile and aerobic stability of whole corn and sorghum silages.
Effects of conservation period and Lactobacillus hilgardii inoculum on the fermentation profile and aerobic stability of whole corn and sorghum silages.
Effects of Lactobacillus hilgardii 4785 and Lactobacillus buchneri 40788 on the bacterial community, fermentation, and aerobic stability of high-moisture corn silage.
Effects of inoculation of corn silage with Lactobacillus hilgardii and Lactobacillus buchneri on silage quality, aerobic stability, nutrient digestibility, and growth performance of growing beef cattle.
The effect of Propionibacterium acidipropionici, with or without Lactobacillus plantarum, on the fermentation and aerobic stability of wheat, sorghum, and maize silages.
), and the long ensiling duration required for improvement of aerobic stability by LB, there is a need for more research on the potential of using LH with or without LB to hasten and improve aerobic stability of silage. This could reduce silage DM, energy, and nutrient losses on US dairy farms, which are worth over $2 billion annually (
Changes in forage quality during harvest and storage.
in: Fahey G.C. Collins M. Mertens D.R. Moser L.E. Forage quality, evaluation, and utilization. Natl. Conf. on Forage Quality, Evaluation, and Utilization, Lincoln, NE. American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America,
1994: 828-868
The objective of this study was to determine the effect of applying LH with or without LB on the fermentation, chemical composition, and aerobic stability of sorghum silage harvested at a normal maturity stage and corn silage harvested at normal and late maturity stages. We hypothesized that application of LH with or without LB would result in earlier and greater aerobic stability than in uninoculated or LB-treated sorghum and corn silages.
MATERIALS AND METHODS
Experiment 1: Effect of LH, LB, or the Combination on Fermentation Profile and Aerobic Stability of Sorghum Silage (27% DM)
A forage sorghum hybrid (SuperSugar DM; Gayland Ward Seed) was grown on 4 plots within different blocks on an irrigated field at the Plant Science Research and Education Center, University of Florida in Citra, Florida. Sorghum plants from each plot were harvested at 27% DM (soft dough stage) as recommended by
using an adapted one-row pull-type forage harvester (New Holland model 707; New Holland Agriculture), set to a chop length of 16 mm. Inoculants were dissolved in 480 mL of deionized water and sprayed in a fine mist on 16 kg of representative forage from each plot under constant mixing. Plot was considered the experimental unit and treatments applied in quadruplicate were (1) deionized water (uninoculated); (2) Lactobacillus buchneri NCIMB 40788 (1.5 × 105 cfu/g of fresh weight; LB); (3) Lactobacillus hilgardii CNCM I-4785 (1.5 × 105 cfu/g of fresh weight; LH); and (4) Lactobacillus buchneri and Lactobacillus hilgardii (1.5 × 105 cfu/g of fresh weight of each inoculant; LB+LH). To verify the viability of the inoculants and the inoculation rate, the lactic acid bacteria (LAB) were plated as described below and counted before inoculation of forages and then stored as recommended to retain viability and prevent multiplication. Forage from each experimental unit was ensiled (4.5 kg of forage) in each of 2 vacuum-heat sealed nylon-polyethylene standard barrier bags (0.09-mm thickness, 40 × 61 cm; Doug Care Equipment Inc.). One bag was ensiled for 30 d, and the other one was ensiled for 90 d at room temperature (23°C), such that 4 replicate bags per treatment per ensiling duration were available. Air was withdrawn from silos immediately before they were sealed with an external clamp vacuum pump (Bestvac; distributed by Doug Care Equipment Inc.). Extracts of silage samples from d 0, 30, and 90 were obtained by mixing 25 g of forage (0 d) or silage (30 and 90 d) with 225 mL of 0.1% peptone water in a stomacher (Lab-blender 400; Tekmor company) for 1 min. The solution was filtered through 2 layers of sterilized cheesecloth, and 1 aliquot was immediately used for measuring total LAB, yeast, and mold counts. Briefly, yeasts and molds were analyzed using the pour plate method with malt extract agar (Thermo Fisher Scientific-Oxoid CM0059B) and incubated at 32°C for 72 h (2 mL of 85% lactic acid per 400 mL of liquid agar medium to inhibit growth of bacteria); LAB were analyzed using the pour plate method with de Man Rogosa Sharpe agar (Thermo Fisher Scientific R01585) and incubated aerobically at 32°C for 48 h. Another silage extract aliquot was used for measuring pH (using a digital probe; Accumet AB15, Fisher Scientific). After pH measurement, 40 mL of silage extract aliquots were acidified with 1% of 50% sulfuric acid centrifuged at 7,000 × g for 15 min at 4°C. One part of this supernatant was retained for ammonia-N analysis (using a Technicon Auto Analyzer, Technicon) and another part was filtered using 0.22-µm filters and transferred to vials for organic acids analysis (using a HPLC system, Hitachi, L2400), a UV detector, and an Aminex HPX-87H column (Bio-Rad Laboratories) with 0.015 M sulfuric acid mobile phase and a flow rate of 0.7 mL/min at 47°C. Ethanol and 1,2 propanediol were similarly analyzed but with a refractive index detector. Samples from 0, 30, and 90 d were dried at 60°C for 48 h in a forced-air oven, ground to pass the 1-mm screen of a Wiley mill and analyzed for DM, ash (method 942.05;
and a YSI analyzer (YSI 2700 SELECT Biochemistry Analyzer).
Losses of DM were calculated using the dry weight of samples at ensiling and after silo opening. Aerobic stability was determined after 30 and 90 d of ensiling by placing temperature sensors (Onset Computer Corporation) at the center of 20-L plastic buckets containing 3 kg of silage and set to record temperatures every 30 min for 21 d; 2 additional sensors were placed in the room to record ambient temperature (23°C). Aerobic stability was determined as the time that elapsed before silage, and ambient temperature differed by at least 2°C.
For the estimation of apparent in vitro DM digestibility (IVDMD) and NDF degradability (IVNDFD), rumen fluid was collected from 3 lactating, ruminally-cannulated Holstein cows approximately 2 h after the morning feeding. Donor cows were fed a TMR containing (DM basis) corn silage (38.2%), alfalfa hay (4%), ground corn (27.3%), soybean meal 44% (14.5%), citrus pulp (9.2%), mineral mix and clay (5%), and rumen-inert fat (1.8%). Diets were offered twice every day (0600 and 1400 h). All animal handling and rumen fluid sampling procedures were performed according to a protocol approved by the University of Florida Animal Care Research Committee; protocol number 202009849. Rumen fluid inoculum was then added to buffered pre-warmed (39°C) medium (
) in a 1:2 ratio (rumen fluid:artificial saliva). The medium was pre-bubbled with CO2 before mixing with rumen inoculum. The medium was continuously infused with CO2 to maintain the anaerobic environment for the rumen fluid inoculum. Substrate (0.5 g) was weighed in triplicate into F-57 Ankom bags (Ankom Technology) and incubated with 52 mL of buffered ruminal fluid (
) in 125-mL glass serum bottles gassed with CO2 at 39°C for 24 h. After 24 h of incubation, bags with residue samples were washed and oven-dried at 60°C for 48 h and weighed. Dried residue samples were analyzed for NDF using an Ankom Fiber Analyzer (Ankom Technology). Residue weights and their NDF concentrations were used to calculate IVDMD and IVNDFD. The in vitro assay was performed in 3 runs.
Experiment 2: Effect of LH, LB, or the Combination on Fermentation Profile and Aerobic Stability of Late-Harvested Corn Silage (43.8% DM)
A conventional corn hybrid (Croplan Genetics 5900VT2P) was grown on 4 plots in each of 4 blocks on an irrigated field at the Plant Science Research and Education Center (Citra, FL). A hurricane prevented timely harvest of the corn plants from each plot at 35% DM. Consequently, corn was eventually harvested at 43.8% DM using an adapted one-row pull-type forage harvester (New Holland model 707), set to a chop length of 16 mm. Forage from the respective plots were treated as mentioned in experiment 1, ensiled for 30 and 90 d, and analyzed for fermentation profile, IVDMD and IVNDFD, and aerobic stability. Plots were considered the experimental unit. The DM at harvest of the forage in experiment 2 was typical of late-harvested corn forage destined for silage (
Experiment 3: Effect of LH, LB, or the Combination on the Fermentation Profile and Aerobic Stability of Corn Silage Harvested at Normal Maturity (34% DM)
Due to the late harvest of the corn forage ensiled in experiment 2, the experiment was repeated with corn forage harvested at a normal maturity stage for corn silage, but the forage was from a commercial farm because it was no longer possible to source sufficient forage at the right maturity from the replicated plots. A conventional corn hybrid (Pioneer 1794VYHR) grown on an irrigated field at the Alliance Dairies farm (Trenton, FL) was harvested at 34% DM using a Claas Jaguar 980 (Claas of America LLC) self-propelled forage harvester with a theoretical chop length of approximately 13 mm. The harvested forages were separated into 4 replicate piles per treatment. Forages were treated as mentioned in experiment 1, ensiled for 30 and 90 d, and analyzed for fermentation profile, IVDMD and IVNDFD, and aerobic stability. Replicate pile was considered the experimental unit.
Statistical Analysis
All 3 experiments were analyzed as randomized complete block designs with a 2 × 2 × 2 factorial treatment arrangement with 4 replicates per treatment combination. The model included the fixed effects of LB, LH, ensiling period (days), and all possible 2-way and 3-way interactions. Data for each experiment were analyzed separately using the GLIMMIX procedure of SAS version 9.4 (SAS Institute Inc.). The Bonferroni pairwise multiple comparison test was used for mean separation. Significance was declared at P < 0.05 and tendencies at 0.05 ≤ P ≤ 0.10. The 3-way interaction tables are shown in Supplemental Tables S1 to S6 (https://doi.org/10.7910/DVN/6FCKA6). When the day effect in 3-way (LB × LH × day) interactions was statistically significant, data were reanalyzed as a 2 × 2 factorial for each ensiling duration and presented in Figure 1 and Supplemental Figures S1, S2, S4, S5, S6, S8 (https://doi.org/10.7910/DVN/6FCKA6). When the day effect in 3-way (LB × LH × day) interactions was not significant, data were averaged over d 30 and 90 and presented in Tables 1, 2, and 3.
Figure 1Effect of Lactobacillus buchneri (LB), Lactobacillus hilgardii (LH) or the combination (+LB +LH) on aerobic stability of sorghum silage (27% DM; experiment 1) ensiled for 30 (a; all silages were stable for >500 h) or 90 d (b; LB × LH, P = 0.03; SEM = 47.9; all treated silages were stable for >576 h). Bars with unlike letters (a,b) differ (P < 0.05). Error bars indicate SEM.
WSC = water soluble carbohydrates; LAB = lactic acid bacteria; IVDMD = 24-h in vitro DM digestibility; IVNDFD = 24-h in vitro NDF degradability. Molds counts were not detected at either ensiling duration.
LB = Lactobacillus buchneri; LH = Lactobacillus hilgardii; treatments were −LB−LH = uninoculated; −LB +LH = inoculated with LH; +LB−LH = inoculated with LB; and +LB+LH = inoculated with LB and LH. Data in the table were averaged for d 30 and 90 as treatment × day interactions were not significant (P > 0.10).
Means within the same row with different superscripts differ (P < 0.05).
0.11
<0.01
<0.01
<0.01
Yeast, log cfu/g of fresh weight
1.18
0.68
0
0.31
0.57
0.18
0.87
0.48
a,b Means within the same row with different superscripts differ (P < 0.05).
1 WSC = water soluble carbohydrates; LAB = lactic acid bacteria; IVDMD = 24-h in vitro DM digestibility; IVNDFD = 24-h in vitro NDF degradability. Molds counts were not detected at either ensiling duration.
2 LB = Lactobacillus buchneri; LH = Lactobacillus hilgardii; treatments were −LB−LH = uninoculated; −LB +LH = inoculated with LH; +LB−LH = inoculated with LB; and +LB+LH = inoculated with LB and LH. Data in the table were averaged for d 30 and 90 as treatment × day interactions were not significant (P > 0.10).
LB = Lactobacillus buchneri; LH = Lactobacillus hilgardii; treatments were −LB−LH = uninoculated; −LB+LH = inoculated with LH; +LB−LH = inoculated with LB; and +LB+LH = inoculated with LB and LH. Data in the table were averaged for d 30 and 90 as treatment × day interactions were not significant (P > 0.10).
2 LB = Lactobacillus buchneri; LH = Lactobacillus hilgardii; treatments were −LB−LH = uninoculated; −LB+LH = inoculated with LH; +LB−LH = inoculated with LB; and +LB+LH = inoculated with LB and LH. Data in the table were averaged for d 30 and 90 as treatment × day interactions were not significant (P > 0.10).
Table 3Chemical composition, DM loss fermentation profile, microbial counts, and aerobic stability of corn silage harvested at normal maturity (34% DM; experiment 3)
LB = Lactobacillus buchneri; LH = Lactobacillus hilgardii; treatments were −LB−LH = uninoculated; −LB+LH = inoculated with LH; +LB−LH = inoculated with LB; and +LB+LH = inoculated with LB and LH. Data in the table were averaged for d 30 and 90 as treatment × day interactions were not significant (P > 0.10).
Means within same row with different superscripts differ (P < 0.05).
20.3
<0.01
0.06
0.03
a,b Means within same row with different superscripts differ (P < 0.05).
1 LB = Lactobacillus buchneri; LH = Lactobacillus hilgardii; treatments were −LB−LH = uninoculated; −LB+LH = inoculated with LH; +LB−LH = inoculated with LB; and +LB+LH = inoculated with LB and LH. Data in the table were averaged for d 30 and 90 as treatment × day interactions were not significant (P > 0.10).
2 Butyrate was detected only at 30 d.
3 Yeasts were only detected at 90 d and no molds were detected at either ensiling duration.
Experiment 1: Effect of LH, LB, or the Combination on Fermentation Profile and Aerobic Stability of Sorghum Silage (27% DM)
The average concentrations of NDF, ADF, WSC and starch in the sorghum were 61.2, 38.4, 18.2, and 5.0% respectively (Table 4). Average forage pH was 5.74, and LAB count was 6.51 log cfu/g. Ammonia-N concentration tended (P = 0.08) to increase by LB inoculation (Table 1). Inoculation with LB and LH (P < 0.01) increased concentrations of NDF and ADF and reduced IVDMD (P < 0.01) but not IVNDFD. Dry matter loss tended to be greater with LB inoculation (P = 0.06); a similar tendency was evident when LH was applied alone but not with LB (LB × LH, P = 0.09). Lactobacillus hilgardii inoculation tended to increase CP concentration (P = 0.05) and increased ethanol concentration (P = 0.04). Inoculation with LB reduced WSC concentration (P < 0.01), and LH had a similar tendency (P = 0.07).
Table 4Chemical composition and bacterial counts of sorghum forage treated with or without bacterial inoculants before ensiling (experiment 1; values presented as mean ± SD; n = 4)
Acetate concentration was increased (P < 0.01; P < 0.01) and lactate concentration was reduced (P = 0.02; P = 0.04) by LB or LH inoculation, respectively. There was an interaction between LB and LH on lactate-to-acetate ratio (LB × LH; P < 0.01) such that inoculation with LH or LB or the combination reduced the lactate-to-acetate ratio by similar (P > 0.05) amounts. Counts of LAB were similarly increased by all inoculants (LB × LH; P < 0.01). The 1,2 propanediol concentration was increased by LB regardless of ensiling duration (Supplemental Table S2, https://doi.org/10.7910/DVN/6FCKA6), but for LH, it was increased after 90 d but not after 30 d of ensiling (LH × day; P = 0.02; Figure 2). The LB and LH inoculants improved aerobic stability of sorghum silage after 90 d of ensiling (P = 0.03; Figure 1b) but not 30 d (Figure 1a).
Figure 2Effect of Lactobacillus hilgardii (LH) and ensiling duration on 1,2 propanediol concentration of sorghum silage (27% DM; experiment 1; LH × day, P = 0.02; SEM = 0.31). Bars with unlike letters (a,b) differ (P < 0.05). Error bars indicate SEM.
Experiment 2: Effect of LH, LB, or the Combination on Fermentation Profile and Aerobic Stability of Late-Harvested Corn Silage (43.8% DM)
Corn forage was harvested at 43.8% DM. The mean concentrations of NDF, ADF, WSC, and starch were 48, 23, 3.9, and 33%, respectively (Table 5). Forage pH was 6.3, and LAB counts were 7.25 log cfu/g of fresh weight. Ash concentration tended to be greater with LH inoculation (P = 0.09; Table 2). Ammonia-N concentration tended to increase when LB was applied with or without LH (LB × LH; P = 0.05). Inoculation with LB (P = 0.03) increased DM loss but LH (P = 0.002) decreased DM loss. There was an interaction between LB and LH on IVDMD (LB × LH; P = 0.03); inoculation with LH reduced IVDMD when it was applied separately but not with the combination of LB and LH. Neutral detergent fiber degradability was reduced by LH; however, combination of LB and LH increased IVNDFD compared with LB alone (LB × LH; P = 0.005; Supplemental Table S3, https://doi.org/10.7910/DVN/6FCKA6). Inoculation reduced WSC after 90 d (P = 0.002; Supplemental Figure S1b); however, no effects were observed after 30 d (P = 0.22; Supplemental Figure S1a, https://doi.org/10.7910/DVN/6FCKA6). The IVNDFD tended to be increased by inoculation after 30 d (P = 0.09; Supplemental Figure S2a), but after 90 d, LB and LH separately reduced IVNDFD (P < 0.01; Supplemental Figure S2b, https://doi.org/10.7910/DVN/6FCKA6). Lactobacillus buchneri inoculation increased silage pH (P = 0.001; Table 2), and LH had a similar tendency (P = 0.08). Though lactate concentration was decreased by LB with or without LH, LH alone had no effect (LB × LH, P = 0.04). Acetate concentration was increased by LB inoculation or tended to be increased by LH inoculation separately but not with the combination of LB and LH (LB × LH, P = 0.08). Lactate-to-acetate ratio was reduced by LB or LH or the combination, with the response being greater for LB than LH (LB × LH; P = 0.009). Both LB and LH alone or the combination increased 1,2 propanediol concentration (LB × LH; P = 0.009) and LAB counts; however, for LAB counts, the LB response was greater, and combining the inoculants increased the response relative to LH but not LB (LB × LH; P < 0.01). Yeast counts were similarly (P < 0.05) reduced by both LB and LH and the combination (LB × LH; P = 0.04), but aerobic stability was not affected (P = 0.11). Inoculation with LH increased acetate concentration after 90 d but not 30 d of ensiling (LH × day, P = 0.04; Figure 3). Inoculation with LB increased 1,2 propanediol concentration after 90 d but not 30 d of ensiling (LB × day; P = 0.004; Figure 4). Inoculation with LB increased ammonia-N concentration after 90 d but not 30 d (LB × day; P = 0.02; Supplemental Figure S3a, https://doi.org/10.7910/DVN/6FCKA6) and increased LAB counts to a greater extent after 90 versus 30 d of ensiling (LB × day; P < 0.01; Supplemental Figure S3b).
Table 5Chemical composition and bacterial counts of late-harvested corn forage (43.8% DM) treated with or without bacterial inoculants before ensiling (experiment 2; values presented as mean ± SD; n = 4)
Experiment 3: Effect of LH, LB, or the Combination on Fermentation Profile and Aerobic Stability of Corn Silage Harvested at Normal Maturity (34% DM)
Corn forage was harvested at 34% DM. The mean concentrations of NDF, ADF, WSC, and starch were 51.1, 28.0, 4.6, and 25.2%, respectively (Table 6). Inoculation with LH reduced ash concentration (P = 0.02) or tended (P = 0.09) to reduce DM loss of corn silage harvested at 34% DM (Table 3). There were LB × LH × day interactions (Supplemental Table S5, https://doi.org/10.7910/DVN/6FCKA6) for WSC concentration (P = 0.03), IVDMD (P < 0.01), and IVNDFD (P < 0.01). Inoculation had no effect on WSC after 30 d of ensiling (P = 0.34; Supplemental Figure S4a), but after 90 d, LH alone increased WSC (P = 0.01; Supplemental Figure S4b, https://doi.org/10.7910/DVN/6FCKA6). Inoculation had no effect on IVDMD after 30 d of ensiling (P = 0.16; Supplemental Figure S5a) but after 90 d, the combined inoculant increased IVDMD (P < 0.01; Supplemental Figure S5b, https://doi.org/10.7910/DVN/6FCKA6). All inoculants increased IVNDFD after 30 d (P = 0.02; Supplemental Figure S6a) but no effects were observed after 90 d (P = 0.07; Figure S6b, https://doi.org/10.7910/DVN/6FCKA6).
Table 6Chemical composition and bacterial counts of corn forage harvested at normal maturity (34% DM) treated with or without bacterial inoculants before ensiling (experiment 3; values presented as mean ± SD; n = 4)
Silage pH decreased with LH inoculation (P < 0.01; Table 3) but not LB inoculation (P = 0.36). Lactate concentration and lactate-to-acetate ratio were lower (P ≤ 0.04) and total acids tended (P = 0.06) to be lower with LB inoculation but not LH inoculation (P > 0.10). Concentration of 1,2 propanediol was increased by LH (P = 0.004) but not LB inoculation (P = 0.73). Butyrate and yeast were only detected on d 30, and molds were not detected. Yeast counts on d 30 tended to be reduced by LH (P = 0.06; Supplemental Table S6, https://doi.org/10.7910/DVN/6FCKA6) but were unaffected by LB (P = 0.83). Aerobic stability was similarly increased by all inoculants (LB × LH; P = 0.03; Table 3). Inoculation with LB reduced silage pH after 30 d but not 90 d of ensiling (LB × day; P < 0.01; Supplemental Figure S7a), and a similar trend was evident for LH (LH × day; P < 0.01; Figure S7b, https://doi.org/10.7910/DVN/6FCKA6). Inoculation with LH increased 1, 2 propanediol after 90 d but not after 30 d of ensiling (LH × day; P < 0.01; Figure 5). Lactic acid bacteria counts were not affected after 30 d of ensiling (P = 0.79; Supplemental Figure S8a) but were similarly increased by all inoculants after 90 d of ensiling (P = 0.002; Figure S8b, https://doi.org/10.7910/DVN/6FCKA6).
Figure 5Effect of Lactobacillus hilgardii (LH) and ensiling duration on 1,2 propanediol concentration of corn silage harvested at normal maturity (34% DM; experiment 3; LH × day, P < 0.01; SEM = 0.02). Bars with unlike letters (a,b) differ (P < 0.05). Error bars indicate SEM.
The respective forage nutritional compositions are in agreement with typical values from harvests at the corresponding DM concentrations for sorghum forage (27%;
Effects of whole-plant corn silage hybrid type on intake, digestion, ruminal fermentation, and lactation performance by dairy cows through a meta-analysis.
). Those for the other forages were at the low end of the range. The generally low IVNDFD values were partly because the forages were from Florida, and high temperatures typically increase cell wall deposition and acid detergent insoluble CP concentration, and reduce starch accumulation in forages, which collectively reduce IVNDFD (
). The lower values for the normal-harvested corn forage may be related to the hybrid or may reflect longer exposure of the hybrid to high temperatures due to harvesting much later in the summer than the other forages.
General Inoculant Effects
The hypothesis that LH would confer aerobic stability earlier than LB was not supported by the results.
Effects of conservation period and Lactobacillus hilgardii inoculum on the fermentation profile and aerobic stability of whole corn and sorghum silages.
reported that LH increased aerobic stability of corn silage in 1 of 2 trials after 30 d of ensiling, but not sorghum silage. However, for LB, longer ensiling periods (100 or 250 d) were required. A recent study reported that inoculating high-moisture corn with LH, LB, or LH+LB resulted in greater aerobic stability at 10 d, and the response was highest for LH+LB, intermediate for LH, and lowest for LB (
Effects of Lactobacillus hilgardii 4785 and Lactobacillus buchneri 40788 on the bacterial community, fermentation, and aerobic stability of high-moisture corn silage.
). The effects of LH or LH+LB inoculation on aerobic stability have varied among studies partly due to differences in the crops examined. Earlier onset of aerobic stability with LH or LH+LB compared with LB was not supported in this study, perhaps due to the low yeast counts detected. Nevertheless, in the current study, all inoculants improved 1,2 propanediol concentrations, increased acetate, or reduced the lactate-to-acetate ratio. Consequently, aerobic stability improved after the same ensiling period for all inoculants for sorghum (90 d) and normal maturity (34% DM) corn silage (30 or 90 d). However, no similar trend was observed for late-harvested (43.8% DM) corn silage was observed. Therefore, more research is needed to understand how to improve the consistency of effects of inoculation with LB and LH and how to hasten the response across forage types.
showed that LB increased DM losses of grass and small grain silages, and corn silage when applied at 1 × 105 and 1 × 106 cfu/g, respectively. In the current study, LB or LH alone increased DM losses from sorghum silage, but only LH increased that of normal corn silage. However, LB increased that of late corn silage, but LH reduced the response. Using the combined inoculant consistently prevented increased DM losses, perhaps by increasing the efficiency of the fermentation relative to either inoculant alone (
Dynamic succession of microbiota during ensiling of whole plant corn following inoculation with Lactobacillus buchneri and Lactobacillus hilgardii alone or in combination.
Fermentation characteristics and aerobic stability of grass silage inoculated with Lactobacillus buchheri, with or without homofermentative lactic acid bacteria.
), were generally low, particularly in sorghum silage, for unknown reasons. This may be related to the use of vacuum bags, which reduced yeast counts in a previous study (
). The low yeast counts in this study likely explain the high aerobic stability of uninoculated silages as well as the relatively small increases in aerobic stability by inoculation.
Inoculant Effect on Sorghum Silage (27% DM)
Concentrations of NDF and ADF were increased, and IVDMD was decreased in sorghum silage inoculated with LB for 90 d or with LH for 30 or 90 d. This contradicts
The effect of Lactobacillus buchneri, with or without homofermentative lactic acid bacteria, on the fermentation, aerobic stability and ruminal degradability of wheat, sorghum and maize silages.
) who reported that inoculation with LB did not affect IVDMD of sorghum silage. In the current study, the greater fiber concentrations of the inoculated forages, which contributed to their lower IVDMD, may have been due to the high WSC (18%) concentration of the “Super Sugar” hybrid. Greater fermentation of such sugars by inoculants versus epiphytic bacteria, would have resulted in relatively higher fiber concentrations in inoculated silages.
The ratio of lactate to acetate is a good indicator of changes in fermentation profiles with heterofermentative bacterial inoculation (
Dynamic succession of microbiota during ensiling of whole plant corn following inoculation with Lactobacillus buchneri and Lactobacillus hilgardii alone or in combination.
). Both LB and LH alone and the combination (LB+LH) reduced lactate-to-acetate ratio, regardless of the ensiling duration. This confirmed the efficacy of both LH and LB at promoting a heterofermentative pathway, but combining the inoculants did not improve or hasten the response.
Greater acetate concentrations typically inhibit or kill spoilage organisms such as yeasts and molds by decreasing intracellular pH after the acid enters and dissociates in the microbial cell, leading to increased aerobic stability of silage (
Dynamic succession of microbiota during ensiling of whole plant corn following inoculation with Lactobacillus buchneri and Lactobacillus hilgardii alone or in combination.
). Nevertheless, both inoculants improved aerobic stability of sorghum silage, indicating that their antibacterial and antifungal compounds such as bacteriocins and acetate, respectively (
), inhibited the growth of the spoilage microorganisms. This agrees with previous reports that LB increased sorghum silage aerobic stability after 90 d or more of storage (
The effect of Lactobacillus buchneri and Lactobacillus plantarum on the fermentation, aerobic stability, and ruminal degradability of low dry matter corn and sorghum silages.
The effect of Lactobacillus buchneri, with or without homofermentative lactic acid bacteria, on the fermentation, aerobic stability and ruminal degradability of wheat, sorghum and maize silages.
Clostridia spore formation during aerobic deterioration of maize and sorghum silages as influenced by Lactobacillus buchneri and Lactobacillus plantarum inoculants.
J. Appl. Microbiol.2009; 107 (19457043): 1632-1641
Effects of conservation period and Lactobacillus hilgardii inoculum on the fermentation profile and aerobic stability of whole corn and sorghum silages.
). The contrasting aerobic stability responses across studies may be due to differences in sorghum hybrid and maturity as well as the inoculant application rate, strain, and viability. More research is needed to understand why these results of LB and LH inoculation are inconsistent.
Inoculant Effects on Late-Harvested Corn Silage (43.8% DM)
Few studies have evaluated the effect of inoculants, particularly LB or LH, on corn silage harvested above 40% DM (
The effect of Lactobacillus buchneri 40788 or Lactobacillus plantarum MTD-1 on the fermentation and aerobic stability of corn silages ensiled at two dry matter contents.
The effects of Lactobacillus buchneri with or without a homolactic bacterium on the fermentation and aerobic stability of corn silages made at different locations.
in: Buxton D.R. Muck R.E. Harrison J.H. Silage and technology. American Society of Agronomy, Crop Science Society of America, Soil Science Society of America,
2003: 199-250
Influence of harvest date of primary growth on microbial flora of grass herbages and haylage, and on fermentation and aerobic stability of haylage conserved in laboratory silos.
). Inoculation of late-harvested corn silage (43.8% DM) with LB increased silage pH, and LH had a similar tendency. These increases resulted in pH values greater than 4.0, possibly due to the high maturity of the forage. Low lactate concentrations occur in mature or dry corn silages because they lack sufficient fermentable sugars, particularly glucose (
suggested that corn silage pH levels higher than 4.2 could be due to ensiling mature or dry silages (>42% DM).
As for sorghum silage, LB, LH, and LB+LH increased acetate concentration, and thus reduced lactate-to-acetate ratio, indicating that they promoted a heterofermentative pathway. Yet, the inoculants did not improve aerobic stability of late corn silage. This contradicts one previous study (
The effect of Lactobacillus buchneri 40788 or Lactobacillus plantarum MTD-1 on the fermentation and aerobic stability of corn silages ensiled at two dry matter contents.
The effects of Lactobacillus buchneri with or without a homolactic bacterium on the fermentation and aerobic stability of corn silages made at different locations.
, 40% DM). Aerobic stability of late corn silage was probably not improved because late harvesting makes corn forage more susceptible to aerobic spoilage due to increased potential for air pockets in which spoilage-causing yeasts and molds grow (
). Factors such as the low yeast counts and low water activity may have prevented inoculation from resulting in greater aerobic stability. Neither the hypothesis that LH would confer greater aerobic stability than other inoculants, nor the one that LH would confer aerobic stability earlier than LB (after 30 vs. 90 d) was supported by the results.
Inoculant Effects on Corn Silage Harvested at Normal Maturity (34% DM)
Inoculation of normal maturity corn forage resulted in a trend for a more heterofermentative pathway as shown by the reduction in the lactate-to-acetate ratio by LB and the similar numerical trend for LH. In addition, LH increased 1,2 propanediol and reduced yeast counts. These results likely contributed to the reduction in aerobic stability by LB and the similar tendency by LH inoculation regardless of ensiling duration. Likewise,
reported an improvement in aerobic stability when corn silage (harvested at 32.8% DM) was treated with 2 different strains of LB at 2 different levels and ensiled for 108 d (yr 2). In addition,
Effects of conservation period and Lactobacillus hilgardii inoculum on the fermentation profile and aerobic stability of whole corn and sorghum silages.
reported greater aerobic stability when corn silage (harvested at 34% DM) was inoculated with LH or LB+LH and ensiled for 30 d, but only LB+LH increased aerobic stability when the forage was ensiled for 100 and 250 d. Collectively, these results support the notion that treatment with LB or LH can increase aerobic stability of corn silage harvested at or around the recommended maturity stage of 35% DM. In support, similar to that of
Meta-analysis of effects of inoculation with Lactobacillus buchneri with or without other bacteria, on silage fermentation, aerobic stability, and performance of dairy cows.
speculated that LAB may compete with lactate-producing microorganisms for rapidly fermentable carbohydrates including sugars. This competition may reduce the rate of ruminal lactate production, resulting in elevated ruminal pH (
), and higher activity of cellulolytic microbial population, resulting in improved fiber degradation. Across the 3 experiments, the hypothesis that inoculation with LH would increase aerobic stability to a greater extent and within a shorter ensiling duration than LB or LB+LH was not supported.
CONCLUSIONS
Inoculant effects on silage fermentation characteristics, DM losses, and aerobic stability varied with forage and inoculant type and ensiling duration. Yeast counts were low, particularly for sorghum, and these may have contributed to the relatively small increases in aerobic stability by inoculation in the study. Using LH did not result in consistent improvements in fermentation or aerobic stability over using LB. Inoculating sorghum forage with LB or LH increased 1,2 propanediol concentration, reduced lactate-to-acetate ratio, and increased aerobic stability after 90 d but not 30 d of ensiling. Inoculating late-harvested corn silage with LB or LH increased 1,2 propanediol concentration, increased or tended to increase acetate acid concentration, and decreased lactate-to-acetate ratio but did not affect aerobic stability. Inoculating corn silage harvested at normal maturity with LH increased 1,2 propanediol; inoculating with LB or LH separately increased or tended to increase aerobic stability, respectively, regardless of ensiling duration. Aerobic stability responses did not differ with inoculant type or ensiling duration, and combining LB and LH neither hastened nor increased the response. Except for preventing increased DM losses, no other consistent benefits of using the combined inoculant instead of single inoculants was detected.
ACKNOWLEDGMENTS
We gratefully acknowledge funding for this study by Lallemand Animal Nutrition (Milwaukee, WI). We are thankful for the assistance of staff of the University of Florida Plant Science Research and Education Center (Citra, FL) and Alliance Dairies (Trenton, FL) for their assistance with the study and for provision of experiment 3 forage, respectively. The authors have not stated any conflicts of interest.
Meta-analysis of effects of inoculation with Lactobacillus buchneri with or without other bacteria, on silage fermentation, aerobic stability, and performance of dairy cows.
in: Buxton D.R. Muck R.E. Harrison J.H. Silage and technology. American Society of Agronomy, Crop Science Society of America, Soil Science Society of America,
2003: 199-250
Effects of Lactobacillus hilgardii 4785 and Lactobacillus buchneri 40788 on the bacterial community, fermentation, and aerobic stability of high-moisture corn silage.
Fermentation characteristics and aerobic stability of grass silage inoculated with Lactobacillus buchheri, with or without homofermentative lactic acid bacteria.
Dynamic succession of microbiota during ensiling of whole plant corn following inoculation with Lactobacillus buchneri and Lactobacillus hilgardii alone or in combination.
Effects of whole-plant corn silage hybrid type on intake, digestion, ruminal fermentation, and lactation performance by dairy cows through a meta-analysis.
Effects of conservation period and Lactobacillus hilgardii inoculum on the fermentation profile and aerobic stability of whole corn and sorghum silages.
The effect of Lactobacillus buchneri and Lactobacillus plantarum on the fermentation, aerobic stability, and ruminal degradability of low dry matter corn and sorghum silages.
The effect of Lactobacillus buchneri, with or without homofermentative lactic acid bacteria, on the fermentation, aerobic stability and ruminal degradability of wheat, sorghum and maize silages.
The effect of Propionibacterium acidipropionici, with or without Lactobacillus plantarum, on the fermentation and aerobic stability of wheat, sorghum, and maize silages.
The effect of Lactobacillus buchneri 40788 or Lactobacillus plantarum MTD-1 on the fermentation and aerobic stability of corn silages ensiled at two dry matter contents.
Influence of harvest date of primary growth on microbial flora of grass herbages and haylage, and on fermentation and aerobic stability of haylage conserved in laboratory silos.
Effects of inoculation of corn silage with Lactobacillus hilgardii and Lactobacillus buchneri on silage quality, aerobic stability, nutrient digestibility, and growth performance of growing beef cattle.
Changes in forage quality during harvest and storage.
in: Fahey G.C. Collins M. Mertens D.R. Moser L.E. Forage quality, evaluation, and utilization. Natl. Conf. on Forage Quality, Evaluation, and Utilization, Lincoln, NE. American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America,
1994: 828-868
The effects of Lactobacillus buchneri with or without a homolactic bacterium on the fermentation and aerobic stability of corn silages made at different locations.
Clostridia spore formation during aerobic deterioration of maize and sorghum silages as influenced by Lactobacillus buchneri and Lactobacillus plantarum inoculants.
J. Appl. Microbiol.2009; 107 (19457043): 1632-1641
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