The use of Lentilactobacillus buchneri PJB1 and Lactiplantibacillus plantarum MTD1 on the ensiling of whole-plant corn silage, snaplage, and high-moisture corn.

Experiments were conducted over a 3-yr period to evaluate the effects of bacterial inoculants on the fermentation profile and aerobic stability of whole-plant corn silage (WPC), snaplage (SNA), and high-moisture corn (HMC). Whole-plant corn was inoculated with Lentilactobacillus buchneri PJB1 in combination with Lactiplantibacillus plantarum MTD1 or with Lpb. plantarum alone (experiments 1 and 2). Snaplage (experiment 3) and HMC (experiments 4 and 5) were inoculated with Len. buchneri in combination with Lpb. plantarum or with Len. buchneri alone. After inoculation, the feedstuffs were ensiled in 7.57-L silos and stored at 21 ± 2°C for 30 or 90 d. In experiment 5, silage was subjected to air stress for 2 h every 2 wk through 42 d and then for 2 h/wk until 90 d and had samples analyzed for their bacterial community composition by metagenomics. Overall, in all experiments, silages inoculated with Len. buchneri alone or in combination with Lpb. plantarum had more acetic acid and 1,2-propanediol and fewer yeasts than uninoculated silages. After 30 d of ensiling, inoculation with Len. buchneri alone or in combination with Lpb. plantarum did not affect the aerobic stability of SNA, but it slightly increased the stability of WPC and markedly improved the stability of HMC. After 90 d of ensiling, inoculation with Len. buchneri alone or in combination with Lpb. plantarum markedly improved the aerobic stability of WPC, SNA, and HMC. In experiment 5, inoculation increased the relative abundance (RA) of Lactobacillaceae and reduced the RA of Enterobacteriaceae and Leuconostocaceae in HMC at 30 and 90 d and the RA of Clostridiaceae in non-air-stressed HMC at 90 d. Air-stressed HMC inoculated with Len. buchneri had less lactic acid, more acetic acid and 1,2-propanediol, and markedly greater aerobic stability than uninoculated air-stressed HMC at 90 d. In conclusion, inoculation with Len. buchneri PJB1 alone or in combination with Lpb. plantarum MTD1 increased the production of acetic acid and 1,2-propanediol, inhibited yeasts development, and improved the aerobic stability of WPC, SNA, and HMC. In HMC, inoculation markedly improved aerobic stability as soon as after 30 d of ensiling, and after 90 d, inoculation improved stability even under air stress conditions.

Application of 3-nitrooxypropanol and canola oil to mitigate enteric methane emissions of beef cattle results in distinctly different effects on the rumen microbial community.

BACKGROUND: The major greenhouse gas from ruminants is enteric methane (CH4) which in 2010, was estimated at 2.1 Gt of CO2 equivalent, accounting for 4.3% of global anthropogenic greenhouse gas emissions. There are extensive efforts being made around the world to develop CH4 mitigating inhibitors that specifically target rumen methanogens with the ultimate goal of reducing the environmental footprint of ruminant livestock production. This study examined the individual and combined effects of supplementing a high-forage diet (90% barley silage) fed to beef cattle with the investigational CH4 inhibitor 3-nitrooxypropanol (3-NOP) and canola oil (OIL) on the rumen microbial community in relation to enteric CH4 emissions and ruminal fermentation. RESULTS: 3-NOP and OIL individually reduced enteric CH4 yield (g/kg dry matter intake) by 28.2% and 24.0%, respectively, and the effects were additive when used in combination (51.3% reduction). 3-NOP increased H2 emissions 37-fold, while co-administering 3-NOP and OIL increased H2 in the rumen 20-fold relative to the control diet. The inclusion of 3-NOP or OIL significantly reduced the diversity of the rumen microbiome. 3-NOP resulted in targeted changes in the microbiome decreasing the relative abundance of Methanobrevibacter and increasing the relative abundance of Bacteroidetes. The inclusion of OIL resulted in substantial changes to the microbial community that were associated with changes in ruminal volatile fatty acid concentration and gas production. OIL significantly reduced the abundance of protozoa and fiber-degrading microbes in the rumen but it did not selectively alter the abundance of rumen methanogens. CONCLUSIONS: Our data provide a mechanistic understanding of CH4 inhibition by 3-NOP and OIL when offered alone and in combination to cattle fed a high forage diet. 3-NOP specifically targeted rumen methanogens and partly inhibited the hydrogenotrophic methanogenesis pathway, which increased H2 emissions and propionate molar proportion in rumen fluid. In contrast, OIL caused substantial changes in the rumen microbial community by indiscriminately altering the abundance of a range of rumen microbes, reducing the abundance of fibrolytic bacteria and protozoa, resulting in altered rumen fermentation. Importantly, our data suggest that co-administering CH4 inhibitors with distinct mechanisms of action can both enhance CH4 inhibition and provide alternative sinks to prevent excessive accumulation of ruminal H2.

Effect of dry matter content on the microbial community and on the effectiveness of a microbial inoculant to improve the aerobic stability of corn silage.

Silage inoculants are commonly used as a tool to improve the fermentation and aerobic stability of corn silage fed to dairy cows. However, their effectiveness can be inconsistent. Our objective was to determine the effect of the dry matter (DM) content of freshly chopped whole-plant corn on its microbial community as affected by an inoculant containing Lentilactobacillus hilgardii, Lentilactobacillus buchneri, and Pediococcus pentosaceus on improving the aerobic stability of silage. Whole-plant corn was harvested at low (31.80%, LDM), medium (33.32%, MDM), or high (39.44%, HDM) DM content and treated with nothing (CTR) or an inoculant (INO) containing L. hilgardii CNCM I-4785 at 150,000 cfu/g fresh forage, L. buchneri NCIMB 40788 at 150,000 cfu/g fresh forage, P. pentosaceus NCIMB12455 at 100,000 cfu/g of fresh forage, ß-glucanase (5,750 IU/g), and xylanase (30,000 IU/g) and ensiled for 20 and 60 d. Data were analyzed as a completely randomized design in a 3-by-2 factorial arrangement of treatments. Fresh LDM forage had a higher concentration of reducing sugars, a less rich, diverse, and even bacterial community, and greater relative abundance of Saccharomycetales than MDM and HDM forages. Silages at 20 and 60 d, inoculated LDM had a more modest proliferation of culturable lactic acid bacteria than inoculated MDM. At 20 d, regardless of treatment, LDM had greater concentrations of lactic and acetic acids. Also at 20 d, LDM had lower numbers of culturable yeasts but greater relative abundance of Enterobacteriaceae than MDM and HDM. For silage at 20 d, HDM silage was more aerobically stable than LDM and MDM and inoculation improved aerobic stability 1.8-fold compared with CTR. For silage at 60 d, there was an interaction between DM content and inoculation. The improvements in stability by inoculation, compared with CTR, were greater in MDM (261 vs. 41 h) and HDM (320 vs. 66 h) silages than in LDM (85 vs. 46 h). The lower DM content and possible slower pH decline in LDM might have facilitated the development of undesirable bacteria and coupled with its greater concentration of reducing sugars and lactic and acetic acids, which are substrates for aerobic microorganisms, might explain the more modest improvements in aerobic stability from inoculation in LDM compared with MDM and HDM. Our findings suggest that the DM content of whole-plant corn affected its epiphytic microbial community and the effectiveness of the inoculant, which improved aerobic stability at all DM but to a greater extent in HDM and MDM than in LDM, especially after 60 d of ensiling.

The effects of Lactobacillus hilgardii 4785 and Lactobacillus buchneri 40788 on the microbiome, fermentation, and aerobic stability of corn silage ensiled for various times.

We evaluated the ability of an inoculant containing a combination of Lactobacillus hilgardii and Lactobacillus buchneri to modify the microbiome and improve the aerobic stability of whole-plant corn silage after various lengths of ensiling. Chopped whole-plant corn at about 33% dry matter (DM) was uninoculated (CTR) or inoculated with L. hilgardii CNCM I-4785 and L. buchneri NCIMB 40788 at 200,000 cfu/g of fresh forage weight each (combined application rate of 400,000 cfu of lactic acid bacteria/g of fresh forage weight; LHLB), L. buchneri NCIMB 40788 at 400,000 cfu/g of fresh forage weight and Pediococcus pentosaceus NCIMB 12455 at 100,000 cfu/g of fresh forage weight, used as a positive control (LB500), L. hilgardii CNCM I-4785 at the application rate used in the LHLB formulation of 200,000 cfu/g of fresh forage weight (LH), or L. buchneri NCIMB 40788 at the application rate used in the LHLB formulation of 200,000 cfu/g of fresh forage weight (LB). Silos were opened after 34 and 99 d of ensiling and analyzed for nutrient composition, fermentation profile, microbiome, and aerobic stability. After 34 d of ensiling, the inoculated silages had greater numbers of culturable lactic acid bacteria, a bacterial community less rich and diverse, greater relative abundance of Lactobacillus, lower relative abundance of Klebsiella, and a greater concentration of propionic acid than uninoculated silages. Inoculation decreased the ratio of lactic acid to acetic acid, except for LB alone. Treatment LHLB resulted in silage with a greater concentration of 1,2-propanediol than LB500 and was the only treatment to have a lower relative abundance of Saccharomycetes compared with uninoculated silage. Treatments LHLB and LB500 improved the aerobic stability compared with CTR, but the individual LH and LB treatments applied at a low dose did not. Whereas LB500 was stable 34 h longer than CTR, LHLB was stable 91 h longer. After 99 d of ensiling, all inoculated silages had markedly greater aerobic stability than uninoculated silage and were stable for more than 360 h. The inoculant containing a combination of L. hilgardii and L. buchneri markedly improved the aerobic stability of corn silage after a relatively short period of ensiling, and such improvements were greater than the ones obtained from inoculation with the combination of L. buchneri and P. pentosaceus. Inoculating with the combination of L. hilgardii and L. buchneri may be helpful to producers that must feed silage shortly after ensiling.

Effect of microbial and chemical additives on the fermentation and aerobic stability of alfalfa silage ensiled at 2 dry matters and subjected to air stress during storage.

We evaluated the effects of different types of additives on the fermentation and aerobic stability of alfalfa (Medicago sativa) ensiled at 2 dry matters (DM). Alfalfa was untreated (CTRL) or treated with sodium benzoate, potassium sorbate, and sodium nitrite (SFE), or microbial inoculants (Lactobacillus plantarum MTD1 [LP] or L. buchneri 40788 and Pediococcus pentocaseus 12455 [LBPP]) at a moderate (38%) and high (46%) DM using a completely randomized design with a 2 × 4 factorial arrangement of treatments. High DM silage was higher (P < 0.01) in pH, had less lactic and acetic acid (P < 0.01) and had more yeasts (P < 0.05) and molds (P < 0.01) than moderate DM silage. Recovery of DM declined (P < 0.01) for CTRL and LP treated silages with increasing DM but was not different between LBPP and SFE treatments. Compared to CTRL, LBPP had a lower (P < 0.01) DM recovery at the moderate DM, but SFE had the greatest (P < 0.01) recovery of all treatments at the high DM. Treatment with LBPP increased (P < 0.05) the concentrations of acetic acid and 1,2 propanediol (PD) compared with other treatments (P < 0.01). Numerically, fewer yeasts were found in additive treated silages compared with CTRL, but they were statistically (P < 0.01) lower only when treated with SFE. Treatment with LP resulted in a small improvement in aerobic stability at the moderate but not high DM. In contrast, treatment with SFE and LBPP markedly improved (P < 0.01) the aerobic stability of alfalfa silage at both DM. Whereas SFE and LBPP were similar in their improvements in aerobic stability at the DM, LBPP was better (P < 0.01) than SFE at the high DM. A higher (P < 0.01) concentration of acetic acid in LBPP compared with other treatments was most likely responsible for better stability. This study showed that LBPP and SFE resulted in increases in the aerobic stability of alfalfa silage and it is the first study showing SFE, can markedly improve the aerobic stability of alfalfa silage.

Combined effects of 3-nitrooxypropanol and canola oil supplementation on methane emissions, rumen fermentation and biohydrogenation, and total tract digestibility in beef cattle.

The individual and combined effects of 3-nitrooxypropanol (3-NOP) and canola oil (OIL) supplementation on enteric methane (CH4) and hydrogen (H2) emissions, rumen fermentation and biohydrogenation, and total tract nutrient digestibility were investigated in beef cattle. Eight beef heifers (mean body weight ± SD, 732 ± 43 kg) with ruminal fistulas were used in a replicated 4 × 4 Latin square with a 2 (with and without 3-NOP) × 2 (with and without OIL) arrangement of treatments and 28-d periods (13 d adaption and 15 d measurements). The four treatments were: control (no 3-NOP, no OIL), 3-NOP (200 mg/kg dry matter [DM]), OIL (50 g/kg DM), and 3-NOP (200 mg/kg DM) plus OIL (50 g/kg DM). Animals were fed restrictively (7.6 kg DM/d) a basal diet of 900 g/kg DM barley silage and 100 g/kg DM supplement. 3-NOP and OIL decreased (P < 0.01) CH4 yield (g/kg DM intake) by 31.6% and 27.4%, respectively, with no 3-NOP × OIL interaction (P = 0.85). Feeding 3-NOP plus OIL decreased CH4 yield by 51% compared with control. There was a 3-NOP × OIL interaction (P = 0.02) for H2 yield (g/kg DM intake); the increase in H2 yield (P < 0.01) due to 3-NOP was less when it was combined with OIL. There were 3-NOP × OIL interactions for molar percentages of acetate and propionate (P < 0.01); individually, 3-NOP and OIL decreased acetate and increased propionate percentages with no further effect when supplemented together. 3-NOP slightly increased crude protein (P = 0.02) and starch (P = 0.01) digestibilities, while OIL decreased the digestibilities of DM (P < 0.01) and neutral detergent fiber (P < 0.01) with no interactions (P = 0.15 and 0.10, respectively). 3-NOP and OIL increased (P = 0.04 and P < 0.01, respectively) saturated fatty acid concentration in rumen fluid, with no interaction effect. Interactions for ruminal trans-monounsaturated fatty acids (t-MUFA) concentration and percentage were observed (P = 0.02 and P < 0.01); 3-NOP had no effect on t-MUFA concentration and percentage, while OIL increased the concentration (P < 0.01) and percentage (P < 0.01) of t-MUFA but to a lesser extent when combined with 3-NOP. In conclusion, the CH4-mitigating effects of 3-NOP and OIL were independent and incremental. Supplementing ruminant diets with a combination of 3-NOP and OIL may help mitigate CH4 emissions, but the decrease in total tract digestibility due to OIL may decrease animal performance and needs further investigation.

Effects of a chemical additive on the fermentation, microbial communities, and aerobic stability of corn silage with or without air stress during storage.

We evaluated the effects of a chemical additive on the microbial communities, fermentation profile, and aerobic stability of whole-plant corn silage with or without air stress during storage. Whole-plant corn was either untreated or treated with a chemical additive containing sodium benzoate, potassium sorbate, and sodium nitrite at 2 or 3 liters/t of fresh forage weight. Ten individually treated and replicated silos (7.5 liters) were made for each treatment. Half of the silos remained sealed throughout a 63-d storage period, and the other half was subjected to air stress for 2 h/wk. The composition of the bacterial and fungal communities of fresh forage and silages untreated or treated with 2 liters/t of fresh forage weight was analyzed by Illumina Miseq sequencing. Treated silage had greater (P < 0.05) aerobic stability than untreated, even when subjected to air stress during storage, but the numbers of yeasts culturable on selective agar were not affected. However, the additive reduced the relative abundance (RA) of the lactating-assimilating yeast Candida tropicalis (P < 0.01). In air-stressed silages, untreated silage had a greater (P < 0.05) RA of Pichia kudriavzevii (also a lactate assimilator) than treated silage, whereas treated silage was dominated by Candida humilis, which is usually unable to assimilate lactate or assimilates it slowly. The additive improved the aerobic stability by specifically preventing the dominance of yeast species that can consume lactate and initiate aerobic spoilage. To the best of our knowledge, this is the first work that identifies the specific action of this additive on shifting the microbial communities in corn silage.

The effect of various doses of an exogenous acid protease on the fermentation and nutritive value of corn silage.

The objective of this experiment was to evaluate the effects of treating whole-plant corn at harvest with various doses of an exogenous acidic protease on fermentation and changes in nutritive value after a short period (45 d) of ensiling. Whole-plant corn (37% dry matter) was chopped and treated with 0, 20, 200, 1,000, or 2,000 mg of protease/kg of wet forage. Forages (~500 g) were packed in bag silos and ensiled at 22 to 23°C for 45 d. Data were analyzed as a 5 × 2 factorial arrangement of treatments with the main effects of the dose of protease, day of ensiling, and their interaction. Treatment with protease did not alter the concentrations of dry matter, neutral detergent fiber, acid detergent fiber, starch, lactic acid, or acetic acid compared with untreated silage, with the exception that the concentration of starch was lower in silage treated with 20 mg of protease/kg compared with untreated silage. However, the 2 highest doses of protease resulted in silages with higher concentrations of ethanol and more yeasts compared with untreated silage. Protease treatment did not affect the ruminal in vitro digestibility of neutral detergent fiber. Concentrations of soluble protein (percentage of crude protein) increased after ensiling for all treatments but was not different between silage treated with the lowest dose of protease and untreated silage. Soluble protein increased in a dose-dependent manner above the low dose of protease in silages. Concentrations of NH3-N were higher only in silages treated with the 2 highest doses of protease compared with untreated silage. Silages treated with the 3 highest doses of protease were higher in ruminal in vitro digestibility of starch compared with untreated silage but were similar to each other. The concentrations of total AA were determined in fresh forage and silages for the untreated and 200 and 2,000 mg/kg doses of protease. Neither amount of added protease affected the total concentrations of essential, nonessential, or total AA in silage. However, of the essential AA, treatment with protease resulted in silages with lower concentrations of lysine and arginine but higher concentrations of leucine compared with untreated silage. The 200 mg/kg dose of protease substantially improved ruminal in vitro starch digestion in corn silage after a short period of ensiling without affecting concentrations or numbers of ethanol and yeasts, respectively.

An evaluation of the effectiveness of a chemical additive based on sodium benzoate, potassium sorbate, and sodium nitrite on the fermentation and aerobic stability of corn silage.

We evaluated the effectiveness of an additive comprising sodium benzoate, potassium sorbate, and sodium nitrite (SSL) as active ingredients for its ability to improve the aerobic stability of corn silages made in North America. In experiment 1, treatment with SSL (1.5 and 2.0 L/t) on whole-plant corn (WPC) was compared with treatment with an additive containing buffered propionic acid and citric acid (BPA; 2 L/t) on corn harvested at 32 and 38% dry matter and ensiled for 120 d. Silage treated with BPA was higher in ammonia-N and propionic acid relative to other treatments. Treatments with all of the additives had numerically, but not statistically, fewer yeasts compared with untreated silage. Both application rates of SSL resulted in lower concentrations of ethanol compared with untreated and BPA silages. Treatment with BPA improved the aerobic stability of silages compared with untreated silage, but the effect from SSL was markedly greater. In experiment 2, WPC was untreated or treated with 2 or 3 L of SSL/t or a microbial inoculant containing Enterococcus faecium M74, Lactobacillus plantarum CH6072, and Lactobacillus buchneri LN1819 (final total lactic acid bacteria application rate of 150,000 cfu/g of fresh forage). Silages were air stressed for 24 h at 28 and 42 d of storage and ensiled for 49 d before opening. Inoculation had no effect on acid end products, ethanol, number of yeasts, or aerobic stability compared with other treatments. Treatment with SSL decreased the amount of ethanol, had no effect on number of yeasts, and improved aerobic stability in a dose-dependent manner compared with other treatments. In experiment 3, WPC was untreated or treated with 2 L of SSL/t and ensiled for 5, 15, and 30 d. Treatment with SSL resulted in silage with fewer yeasts and lower concentrations of ethanol after all times of ensiling compared with untreated silage. In addition, SSL improved aerobic stability after each period of ensiling, but the effect was more at 15 and 30 d compared with 5 d of storage. Treating WPC with SSL can improve the aerobic stability of corn silage made in North America, and the effect can be observed as soon as 5 d after ensiling.

Silage review: Interpretation of chemical, microbial, and organoleptic components of silages.

The goal of making silage is to produce a stable feed with a high recovery of dry matter, energy, and highly digestible nutrients compared with the fresh crop. Microbial fermentation in the silo produces an array of end products and can change many nutritive aspects of a forage. High-quality silage should be void of undesirable compounds that could negatively affect animal performance, the environment, or net farm income. This review discusses the interpretation of the common fermentation end products, microbial populations, organoleptic properties, and changes in nutritive aspects of silages during storage of silages with emphasis on a North American perspective.

Potassium sorbate reduces production of ethanol and 2 esters in corn silage.

The objective of this work was to evaluate the effects of biological and chemical silage additives on the production of volatile organic compounds (VOC; methanol, ethanol, 1-propanol, methyl acetate, and ethyl acetate) within corn silage. Recent work has shown that silage VOC can contribute to poor air quality and reduce feed intake. Silage additives may reduce VOC production in silage by inhibiting the activity of bacteria or yeasts that produce them. We produced corn silage in 18.9-L bucket silos using the following treatments: (1) control (distilled water); (2) Lactobacillus buchneri 40788, with 400,000 cfu/g of wet forage; (3) Lactobacillus plantarum MTD1, with 100,000 cfu/g; (4) a commercial buffered propionic acid-based preservative (68% propionic acid, containing ammonium and sodium propionate and acetic, benzoic, and sorbic acids) at a concentration of 1 g/kg of wet forage (0.1%); (5) a low dose of potassium sorbate at a concentration of 91 mg/kg of wet forage (0.0091%); (6) a high dose of potassium sorbate at a concentration of 1g/kg of wet forage (0.1%); and (7) a mixture of L. plantarum MTD1 (100,000 cfu/g) and a low dose of potassium sorbate (91 mg/kg). Volatile organic compound concentrations within silage were measured after ensiling and sample storage using a headspace gas chromatography method. The high dose of potassium sorbate was the only treatment that inhibited the production of multiple VOC. Compared with the control response, it reduced ethanol by 58%, ethyl acetate by 46%, and methyl acetate by 24%, but did not clearly affect production of methanol or 1-propanol. The effect of this additive on ethanol production was consistent with results from a small number of earlier studies. A low dose of this additive does not appear to be effective. Although it did reduce methanol production by 24%, it increased ethanol production by more than 2-fold and did not reduce the ethyl acetate concentration. All other treatments increased ethanol production at least 2-fold relative to the control, and L. buchneri addition also increased the 1-propanol concentration to approximately 1% of dry matter. No effects of any treatments on fiber fractions or protein were observed. However, L. buchneri addition resulted in slightly more ammonia compared with the control. If these results hold under different conditions, a high dose of potassium sorbate will be an effective treatment for reducing VOC production in and emission from silage. Regulations aimed at reducing VOC emission could be ineffective or even increase emission if they promote silage additives without recognition of different types of additives.