Bacterial diversity and composition of alfalfa silage as analyzed by Illumina MiSeq sequencing: Effects of Escherichia coli O157:H7 and silage additives.

The first objective of this study was to examine effects of adding Escherichia coli O157:H7 with or without chemical or microbial additives on the bacterial diversity and composition of alfalfa silage. The second objective was to examine associations between the relative abundance of known and unknown bacterial species and indices of silage fermentation quality. Alfalfa forage was harvested at 54% dry matter, chopped to a theoretical length of cut of 19 mm, and ensiled in quadruplicate in laboratory silos for 100 d after the following treatments were applied: (1) distilled water (control); (2) 1 × 105 cfu/g of E. coli O157:H7 (EC); (3) EC and 1 × 106 cfu/g of Lactobacillus plantarum (EC+LP); (4) EC and 1 × 106 cfu/g of Lactobacillus buchneri (EC+LB); and (5) EC and 0.22% propionic acid (EC+PA). After 100 d of ensiling, the silage samples were analyzed for bacterial diversity and composition via the Illumina MiSeq platform (Illumina Inc., San Diego, CA) and chemically characterized. Overall, Firmicutes (74.1 ± 4.86%) was the most predominant phylum followed by Proteobacteria (20.4 ± 3.80%). Relative to the control, adding E. coli O157:H7 alone at ensiling did not affect bacterial diversity or composition but adding EC+LP or EC+LB reduced the Shannon index, a measure of diversity (3.21 vs. 2.63 or 2.80, respectively). The relative abundance of Firmicutes (69.2 and 68.8%) was reduced, whereas that of Proteobacteria (24.0 and 24.9%) was increased by EC+LP and EC+PA treatments, relative to those of the control (79.5 and 16.5%) and EC+LB (77.4 and 18.5%) silages, respectively. Compared with the control, treatment with EC+LP increased the relative abundance of Lactobacillus, Sphingomonas, Pantoea, Pseudomonas, and Erwinia by 426, 157, 200, 194, and 163%, respectively, but reduced those of Pediococcus, Weissella, and Methylobacterium by 5,436, 763, and 250%, respectively. Relative abundance of Weissella (9.19%) and Methylobacterium (0.94%) were also reduced in the EC+LB silage compared with the control (29.7 and 1.50%, respectively). Application of propionic acid did not affect the relative abundance of Lactobacillus, Weissella, or Pediococcus. Lactate concentration correlated positively (r = 0.56) with relative abundance of Lactobacillus and negatively (r = -0.41) with relative abundance of Pediococcus. Negative correlations were detected between ammonia-N concentration and relative abundance of Sphingomonas (r = -0.51), Pantoea (r = -0.46), Pseudomonas (r = -0.45), and Stenotrophomonas (r = -0.38). Silage pH was negatively correlated with relative abundance of Lactobacillus (r = -0.59), Sphingomonas (r = -0.66), Pantoea (r = -0.69), Pseudomonas (r = -0.69), and Stenotrophomonas (r = -0.50). Future studies should aim to speciate, culture, and determine the functions of the unknown bacteria detected in this study to elucidate their roles in silage fermentation.

Fate of Escherichia coli O157:H7 and bacterial diversity in corn silage contaminated with the pathogen and treated with chemical or microbial additives.

Inhibiting the growth of Escherichia coli O157:H7 (EC) in feeds may prevent the transmission or cycling of the pathogen on farms. The first objective of this study was to examine if addition of propionic acid or microbial inoculants would inhibit the growth of EC during ensiling, at silo opening, or after aerobic exposure. The second objective was to examine how additives affected the bacterial community composition in corn silage. Corn forage was harvested at approximately 35% dry matter, chopped to a theoretical length of cut of 10 mm, and ensiled after treatment with one of the following: (1) distilled water (control); (2) 1 × 105 cfu/g of EC (ECCH); (3) EC and 1 × 106 cfu/g of Lactobacillus plantarum (ECLP); (4) EC and 1 × 106 cfu/g of Lactobacillus buchneri (ECLB); and (5) EC and 2.2 g/kg (fresh weight basis) of propionic acid, containing 99.5% of the acid (ECA). Each treatment was ensiled in quadruplicate in laboratory silos for 0, 3, 7, and 120 d and analyzed for EC, pH, and organic acids. Samples from d 0 and 120 were also analyzed for chemical composition. Furthermore, samples from d 120 were analyzed for ammonia N, yeasts and molds, lactic acid bacteria, bacterial community composition, and aerobic stability. The pH of silages from all treatments decreased below 4 within 3 d of ensiling. Escherichia coli O157:H7 counts were below the detection limit in all silages after 7 d of ensiling. Treatment with L. buchneri and propionic acid resulted in fewer yeasts and greater aerobic stability compared with control, ECCH, and ECLP silages. Compared with the control, the diversity analysis revealed a less diverse bacterial community in the ECLP silage and greater abundance of Lactobacillus in the ECLP and ECA silages. The ECLB silage also contained greater abundance of Acinetobacter and Weissella than other silages. Subsamples of silages were reinoculated with 5 × 105 cfu/g of EC either immediately after silo opening or after 168 h of aerobic exposure, and EC were enumerated after 6 or 24 h, respectively. All silages reinoculated with EC immediately after silo opening (120 h) had similar low pH values (<4.0) and EC counts were below the detection limit. The ECCH and ECLP silages reinoculated with EC after 168 h of aerobic exposure had relatively high pH values (>5.0) and EC counts (5.39 and 5.30 log cfu/g, respectively) 24 h later. However, those treated with L. buchneri or propionic acid had lower pH values (4.24 or 3.96, respectively) and lower EC counts (1.32 log cfu/g or none, respectively). During ensiling, EC was eliminated from all silages at pH below 4.0. During aerobic exposure, the growth of EC was reduced or prevented in silages that had been treated with L. buchneri or propionic acid at ensiling, respectively.

An in vitro model to study interactions between Escherichia coli and lactic acid bacterial inoculants for silage in rumen fluid.

UNLABELLED: Previous studies have shown that silages treated with lactic acid bacteria (LAB) inoculants enhance ruminants' performance. The objective of the current experiments was to develop an in vitro model to study interactions between LAB silage inoculants and inoculated silages and Escherichia coli (EC) in rumen fluid (RF). Our hypothesis was that some inoculants inhibit EC in RF. For that purpose buffered RF was incubated under anaerobic conditions at 39°C with commercial strains of LAB silage inoculants or with laboratory corn and wheat silages treated with these LAB, an EC strain and with various ruminant feed ingredients. The EC strain was originally isolated from cattle manure and tagged with a plasmid expressing the green fluorescence protein and kanamycin and streptomycin resistance. Results indicate that the LAB or the treated silages did not suppress EC numbers in the RF. When the pH of the RF decreased below 5·0 the EC disappeared. We conclude that both LAB inoculants for silage and EC survived in RF for several days; however, the inoculants and silages treated with such inoculants did not inhibit EC in RF in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: Forage crops, silage and hay are initial stages of the food chain for humans. Cattle harbours and sheds enterobacteria regularly, some strains of which are pathogens. These can contaminate forage crops through field fertilization with cattle manure. The objective of this study was to develop an in vitro model to test whether lactic acid bacteria, which are used in silage inoculants, alone or in treated silages can inhibit Escherichia coli in rumen fluid. This study presents safety aspects and it is also part of a broad research effort aimed at finding out how LAB silage inoculants and inoculated silages enhance ruminant performance or exert probiotic effects in ruminants.

Control of Escherichia coli O157:H7 in contaminated alfalfa silage: Effects of silage additives.

This study was conducted to examine if adding microbial inoculants or propionic acid to alfalfa silages contaminated with Escherichia coli O157:H7 would inhibit the growth of the pathogen during or after ensiling. Alfalfa forage was harvested at the early bloom stage, wilted to a dry matter concentration of 54%, chopped to 19-mm lengths, and ensiled after treatment with one of the following: (1) distilled water (control); (2) 1×10(5) cfu/g of E. coli O157:H7 (EC); (3) EC and 1×10(6) cfu/g of Lactobacillus plantarum (EC+LP); (4) EC and 1×10(6) cfu/g of Lactobacillus buchneri (EC+LB); and (5) EC and 2.2g/kg of propionic acid (EC+PA). Each treatment was ensiled in quadruplicate in laboratory silos for 0, 3, 7, 16, and 100d and analyzed for EC counts, pH, and organic acids. In addition, samples from d 100 were analyzed for chemical composition, ammonia-N, counts of yeasts and molds, and aerobic stability. Escherichia coli O157:H7 was detected in all silages until d 7, but by d 16 it was not detected in those treated with EC+LB and EC+LP, though it was still detected in EC and EC+PA silages. However, by d 100, the pathogen was not detected in any silage. The rate of pH decrease to 5.0 was fastest for the EC+LP silage (7d), followed by the EC+LB silage (16d). Nevertheless, all silages had attained a pH of or less than 5.0 by d 100. The rapid decrease in pH in EC+LP and EC+LB silages was observed due to higher lactate and acetate concentrations, respectively, relative to the other silages during the early fermentation phase (d 3-16). Propionic acid was only detected in the EC+PA silage. Yeast counts were lowest in EC+LB and EC+PA silages. Subsamples of all d-100 silages were reinoculated with 1×10(5) cfu/g of EC immediately after silo opening. When the pathogen was subsequently enumerated after 168h of aerobic exposure, it was not detected in silages treated with EC+PA, EC+LB, or EC+LP, which all had pH values less than 5.0. Whereas the EC silage had a pH value of 5.4 and 2.3 log cfu/g of the pathogen. Certain bacterial inoculants can hasten the inhibition of E. coli O157:H7 during ensiling, such as propionic acid, and they can also prevent its growth on silage contaminated with the pathogen after ensiling.

The effect of relocation of whole-crop wheat and corn silages on their quality.

Whole-crop wheat and corn silages in 1.5-L anaerobic jars were exposed to air for 0 up to 48 h during their anaerobic storage period to simulate relocation of silages. Ensiling treatments included control (no additives) and either Koffosil T (Koffolk Inc., Petah Tikva, Israel) comprising a mixture of organic acids or Lactobacillus plantarum MTD1 (Ecosyl Products Ltd., Stokesley, UK). In the first set of experiments, the duration of exposure to air had little effect on ensiling parameters or on the aerobic stability of the final silages. In the second set of experiments, both the inoculant and duration of exposure to air had an effect on various fermentation parameters and on the aerobic stability of the final silages. We concluded that if the silages are of good quality, the duration of the relocation process has little effect on silage quality or its aerobic stability. However, if the silage contains any factor that may affect its aerobic stability, it is more sensitive to the time it takes to re-ensile the forage.

Novel use of the wild species Cephalaria joppensis for silage preparation and its nutritive value for feeding lactating dairy cows.

This study presents a novel method for use of the wild plant species Cephalaria joppensis (CJ) as agricultural forage for ruminants. Domesticated CJ tends to have higher crop mass yield per hectare than a commercial wheat variety (W) but is similar in in vitro dry matter (DM) digestibility. This study was composed of 3 experiments. Experiment 1 aimed to measure effects of ensiling CJ versus W in packed polyethylene-wrapped bales. Three types of ensiled bales were produced for each plant: 1) direct-cut CJ versus W packed solely; 2) direct-cut CJ versus W mixed as sole roughage source together with dietary ingredient and packed in bales to create CJ total mixed ration (CJ-TMR) or W-TMR; 3) CJ silage versus W silage mixed as one-third of dietary roughage source together with two-thirds sorghum (S) silage and additional dietary ingredients and packed in bales to create CJ-S-TMR or W-S-TMR. Data showed that packing and wrapping created anaerobic conditions within the 4 types of TMR bales while reducing pH (4.12 to 4.37). Dry matter loss during ensilage was higher for the 2 types of TMR containing W compared with CJ. Ensilage decreased soluble nitrate content as well as yeast and mold contamination, and the 4 types of TMR bales were characterized by a long outdoor shelf life (3 mo) and high stability under aerobic exposure. Experiment 2 aimed to measure the intake and digestibility by sheep of the 4 types of packed TMR after 90 d of ensiling. Data demonstrated higher voluntary intake of the CJ-TMR compared with the other TMR types. The CJ-TMR was characterized by higher digestibility of DM, crude protein, and neutral detergent fiber components compared with the CJ-S-TMR. Experiment 3 examined intake, digestibility, and milk production by 21 pairs of lactating cows individually fed CJ-S-TMR versus W-S-TMR. Similar intake (21.6 to 22.0 kg/d) and digestibility of DM and crude protein were observed in cows fed the 2 TMR types (68 to 69% and 66 to 68%, respectively). However, neutral detergent fiber and cellulose digestibility were slightly higher in the cows fed W-S-TMR and this was reflected in a small increase in their milk and energy-corrected milk yield (36.5 and 31.4 kg/cow per day, respectively) compared with cows fed CJ-S-TMR (35.5 and 30.4 kg/cow per day, respectively). Results demonstrate that direct-cut CJ used as is, or CJ silage can be included and ensiled in TMR bales for feeding productive ruminants as a substitute for wheat silage.

The quality of commercial wheat silages in Israel.

Wheat silages are the major roughage for high-producing lactating dairy cows in Israel; therefore, their quality is important. The main objective of the current study was to determine the preservation status and nutritional quality of commercial wheat silages in Israel. An additional objective was to develop predictive equations for dry matter digestibility (DMD) and neutral detergent fiber digestibility (NDFD) based on chemical composition of the silages, which would permit estimation of the digestibility from chemical composition. A total of 143 commercial wheat silages were sampled and analyzed for 3 yr. Fourteen random samples that were not included in the regression analysis were used to validate the equations by the bias and error of the model. Results revealed that wheat silages were quite sensitive to aerobic exposure; additives resulted in some improvement of the aerobic stability. After choosing the significant terms from ash, crude protein (CP), NDF, acid detergent fiber (ADF), and acid detergent lignin (ADL) by PROC STEPWISE of SAS, the following prediction equations were obtained from all 143 samples: DMD = 86.3 + (0. 70 x CP) - (0.46 x ADF) - (1.67 x ADL); and NDFD = 20.3 + (1.00 x CP) + (1.16 x NDF) - (0.88 x ADF) - (2.25 x ADL). The bias and the error of the prediction model for DMD were approximately 0.006 and 0.065, respectively; the bias and error for NDFD were approximately 0.007 and 0.118, respectively. It was concluded that the prediction model for DMD was quite adequate, whereas that for NDFD was less acceptable.

Ensiling olive cake with and without molasses for ruminant feeding.

The purpose of the current experiment was to study the ensiling properties of olive cake with and without added molasses at 2-6% (w/w). The results indicate that molasses enhanced the ensiling fermentation of olive cake, as evidenced from higher lactic acid content and higher lactic acid bacteria numbers at 4% and 6% added molasses. However, when applied at 4% and 6% molasses increased fermentation losses up to 9.4%, probably due to larger yeast population. Polyphenols which could interfere with protein utilization by ruminants, decreased during ensiling by about 40%. It is concluded that molasses added at 3% could improve the ensiling fermentation of olive cake without substantial losses.

Effect of lactic acid bacteria inoculants on in vitro digestibility of wheat and corn silages.

The aim of the study was to determine the effect of 10 sources of lactic acid bacteria (LAB) on dry matter digestibility (DM-D) and neutral detergent fiber digestibility (NDF-D), in various combinations with starch, in vitro. The soluble starch represented a concentrate feed, whereas silage represented feeding only roughage. The DM-D and NDF-D were determined after 24 and 48 h of incubation to represent effective (24 h) and potential (48 h) digestibility. Addition of LAB was both by direct application of the inoculants to rumen fluid (directly fed microbials) and by the use of preinoculated silages. For each feed combination, tubes without added LAB served as controls. The results indicate that, overall, some LAB inoculants applied at ensiling or added directly to the rumen fluid had the potential to increase the DM-D and NDF-D. The major significant inoculant effect on NDF-D was obtained after 24 h of incubation, whereas the effect after 48 h was mainly nonsignificant. The effective inoculants seemed to minimize the inhibitory effect of the starch on NDF-D within 24 h, perhaps by competition with lactate-producing rumen microorganisms.

Enhancement of polyphenol recovery from rosemary (Rosmarinus officinalis) and sage (Salvia officinalis) by enzyme-assisted ensiling (ENLAC).

The efficacy of enzyme-assisted ensiling (ENLAC) in the recovery of polyphenols from rosemary and sage was tested. Fresh rosemary and sage were chopped and ensiled in 0.5-L anaerobic jars. Treatments comprised control (no additives), 0.5% glucose and lactic acid bacteria, and 1% cellulase plus 1% hemicellulase plus pectinase. Following storage at room temperature for 45 days (experiment 1) and 26 days (experiment 2), polyphenols were extracted from the silages in ethanol either by direct blending or by cold extraction. The enzyme treatment resulted in silages with the lowest pH values, lowest fiber content, highest water-soluble sugar content, and highest polyphenol recovery; this treatment resulted in increased polyphenol recovery from rosemary and sage, by 100 and 20%, respectively. Comparison between direct blending and cold extraction revealed similar efficiency of polyphenol recovery.

Changes in essential oil during enzyme-assisted ensiling of lemongrass (Cymbopogon citratus Stapf.) and lemon eucalyptus (Eucalyptus citriodora Hook).

Changes in essential oil during ensiling of lemongrass and lemon eucalyptus were studied. Wilted lemongrass and eucalyptus leaves were ensiled in 0.25-L anaerobic jars. Samples consisted of a control (no additives) and a treated sample (0.5% glucose and lactic acid bacteria and 1% cellulase plus 1% hemicellulase plus pectinase). Three jars per treatment were sampled on days 2, 6, 10, and 36 for analysis of essential oil. Essential oil was obtained by extraction and by hydrodistillation. Extraction efficacy of essential oil from the lemongrass was improved by the enzyme treatment, but it was much lower than the amount obtained by distillation. The major components of the essential oil were neral and geranial. In the eucalyptus, total essential oils obtained by distillation decreased during ensiling, and the amount was similar to the amount obtained by extraction. Citronellal, which was the major component of the essential oil in the fresh eucalyptus leaves, decreased, whereas isopulegol and 3,8-terpinolhydrate increased during ensiling.

Inhibition of Escherichia coli in cultivated cattle manure.

A common practice on Israeli dairy barns comprises daily cultivation of the manure. Cultivation is a mechanical process used to break up and till the manure bedding and it results in a drier and aerated bedding and cleaner cows, which consequently reduces the incidence of mastitis. Cultivation was associated with a shorter survival of Escherichia coli in cultivated manure as compared with noncultivated manure. The objective of the current study was to elucidate the mechanism responsible for the shorter survival duration of E. coli in the cultivated manure. We hypothesized that microorganisms that are antagonistic to E. coli, developing in the cultivated manure, are responsible for this phenomenon. A cow manure derived E. coli strain expressing the green fluorescence protein and antibiotic resistance markers was used to inoculate cow manure in 1.5-L jars. Manure treatments included cultivated and noncultivated manure. Half the jars of each cultivation treatment were autoclave sterilized at 121°C for 1 h on 3 successive days to eliminate from the manure antagonistic microorganisms. Each cultivation-sterilization treatment was performed in triplicate jars. Following sterilization, E. coli numbers in the cultivated and noncultivated manure were comparable, while in the nonsterilized manure the numbers were lower in the cultivated compared with the noncultivated manure. Several fungi isolated from the cultivated manure samples displayed inhibition effect on the tagged E. coli. Antagonistic fungi were also isolated from large-scale cultivated manure samples collected on several dairy farms in Israel. These findings support the notion that manure cultivation might facilitate the development of microorganisms that are antagonistic to E. coli, thus contributing to the general hygiene of the cattle. Identifying the mechanisms by which the antagonistic fungi affect the survival of E. coli in manure could be exploited for improvement of the animal health and for limiting the transmission of zoonotic pathogens to food and water.

Fate of inoculated Escherichia coli in hay.

AIMS: To monitor the fate of inoculated Escherichia coli in dry and moist hay of various types, under laboratory conditions. METHODS AND RESULTS: Wheat, vetch and clover hay were used as received or wetted to 250-300 g kg(-1) moisture. The hay was inoculated at about 10(6) CFU g(-1) with a kanamycin-resistant E. coli strain that expresses the green fluorescence protein, and was stored in small open glass jars that were covered with aluminium foil. Three jars per treatment were sampled on days 1 and 3, or 4 and 7, or 8, 20 and 50, respectively, after the initiation of the experiments, and the numbers of E. coli in the hay were determined. The results indicated that E. coli disappeared from both dry and moist hay by 7-8 days after inoculation. However, in a few cases colonies that were presumed to be E. coli developed after incubation in Luria broth medium. CONCLUSIONS: The tagged E. coli strain usually disappeared rapidly from both the dry and the moist hay, in spite of the high level of inocula used. However, in some cases a few, possibly injured E. coli might have persisted, and could be detected after incubation in a rich growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is part of a risk assessment associated with sewage irrigation of forage crops in Israel. The results indicate that E. coli added to the hay is not likely to pose a health risk to cattle or to humans. Nevertheless, more research with natural strains of E. coli and other enteric pathogens that might be more adapted to forage conditions is warranted in order to ensure the safety of sewage-irrigated crops.

Antibacterial activity of lactic acid bacteria included in inoculants for silage and in silages treated with these inoculants.

AIMS: To determine antibacterial activity in lactic acid bacteria (LAB) silage inoculants and in wheat and corn silages which were treated with these inoculants. METHODS AND RESULTS: Wheat and two corn silages were prepared in 0.25 l sealed glass jars. Inoculant treatments were prepared for each type of silage with each of 10 LAB silage inoculants at inoculation rate of 10(6) CFU g(-1). Untreated silages served as controls. Antibacterial activity was determined in the inoculants and in their respective silages with Micrococcus luteus and Pseudomonas aeruginosa. Antibacterial activity was detected in nine of the 10 inoculants whereas such activity in the silages varied. Control silages did not have antibacterial activity. CONCLUSIONS: Many LAB silage inoculants have antibacterial activity and in some cases this activity is imparted on inoculated silages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was conducted as part of a broader research objective, which is to find out how LAB silage inoculants enhance ruminant performance. The results of this study indicate that LAB silage inoculants produce antibacterial activity, and therefore, have a potential to inhibit detrimental micro-organisms in the silage or in the rumen.

Fate of Escherichia coli during ensiling of wheat and corn.

A recombinant Escherichia coli strain carrying a plasmid with an antibiotic resistance marker and expressing the green fluorescent protein was inoculated at a concentration of 3.8 x 10(8) CFU/g into direct-cut wheat (348 g of dry matter kg(-1)), wilted wheat (450 g of dry matter kg(-1)), and corn (375 g of dry matter kg(-1)). The forages were ensiled in mini-silos. The treatments included control (no E. coli added), application of tagged E. coli, and delayed sealing of the inoculated wheat. Three silos per treatment were sampled on predetermined dates, and the numbers of E. coli were determined on Chromocult TBX medium with or without kanamycin. Colonies presumptively identified as E. coli were also tested for fluorescence activity. Addition of E. coli at the time of ensiling resulted in a more rapid decrease in the pH but had almost no effect on the chemical composition of the final silages or their aerobic stability. E. coli disappeared from the silages when the pH decreased below 5.0. It persisted longer in silages of wilted wheat, in which the pH declined more slowly. Control silages of all crops also contained bacteria, presumptively identified as E. coli, that were resistant to the antibiotic, which suggests that some epiphytic strains are naturally resistant to antibiotics.

The effects of temperature on the aerobic stability of wheat and corn silages.

The aim of this work was to study the effects of temperature on the aerobic stability of wheat and corn silages. Three silage samples from each crop were taken from the faces of six different commercial bunker silos immediately after unloading them. The samples were exposed to air for 3 or 6 days at 10, 20, 30 or 40 degrees C. The most intensive deterioration occurred at 30 degrees C. Samples incubated at 30 degrees C had the highest yeast counts, most prolific CO(2) production and greatest increases in pH. Silage samples exposed to 10 or 40 degrees C remained stable. The duration of exposure had a significant effect on aerobic stability, especially at 30 degrees C. Temperature has a significant effect on silage aerobic stability. In a warm climate, special care should be taken during unloading of silage in order to prevent intensive aerobic deterioration.

The passage of lactic acid bacteria from silage into rumen fluid, in vitro studies.

Inoculated silages sometimes improve cattle performance, possibly because of probiotic effects of lactic acid bacteria (LAB) silage inoculants. The cause of improved animal performance following feeding with inoculated silage is unclear. One issue in studying this phenomenon is to find out whether LAB pass from silage into the rumen fluid and survive in it. The purpose of the present study was to determine whether LAB from inoculated and uninoculated silages pass into the rumen fluid in vitro. Wheat and corn silages, uninoculated or inoculated with 1 of 10 commercial silage inoculant LAB, were prepared in glass jars. After ensiling, a 2.5-g silage sample was added to 25 mL of heat-sterilized or strained rumen fluid together with 5 g/L glucose, and incubated for 48 h at 39 degrees C. Analysis of the incubated rumen fluid included pH measurement, enumeration of LAB, and determination of lactic acid and volatile fatty acids (VFA). The pH of the rumen fluid decreased during incubation; both heat-sterilized and strained rumen fluid contained large numbers of LAB. The heat-sterilized rumen fluid contained lactic acid in addition to VFA, whereas the strained rumen fluid contained only VFA. The results indicate that LAB pass from silage samples into the rumen fluid in vitro and survive there. Their interactions with rumen microorganisms should be studied further to understand how some silage inoculant LAB exhibit probiotic effects in dairy cattle.

The survival of silage inoculant lactic acid bacteria in rumen fluid.

AIMS: To determine whether lactic acid bacteria (LAB) used in inoculants for silage can survive in rumen fluid (RF), and to identify those that survive best. METHODS AND RESULTS: Twelve commercial silage inoculants were added at 107 CFU ml-1 to strained RF (SRF) taken from dairy cows, with and without 5 g l-1 glucose and incubated in vitro at 39 degrees C. Changes in pH, LAB numbers and fermentation products were monitored for 72 h. In the inoculated RF with glucose, the pH decreased and numbers of LAB increased. The inoculants varied with regard to their effect on pH change and growth. In the SRF, both with and without glucose, the pH values of the inoculated samples were generally higher than those of the uninoculated controls throughout most of the incubation period. This may suggest a positive effect on the rumen environment. CONCLUSIONS: LAB used in silage inoculants can survive in RF in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first step in studying the probiotic potential of silage LAB inoculants for dairy cattle. The survival of these LAB in RF may enable them to interact with rumen microorganisms and to affect rumen functionality.

Stabilization of returned dairy products by ensiling with straw and molasses for animal feeding.

Returned dairy products which are transferred to landfills might add to the environmental pollution. Such products have a high nutritional value for ruminants, but they should be stabilized to enable their use as cattle feed. The purpose of the current study was to examine stabilization of returned dairy products by ensiling in combinations with straw and molasses for animal feeding. Treatments included combinations of milk and cottage cheese with straw and molasses. Results indicate that such products ensile well with straw, and after 3 d of ensiling the pH decreased to around 4.0. It was necessary to supplement cottage cheese with molasses, to supply a carbohydrate source for the lactic acid fermentation. The major fermentation product was lactic acid. Percentage of ammonia N (of total N) was generally higher in the silages made with cottage cheese than in those made with milk; the highest percentage (16%) was measured in the second experiment in the silages prepared with cottage cheese and straw. The study indicates the potential of stabilizing returned dairy products for animal feeding along with straw and molasses. There may also be potential for large dairy farms, or groups of smaller farms, to ensile waste milk with straw for later use as feed.

The effect of temperature on the ensiling process of corn and wheat.

AIMS: The purpose of this work was to study the effect of temperature on the ensiling process and aerobic stability of corn and wheat silages. METHODS AND RESULTS: The crops were ensiled in 1.5 l anaerobic jars, with and without an inoculant, at room or elevated temperatures (37-41 degrees C). After two months of ensiling, the silages were subjected to an aerobic stability test at room and elevated (33 degrees C) temperature. The results indicate that ensiling at elevated temperatures resulted in higher pH values, less lactic acid and higher losses. The silages which were stored at elevated temperatures were more susceptible to aerobic spoilage than those stored at room temperature, especially when the test was performed at elevated temperature. CONCLUSION: High temperatures are detrimental to both the ensiling process and the aerobic stability of silages. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of the current study suggest that in a warm climate, special care should be taken during silage making and storage in order to avoid heating as much as possible. In addition, in a warm climate, silages are more susceptible to aerobic deterioration and therefore, special care should be taken during unloading.