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A meta-analysis of 158 peer-reviewed articles was conducted to examine effects of inoculation with Lactobacillus buchneri (LB)-based inoculants (LBB) that did or did not include homolactic or obligate heterolactic bacteria on silage fermentation and aerobic stability. A complementary meta-analysis of 12 articles examined LBB inoculation effects on dairy cow performance. Raw mean differences between inoculant and control treatment means weighted by inverse variance were compared with a hierarchical effects model that included robust variance estimation. Meta-regression and subgrouping analysis were used to identify effects of covariates including forage type, application rate (≤104, 105, 106, or ≥ 107 cfu/g as fed), bacteria type (LB vs. LB plus other bacteria), enzyme inclusion, ensiling duration, and silo type (laboratory or farm scale). Inoculation with LBB increased acetate (62%), 1, 2 propanediol (364%) and propionate (30%) concentration and aerobic stability (73.8%) and reduced lactate concentration (7.2%), yeast counts (7-fold) and mold counts (3-fold). Feeding inoculated silage did not affect milk yield, dry matter intake, and feed efficiency in lactating dairy cows. However, forage type, inoculant composition, and dose effects on silage quality measures were evident. Inoculation with LBB increased aerobic stability of all silages except tropical grasses. Adding obligate homolactic or facultative heterolactic bacteria to LB prevented the small increase in DM losses caused by LB alone. The 105 and 106 cfu/g rates were most effective at minimizing DM losses while aerobic stability was only increased with 105, 106, and ≥ 107 cfu/g rates. Inoculation with LBB increased acetate concentration, reduced yeast counts and improved aerobic stability but did not improve dairy cow performance.

Dairy farmers are often challenged with the need to feed high-moisture corn (HMC) after less than 30 d of fermentation. The objective this study was to assess the effects of microbial inoculation and particle size on fermentation profile, aerobic stability, and ruminal in situ starch degradation of HMC ensiled for a short period. High-moisture corn was harvested, coarsely ground (3,798 ± 40 µm, on average) or finely ground (984 ± 42 µm, on average), then ensiled in quadruplicate vacuum pouches untreated (CON) or with the following treatments: Lactobacillus plantarum CH6072 at 5 × 104 cfu/g and Enterococcus faecium CH212 at 5 × 104 cfu/g of fresh forage (LPEF); or Lactobacillus buchneri LB1819 at 7.5 × 104 cfu/g and Lactococcus lactis O224 at 7.5 × 104 cfu/g (LBLL). Silos were allowed to ferment for 14 or 28 d. Ruminal in situ starch degradation increased when HMC was finely ground. In addition, in situ starch degradation was greater and aerobic stability increased approximately 5-fold with LBLL compared with CON and LPEF. An interaction between microbial inoculation and storage length occurred for lactic acid. At 14 d, concentrations of lactic acid were greatest in LPEF and lowest in LBLL. Lactic acid concentrations increased from 14 to 28 d with CON and LPEF, but decreased with LBLL. At 28 d, concentrations of lactic acid were lower in LBLL compared with CON and LPEF. An interaction between particle size, microbial inoculation, and storage length occurred for acetic acid and ammonia-N. At 14 and 28 d, acetic acid concentrations were greatest in finely ground LBLL followed by coarsely ground LBLL. Ammonia-N concentrations increased across all treatments from 0 to 28 d. At 14 and 28 d, concentrations of ammonia-N were greatest in finely ground LBLL and lowest in coarsely ground CON and coarsely ground LPEF. Results from this study suggest that L. buchneri LB1819 can produce acetic acid in as little as 14 d, and that by 28 d, it has the potential to improve the aerobic stability of HMC. Additionally, results indicate that L. buchneri LB1819 has the potential to improve ruminal degradation of starch by 28 d of storage. Finally, results confirm enhanced fermentation and improved ruminal starch degradation with finely ground HMC by 28 d of storage.

Few characteristics are more important to a bacterium than the substrates it consumes. It is hard to identify what substrates are consumed by bacteria in natural communities, however, because most bacteria have not been cultured. In this study, we developed a method that uses fluorescent substrate analogs, cell sorting, and DNA sequencing to identify substrates taken up by bacteria. We deployed this method using 2[N-(7-nitrobenz-2-oxa-1,2-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), a fluorescent glucose analog, and bacteria of the bovine rumen. This method revealed over 40 different bacteria (amplicon sequence variants [ASVs]) from the rumen that take up glucose. Nearly half of these ASVs represent previously uncultured bacteria. We attempted to grow these ASVs on agar media, and we confirmed that nearly two-thirds resisted culture. In coculture experiments, the fluorescent label of 2-NBDG was not transferred to nontarget bacteria by cross-feeding. Because it is not affected by cross-feeding, our method has an advantage over stable isotope probing. Though we focus on glucose, many substrates can be labeled with the fluorophore NBD. Our method represents a new paradigm for identifying substrates used by uncultured bacteria. It will help delineate the niche of bacteria in their environment. IMPORTANCE We introduce a method for identifying what substrates are consumed by bacteria in natural communities. Our method offers significant improvement over existing methods for studying this characteristic. Our method uses a fluorescently labeled substrate which clearly labels target bacteria (glucose consumers in our case). Previous methods use isotope-labeled substrates, which are notorious for off-target labeling (due to cross-feeding of labeled metabolites). Our method can be deployed with a variety of substrates and microbial communities. It represents a major advance in connecting bacteria to the substrates they take up.

Increasing the length of storage of whole-plant corn silage (WPCS) has been shown to increase ruminal in vitro starch digestibility by facilitating hydrolysis of the protein matrix surrounding starch granules. It is possible that microbial inoculants could improve fermentation, thereby enhancing proteolytic activity in the silo. Additionally, microbial inoculants have potential to reduce or prevent the growth of toxigenic fungi in silage. Therefore, the objective of this study was to determine the effects of storage length and microbial inoculation with heterofermentative and homofermentative inoculants containing Enterococcus faecium on the fermentation profile, N fractions, and ruminal in situ starch disappearance of whole-plant corn silage, as well as the effect of microbial inoculation on silage mycotoxin concentrations. Whole-plant corn (333 ± 10 g of DM/kg as-fed) was ensiled in quintuplicate vacuum pouches untreated (CON) or after the following treatments: E. faecium at 1.5 × 105 cfu/g (EF); Lactobacillus plantarum at 1 × 105 and E. faecium at 5 × 104 cfu/g of fresh forage (LPEF); and L. buchneri and Lactococcus lactis at 1.5 × 105 cfu/g (LBLL). Silos were stored for 0, 30, 60, 90 or 120 d. Silage pH was greater with LBLL compared to the other three treatments (P = 0.005). Total acids were greater with LPEF than EF (P = 0.005). Ammonia-N (expressed as g/kg of N) was greatest with CON compared to the other treatments (P = 0.001). Concentrations of lactic acid were lower, and concentrations of acetic acid were greater with LBLL compared to the other treatments (P = 0.001). An interaction between microbial inoculation and storage length was observed for soluble CP concentrations as well as ruminal in situ starch disappearance (P = 0.001 and P = 0.012, respectively). Soluble CP (expressed as g/kg of CP) was greater with CON compared to the other treatments at d 30 and 90, but not different at d 60 and 120. Ruminal in situ starch disappearance was reduced for CON compared to the other three treatments at d 60 and 90. However, at d 120, ruminal in situ starch disappearance was similar across all treatments. Overall, the use of microbial inoculants improved fermentation profile. Microbial inoculation also increased starch disappearance in the earlier stages of fermentation but by 120 d of storage, starch disappearance was similar between inoculated silage and CON. Results from this study failed to support the hypothesis that microbial inoculants would reduce mycotoxin contamination.

In recent years, the interest to grow sorghum for silage has increased, with greater prominence in areas where water supply and or availability is an issue for plant growth. Therefore, the objective of this study was to evaluate the effects of sorghum cultivars (forage or sudan), storage length and microbial inoculation with heterofermentative and facultative homofermentative inoculants on the fermentation profile, DM loss, aerobic stability and ruminal in situ starch disappearance of whole-plant sorghum silage. Whole-plant sorghum (279 ± 37 g/kg DM for forage and 232 ± 29 g/kg DM for sudan) was ensiled in quintuplicate 20 L plastic buckets untreated (CON) or after the following treatments: Lactobacillus plantarum CH6072, L. plantarum LSI, and Pediococcus pentosaceus P6 at 1 × 105 cfu/g of fresh forage (LPPP); L. buchneri LB1819 and Lactococcus lactis O224 at 1.5 × 105 cfu/g (LBLL). Silos were allowed to ferment for 0, 15, 30 or 90 d. Lactic acid and total acids were greatest in sudan sorghum silage, and total acids were greatest in LPPP and lowest for LBLL. Ammonia-N (as g/kg of total N) was greatest with sudan sorghum. The acetic acid was greatest in forage sorghum inoculated with LBLL, intermediate in sudan sorghum inoculated with LBLL, and linearly increased in both cultivars with storage length from 15 to 90 d. Silage aerobic stability was greater for LBLL compared to CON and LPPP. Ruminal in situ starch disappearance was increased with ensiling time, and linearly increased from d 15–90. Microbial inoculation with L. buchneri and Lactococcus lactis increased the acetic acid concentrations, improved aerobic stability, and reduced the DM loss by discarded visible spoilage, at the expense of higher DM loss by gases. This effect was more evident in forage sorghum compared to sudan sorghum.

Sixteen strains of propionibacteria were inoculated into in vitro ruminal incubations to evaluate their potential to reduce methane (CH4) production from concentrate and forage diets. Propionibacte...

Objectives of this study were to compare fatty acid (FA) composition of ruminal bacterial (B) and protozoal (P) cells, and to investigate effect of protozoa on FA profile in the rumen of cattle. Three cows were used to prepare ruminal B and P cells. Four faunated and three defaunated cattle (half-siblings) were used to study effect of protozoa on ruminal FA profile. Proportions of C16:0 and C18:0 in total fatty acids in B cells were 20.7% and 37.4%, whereas those in P cells were 33.4% and 11.6%, respectively. Proportions of trans-vaccenic acid (VA) and cis-9, trans-11 conjugated linoleic acid (CLA) in B cells were 3.9% and 1.0%, and those in P cells were 5.5% and 1.6%, respectively, being higher in P cells. Proportions of C18:1, C18:2 and C18:3 in P cells were two to three times higher than in B cells. Proportions of unsaturated fatty acids, VA and CLA in B cells of faunated cattle were higher than those of defaunated. VA and CLA in the ruminal fluid of faunated were also 1.6 to 2.5 times higher than those of defaunated. This tendency was similar for cell-free fraction of ruminal fluid. These results indicate that protozoa contribute greatly in VA and CLA production in the rumen.

In vitro experiments were conducted to examine the synthesis of arginine (Arg) from argininosuccinic acid (ASA) and citrulline (Cit) by crude enzymes of cattle kidney cortex. Kidney samples, collected from Japanese black cattle, were homogenized in KCl solution (ice-cold), and centrifuged at 27 000 × g for 20 min at 4°C, and the supernatant fluid was used as a crude enzyme solution. The enzyme solution was incubated at 39°C in Tris HCl buffer with 15 mmol/L ASA or with 10 mmol/L Cit in the presence of 10 mmol/L aspartic acid (Asp), 10 mmol/L ATP and 5 mmol/L MgCl2 to examine the activities of two enzymes, argininosuccinate lyase and argininosuccinate synthetase, which work at the terminal steps of Arg biosynthesis. The production of Arg from ASA, or ASA and Arg from Cit by argininosuccinate lyase and argininosuccinate synthetase activities, respectively, were determined directly by the HPLC method. The optimum pH for argininosuccinate lyase activity was 7.85. Unfortunately, the optimum pH for argininosuccinate synthetase activity could not be determined because no inhibitor of argininosuccinate lyase was used in the Cit incubation, so the ASA produced from Cit spontaneously converted to Arg during incubation with Cit. The maximum production of ASA from Cit was found at pH 6.45 under these conditions. We observed the optimum pH for the synthesis of Arg from Cit at 7.7. The production of Arg from ASA or Cit was quantitatively determined as 14.4 or 8.83 µmol/g kidney tissue/h, respectively, at the optimal pH values. This suggests that the daily production of Arg from ASA or Cit by the kidney might be sufficient to cover the daily requirement of Arg in cattle.

Three segments of cattle small intestine (duodenum, upper jejuno-ileum and lower jejuno-ileum) were examined in an in vitro system for activity of ornithine carbamoyltransferase (OCT; EC 2.1.3.3) which is involved in the synthesis of citrulline (Cit) from ornithine (Orn). The mucosa of the three segments of small intestine was collected from Japanese black cattle, homogenized and then centrifuged. The supernatant fraction was used as the crude OCT enzyme solution. The OCT activity was assayed by the production of Cit from Orn determined directly by HPLC. The optimal pH and temperature for OCT activities in the duodenum, upper jejuno-ileum and lower jejuno-ileum of cattle small intestine were 7.47 and 39°C. Little difference was observed between the three segments. The OCT activity in cattle kidney was also examined for comparison, and almost no activity was found. The OCT activities in crude enzyme solutions of the three segments of small intestine were stable for up to one month of storage at −20°C in Tris HCl buffer solution. Finally, the role of the small intestine in supplying Cit as a precursor for arginine synthesis in cattle kidney was discussed.

A survey was conducted to investigate the physiological levels of pipecolic acid (Pip) in rumen fluid and plasma of ruminants such as goats and cattle in the presence or absence of rumen protozoa. The concentration of Pip was determined using HPLC. Basal Pip levels in the rumen fluid and plasma of normal faunated animals were 21 ± 8 and 2.3 ± 1.3 µM, respectively, and levels increased 1–2 h after feeding. The Pip levels in the rumen fluid and plasma of faunated goats and cattle were significantly higher than those of defaunated goats and unfaunated cattle. A small amount of Pip was also found in the rumen fluids of the defaunated and unfaunated animals; this appeared to be derived from feeds such as hay cube and corn silage. The results obtained in the present study suggest that a significant amount of rumen-produced Pip is likely to be absorbed into the plasma of the host animals and that rumen protozoa significantly enhance the concentration of Pip in the rumen fluid and plasma of ruminant animals.

The catabolism of arginine (Arg) by mixed rumen bacteria (B), mixed rumen protozoa (P), and their mixture (BP) was quantitatively investigated in an in vitro system in order to confirm the metabolic pathway of Arg and provide basic information for enzymatic and molecular studies as well as an understanding of the quantitative distribution of metabolites. Rumen microbial suspensions (B, P, and BP) collected from fistulated goats were anaerobically incubated with or without 1 mmol/L Arg at 39°C for 12 h. Arg and other related compounds such as citrulline (Cit), ornithine (Orn), proline (Pro) and 5-aminovaleric acid (5AV) in both supernatant and hydrolyzates of B, P, and BP suspensions were analyzed by HPLC. The metabolic pathways of Arg in mixed rumen bacteria and mixed rumen protozoa were considered to be as follows: rumen bacteria, Arg Cit Orn Pro 5AV VFAs + NH3; rumen protozoa, Arg Cit Orn Pro 5AV. The disappearance of Arg (1 mmol/L) was approximately 52.9 and 88.2% in B, 33.9 and 55.6% in P, and 52.8 and 85.2% in BP during 6 and 12 h incubations, respectively. When expressed in units of ‘per gram (g) of microbial nitrogen (MN)’, the net degradation rate of Arg in BP (50.3 µmol/g MN/h) was approximately 46% lower than that of B during a 12 h incubation period. The presence of protozoa tended to inhibit the production of Orn from Cit and the production of 5AV from Pro which were thought to be rate-limiting steps of Arg metabolism in rumen microorganisms. As a result, protozoa appeared to have a saving effect on Arg metabolism, that is, protozoa protected Arg from wasteful exhaustion in the rumen.

An in vitro study was conducted to quantitatively investigate the metabolism of pipecolic acid (Pip), a neuromodulator, by mixed rumen bacteria (B), mixed rumen protozoa (P), a combination of B and P (BP), species-enriched rumen protozoal suspension (Polyplastron sp., Diploplastron sp., entodinia and Entodinium caudatum) and pure cultures of several isolates of rumen bacteria (Prevetolla bryantii, Prevetolla albensis, Streptococcus bovis, Veillonella parvula, Megasphaera elsdenii and Ruminococcus albus). Only P produced Pip from L-lysine (1.0 mmol/L L-Lys) at a rate of 83.5 ± 1.6 µmol/L/h and even in BP, Pip was produced from L-Lys by P and increased at a rate of 31.2 ± 3.8 µmol/L/h. Pip production by P was highest when the substrate (L-Lys) concentration was 6 mmol/L and then the rate was 580 ± 36 µmol/L/h. Pipecolic acid production by P suspension enriched with different species of protozoa showed that Polyplastron sp. had the highest Pip production rate of 0.907 ± 0.092 µmol/L/mg protozoal protein per h, and Diploplastron sp. had the lowest rate of 0.55 ± 0.13 µmol/L/mg protozoal protein per h. The addition of D-Lys (1.0 mmol/L) as a substrate to the P suspension revealed that P were also able to produce Pip from D-Lys, though at a lower rate (1/3) compared with L-Lys (1.0 mmol/L), suggesting the presence of epimerases in P. It was confirmed that B were unable to produce Pip from L- or D-Lys. Only B degraded Pip (1.0 mmol/L) after a lag phase at a rate of 56.0 ± 1.5 µmol/L/h. The B suspension was able to degrade D-Lys, though the products were not identified. Pip degradation by pure culture of some species of rumen bacteria showed that P. bryantii and R. albus had the highest rate followed by P. albensis, S. bovis and M. elsdenii with a low rate of Pip degradation. Veillonella parvula showed no ability to degrade Pip. The results suggest that a fairly large proportion of rumen-produced Pip is likely to be absorbed by the host animal before degradation by rumen bacteria.

Biosynthesis of arginine (Arg) from citrulline (Cit), ornithine (Orn), proline (Pro), and 5-aminovaleric acid (5AV) by mixed rumen bacteria (B), protozoa (P), and their mixture (BP) was quantitatively investigated in an in vitro system from the standpoint of protein nutrition in ruminants. Rumen microorganisms, collected from ruminally fistulated goats, were anaerobically incubated with or without 1 mmol/L each of substrates at 39°C for 12 h. Arginine and other related compounds, produced in both supernatants and acid-hydrolyzates of microorganisms in B, P, and BP suspensions, were analyzed by high-performance liquid chromatography. Arginine production from Cit in BP, when expressed with a unit of ‘μmol/g microbial nitrogen’, was approximately 70% and 94% higher than that in B and P, respectively, in a 12-h incubation period. In the case of Orn, the values were approximately 30% and 75%. Both rumen bacteria and protozoa could produce Cit and Orn from Pro, so it is assumed that they can produce Arg from Pro. Rumen protozoa were unable to degrade 5AV and it was the final product in the metabolism of Cit, Orn and Pro in P suspension. A trace amount of Orn and Pro produced from 5AV in B and BP suspensions indicated that the reversible reaction of 5AV formation was performed only by rumen bacteria. This is the first quantitative report on Arg biosynthesis from its precursors by rumen microorganisms.

Poultry meat and eggs are valuable sources of dietary protein in almost every country in the world. A number of breeding techniques, methods, and technology have been applied to obtain maximum production under different environmental and economic conditions. Indigenous and local breeds share 90 % of the total population of poultry in many developing countries in Asia. However, indigenous chickens are low in productivity. Many studies have found that crossbreeding of exotic with indigenous chickens resulted in birds that performed better, even superior to pure exotic chickens, with respect to body weight, egg production, survivability, fertility, hatchability, and egg quality. There are some other technologies for efficient use of male genetic resource and conservation of rare genetic make-up, namely artificial insemination and chimeric chicken, respectively. It was reported that 25 % of the world's meat supply is derived from poultry, and the proportion is increasing rapidly. The continent of Asia produces almost one third of the world's eggs. However, there are still many scopes to improve the production of poultry in many developing countries in Asia. Therefore, continuous research works would be essential to determine the suitable technologies for more poultry production to feed the increasing habitants on earth.

Objective To investigate the prevalence and mechanisms of fluoroquinolone resistance in Shigella species isolated in Bangladesh and to compare with similar strains isolated in China. Methods A total of 3789 Shigella isolates collected from Clinical Microbiology Laboratory of icddr,b, during 2004–2010 were analyzed for antibiotic susceptibility. Analysis of plasmids, plasmid-mediated quinolone-resistance genes, PFGE, and sequencing of genes of the quinolone-resistance-determining regions (QRDR) were conducted in representative strains isolated in Bangladesh and compared with strains isolated in Zhengding, China. In addition, the role of efflux-pump was studied by using the efflux-pump inhibitor carbonyl cyanide-m-chlorophenylhydrazone (CCCP). Results Resistance to ciprofloxacin in Shigella species increased from 0% in 2004 to 44% in 2010 and S. flexneri was the predominant species. Of Shigella spp, ciprofloxacin resistant (CipR) strains were mostly found among S. flexneri (8.3%), followed by S. sonnei (1.5%). Within S. flexneri (n = 2181), 14.5% were resistance to ciprofloxacin of which serotype 2a was predominant (96%). MIC of ciprofloxacin, norfloxacin, and ofloxacin were 6–32 mg/L, 8–32 mg/L, and 8–24 mg/L, respectively in S. flexneri 2a isolates. Sequencing of QRDR genes of resistant isolates showed double mutations in gyrA gene (Ser83Leu, Asp87Asn/Gly) and single mutation in parC gene (Ser80Ile). A difference in amino acid substitution at position 87 was found between strains isolated in Bangladesh (Asp87Asn) and China (Asp87Gly) except for one. A novel mutation at position 211 (His→Tyr) in gyrA gene was detected only in the Bangladeshi strains. Susceptibility to ciprofloxacin was increased by the presence of CCCP indicating the involvement of energy dependent active efflux pumps. A single PFGE type was found in isolates from Bangladesh and China suggesting their genetic relatedness. Conclusions Emergence of fluoroquinolone resistance in Shigella undermines a major challenge in current treatment strategies which needs to be followed up by using empirical therapeutic strategies.