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Bacterial inoculants can improve the conservation and nutritional quality of silages. Inclusion of the yeast Saccharomycesin the diet of dairy cattle has also been reported to be beneficial. The present study assessed the ability of silage to be used as a means of delivering Saccharomycesstrains to ruminants. Two strains of Saccharomyces cerevisiae(strain 1 and 3) and 1 strain of Saccharomyces paradoxus(strain 2) were inoculated (103cfu/g) individually onto corn forage that was ensiled in mini silos for 90 d. Fermentation characteristics, aerobic stability, and nutritive value of silages were determined and real-time quantitative PCR (RT-qPCR) was used to quantify S. cerevisiae, S. paradoxus, total Saccharomyces, fungal, and bacterial populations. Fermentation characteristics of silage inoculated with S1 were similar to control silage. Although strain 3 inoculation increased ash and decreased OM contents of silage (P= 0.017), no differences were observed in nutrient composition or fermentation profiles after 90 d of ensiling. Inoculation with Saccharomyceshad no detrimental effect on the aerobic stability of silage. In vitro DM disappearance, gas production, and microbial protein synthesis were not affected by yeast inoculation. Saccharomycesstrain 1 was quantified throughout ensiling, whereas strain 2 was detected only immediately after inoculation. Saccharomyces cerevisiaestrain 3 was quantified until d 7 and detectable 90 d after ensiling. All inoculants were detected and quantified during aerobic exposure. Inoculation with Saccharomycesdid not alter lactobacilli populations. Saccharomycetales were detected by RT-qPCR throughout ensiling in all silages. Both S. cerevisiaeand S. paradoxuspopulations increased during aerobic exposure, demonstrating that the density of these yeast strains would increase between the time that silage was removed from storage and the time it was fed.

Little information about the effects of the forage conservation method on the biological activity of condensed tannins (CT) in the rumen is available. The objective of this study was to determine the effects of the forage conservation method and CT in conserved forage on rumen fermentation. Purple prairie clover (PPC; Dalea purpureaVent. ) containing 84.5 CT/kg DM was harvested at full flower and preserved as freeze-dried forage (FD), hay (H), or silage (S). Batch culture incubations were conducted using conserved forages as a substrate with and without inclusion of polyethylene glycol (PEG) which inactivates CT. The treatments were arranged as a 3 × 2 factorial design, and the incubation was repeated twice with quadruplicate fermentation vials for each treatment in each incubation. 15N labeled ammonium sulfate was used as a microbial N marker, and headspace gas was sampled to determine methane production. Concentrations of NDF and ADF were lower (P< 0.01) in FD than in H and S. Ensiling decreased (P< 0.001) total phenolics and extractable CT content but had no effect on total CT. Hay did not affect either extractable or total CT content. Hay and silage had lower (P< 0.01) true DM disappearance (TDMD) and NDF disappearance (NDFD) than S and FD, whereas inclusion of PEG did not affect TDMD or NDFD at 8, 24, or 72 h of incubation. Hay had lower microbial protein (MP) production (P< 0.001) than FD and S, and inclusion of PEG decreased (P< 0.001) MP at 24 or 72 h of incubation. Productions of total gas, methane, and total VFA and the rate of gas production were similar, but ammonia accumulation was higher (P< 0.05) for PEG than non-PEG treatments during 72-h incubation. Incubation with S produced VFA with a lower (P< 0.001) proportion of acetate but higher (P< 0.05) proportion of propionate, resulting in a lower (P< 0.001) acetate:propionate ratio as compared to that of FD or H after 8 h of incubation. No difference was observed between H and S in the VFA profile. Condensed tannins in PPC decreased protein degradation in the rumen but had minimal effects on overall rumen fermentation, and conservation of PPC as hay or silage had little effect on the efficacy of CT in modulating rumen fermentation.

The objective of this study was to determine whether binding a commercial xylanase feed enzyme to the surface of Bacillus subtilisspores would improve feed digestibility compared to free enzyme. An in vivostudy was conducted using 4 treatments: control (CON; no enzyme), B. subtilisspores (SPR; 1.0 × 1010spores/kg DMI), free enzyme (ENZY; 2.5 ml of commercial xylanase enzyme/kg DMI), and spore-bound enzyme (SBE; 2.5 ml of commercial xylanase enzyme bound to 1.0 × 1010spores/kg DMI) applied to alfalfa grass hay. The treatments were fed to 4 ruminally and duodenally cannulated heifers in a 4 × 4 Latin square design, with 21 d experimental periods and a 7 d washout between periods. Ruminal in situ disappearance was determined on d 13 to 18, where triplicate polyester bags containing alfalfa grass hay (5 g) were removed after 0, 4, 8, 12, 24, 36, 48, 72, 96, and 120 h of incubation. Total collection of feces and feed refusals occurred on d 19 to 21 to measure total tract digestibility. Rumen and fecal samples were collected and processed for 16S rRNA gene sequencing to analyze bacterial microbiota. There were no differences in ruminal in situ rates of disappearance of DM, OM, NDF, or ADF (P≥ 0.086). Effective rumen degradability of DM and OM was greater for ENZY, SBE, and SPR compared to CON (P≤ 0.048), but there was no difference between ENZY and SBE (contrast P≥ 0.146). Total tract and ruminal DM and OM digestibilities were not affected by treatment (P≥ 0.067); however, intestinal DM and OM digestibilities were increased for SBE (SBE vs ALL, P≤ 0.043). There was also an increase in total tract digestibility of NDF and ADF for SBE (SBE vs ALL, P≤ 0.048). Rumen microbiota diversity was affected by treatment, with differences observed between CON and SPR (P= 0.050), CON and SBE (P= 0.033), and SPR and SBE (P= 0.009). There was no difference in fecal microbiota diversity between CON and SPR (P= 0.113) or SPR and SBE (P= 0.406), but fecal microbiota was different between CON and SBE (P= 0.009) as well as ENZY and SBE (P< 0.001). These results suggest that spore-bound enzymes are more stable than free enzyme, increasing total tract digestibility of NDF and ADF. The effects likely resulted from enhanced postruminal digestion and alteration of the gastrointestinal microbiota.

Our previous studies have shown that purple prairie clover (PPC) reduced the fecal shedding of Escherichia coliO157:H7 in lambs and generic E. coliin cattle, a response attributed to the presence of condensed tannins (CT). This study assessed the effect of PPC and PPC CT on the composition of the bacterial community in the feces of lambs using high-throughput 16S rRNA gene pyrosequencing. A total of 18 individually fed lambs were randomly divided into three groups and fed alfalfa (Alf), a 40:60 (DM basis; Mix) mixture of Alf and PPC, and Mix with polyethylene glycol (Mix-P) for 18 d. The Mix and Mix-P diets contained about 36 g CT/kg DM. Polyethylene glycol (PEG; MW6, 000) was sprayed onto the Mix-P diet to inactivate the biological activity of CT. Fecal samples were collected on Day 0, 13, and 18 through digital rectal retrieval. The samples were freeze-dried, DNA was extracted, and bacterial 16S rRNA gene amplicons were sequenced using the 454 pyrosequencing technology. Regardless of diet, bacterial communities were dominated by Firmicutes and Bacteroidetes, with a large proportion of OTU within these phyla remaining unclassified at the genus level after analysis. Diet had no effects on the fecal bacterial composition at the phylum level or on α-diversity metrics. Compared with the Alf diet, the Mix diet reduced the number of OTU associated with Butyrivibrio(P= 0.01), Anaeroplasma(P= 0.02), and unclassified bacteria within the families Peptococcaceae (P= 0.03), Christensenellaceae (P= 0.01), Erysipelotrichaceae (P= 0.02), S24-7 (P= 0.02), and Dehalobacteriaceae (P= 0.03). Similar reductions occurred within the orders RF39 (P= 0.05) and ML615J-28 (P= 0.02), but no difference was observed between Mix and Mix-P groups (P> 0.05). In contrast, a greater proportion of genus Prevotella(P= 0.02) was found in the Mix group compared with the Alf group. These results indicate that PPC CT up to 36 g/kg DM in the diet exert only minor effects on the composition of the fecal bacterial community of lambs.

Effects of condensed tannins (CT) on rumen microbes have been well documented, whereas little information exists about their effects on the microbial communities involved in ensiling. The objective of this study was to determine the effects of CT on the composition and diversity of bacterial and fungal core microbiomes associated with ensiling and aerobic spoilage of purple prairie clover (PPC). Purple prairie clover (Dalea purpureaVent.; 60 g CT/kg DM) was harvested at full flower and ensiled in polyvinyl chloride laboratory silos with and without polyethylene glycol (PEG) for 76 d. Silage was then subjected to aerobic exposure for 14 d. Bacterial and fungal core microbiomes in the silage and in the aerobically exposed silage were examined using real-time qPCR and high-throughput sequencing. Real-time qPCR analysis revealed that PPC ensiled without PEG exhibited less (P< 0.01 to approximately 0.001) gene copy numbers of total bacteria, Lactobacillus, yeasts, and fungi than PEG-treated silage. This trend was also observed for d-7 aerobically exposed silage with the exception of Lactobacillus, which had greater (P< 0.05) gene copy numbers for non-PEG than for PEG-treated silage. Metagenome analyses generated a total of 4,273,668 bacterial sequences and 3,455,929 fungal sequences, which were assigned to 225 bacterial and 142 fungal genera, respectively. Addition of PEG increased (P< 0.001) the abundance of Lactobacillusand Pediococcusbut decreased (P< 0.01) that of Lactococcus, Leuconostoc, Agrobacterium, Erwinia, Methylobacterium, Pseudomonas, and Sphingomonas. The abundance of the fungal genera Colletotrichum, Xylogone, Galactomyces, Penicillium, Fusarium, and Cryptococcusin silage were also increased (P< 0.05) by PEG. Diversity measurements of bacterial and fungal communities indicated that addition of PEG decreased (P< 0.01) the number of microbial core genome operational taxonomic units (OTU), ACE, Chao 1, and Shannon indexes. It did not affect the diversity of fungal communities. The PEG-treated silage had a higher (P< 0.001) abundance of Pediococcusbut less (P< 0.001) Lactococcusand Leuconostocthan non-PEG silage after aerobic exposure. The PEG-treated silage also had a greater (P< 0.05 to approximately 0.01) abundance of Colletotrichum, Penicillium, and Fusariumat d 7 and greater (P< 0.05 to approximately 0.001) abundance of Candida, Colletotrichum, Wickerhamomyces, Penicillium, and Pterulaafter aerobic exposure. The observed bacterial and fungal OTU, ACE and Chao 1 were lower (P< 0.05) for PEG-treated than for non-PEG treated silage after aerobic exposure. The results indicated that CT decreased population of majority bacteria, fungus, and yeast but increased bacterial diversity during ensiling and aerobic deterioration but increased fungal diversity only after aerobic exposure.

The objective of this study was to assess the effect of applying a Pediococcus pentosaceusand chitinase mixture (PED+CH) at baling on nutrient digestion and on growth performance of beef cattle. Pure alfalfa was harvested and sun-cured to either 23 to 30% (HMH) or 10 to 13% (NMH) moisture. The HMH (Treatment) was baled with application of PED (6.5 × 1011cfu/tonne)+CH (1.5 g/tonne) and NMH was baled without additives (Control). A crossover experiment (Exp 1) used eight cannulated heifers that were divided into two groups and fed diets containing 77% of control or treated alfalfa hay and 33% concentrate (DM basis). Each period consisted of a 10-d adaption, 2-d for measuring rumen fermentation products and 7-d for measuring total tract digestibility using chromium oxide (Cr2O3) as an indigestible marker. In Exp 2, fifty Angus×Hereford crossed-bred steers (270 ± 1.12 kg) were stratified by BW and allocated randomly to two groups in 10 pens, and fed diets containing 57% (DM basis) of treated or control alfalfa hay for 112 d and DMI, ADG and feed efficiency (FE) were measured. Data were analyzed as a completely randomized design using PROC MIXED procedure of SAS with cattle (Exp 1) or pen (Exp 2) as the statistical unit. Differences among means were identified using LSMEANS with the PDIFF in SAS. Cattle fed both diets had similar (P> 0.05) DM, NDF and ADF digestibility. However, HMH alfalfa treated with PED+CH had lower (P< 0.05) CP digestibility as compared with NMH alfalfa hay. Both groups of cattle had similar rumen pH, VFA and ammonia concentrations and cellulolytic enzyme activity. The two groups of cattle also had similar (P> 0.05) DMI, ADG and FE over the 112-d backgrounding period. The similar rumen fermentation characteristics, nutrient total tract digestibility and growth performance between the 2 groups of cattle indicate that alfalfa HMH preserved with PED+CH exhibited similar ruminal and total tract digestibility and feed value to NMH alfalfa. The PED+CH additive has the potential to conserve high-moisture alfalfa hay so that its nutritive and feeding value is similar to that of sun-dried alfalfa hay.

Immobilization of enzymes on Bacillusspores has been shown to enhance enzyme stability. Binding feed enzymes to spores may therefore protect them in the rumen environment and improve enzyme efficacy. The objective of this study was to determine whether a xylanase feed enzyme bound to the surfaces of Bacillus subtilisor Bacillus coagulansspores would improve in vitro ruminal NDF digestibility compared with free enzyme. Three separate in vitro ruminal batch cultures were performed on different days using the following treatments: B. subtilisspore-bound enzyme (BsubE; 1.0 × 109B. subtilisspores + 0.1 mg xylanase enzyme protein); B. coagulansspore-bound enzyme (BcoaE; 1.0 × 109B. coagulansspores + 0.1 mg xylanase enzyme protein); free enzyme (ENZY; 0.1 mg xylanase enzyme protein); and control (CON; water). The treatments were applied to alfalfa hay (2-mm particle size) 4 h before incubations. Rumen fluid was collected from two cannulated heifers and mixed with Menke's buffer (3:1) under anaerobic conditions to make inoculant. Serum vials containing pre-treated alfalfa hay (0.5 g) were filled with 60 mL of inoculant and then incubated on a shaker (39°C) for 0, 3, 6, 12, 24, and 48 h. Triplicate vials were removed at each time point to measure gas production, methane emission, and alfalfa digestibility. Gas production (ml/g dry matter (DM)) at 48 h was not different between BsubE, BcoaE, or ENZY (P> 0.05); however, it was reduced in CON vials compared with the other treatments (P< 0.001). Methane emissions at 24 and 48 h (ml/g DM) were least for CON (25.2 and 29.8 mL/g, respectively), intermediate for the spore treatments (25.8 and 30.5 g/ml for BsubE; 25.9 and 30.7 g/ml for BcoaE), and greatest for ENZY (35.4 and 39.0 mL/g, respectively; P= 0.011). In vitro DM digestibility was not different at 24 h (P= 0.36), but at 48 h there was a difference between CON (78.9%) and BsubE, BcoaE and ENZY (average 80%; P= 0.018). There was a tendency for greater NDF digestibility at 48 h in the enzyme treatments, compared with CON (P= 0.075). These data showed that the feed enzyme enhanced digestion of alfalfa. However, there was no difference when the enzyme was applied in free-form or bound to spores. Protection of feed enzymes through absorption to Bacillusspores may be more effective when the enzymes are unstable in a ruminal environment.