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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.

Vitamin K (VK) is a ligand of the pregnane X receptor (PXR), which plays a critical role in the detoxification of xenobiotics and metabolism of bile acids. VK1 may reduce the risk of death in patients with chronic liver failure. VK deficiency is associated with intrahepatic cholestasis, and is already being used as a drug for cholestasis-induced liver fibrosis in China. In Japan, to treat osteoporosis in patients with primary biliary cholangitis, VK2 formulations are prescribed, along with vitamin D3. Animal studies have revealed that after bile duct ligation-induced cholestasis, PXR knockout mice manifested more hepatic damage than wild-type mice. Ligand-mediated activation of PXR improves biochemical parameters. Rifampicin is a well-known human PXR ligand that has been used to treat intractable pruritus in severe cholestasis. In addition to its anti-cholestatic properties, PXR has anti-fibrotic and anti-inflammatory effects. However, because of the scarcity of animal studies, the mechanism of the effect of VK on cholestasis-related liver disease has not yet been revealed. Moreover, the application of VK in cholestasis-related diseases is controversial. Considering this background, the present review focuses on the effect of VK in cholestasis-related diseases, emphasizing its function as a modulator of PXR.

Fermented rice bran (FRB) is known to protect mice intestines against dextran sodium sulfate (DSS)-induced inflammation, however, the restoration of post-colitis intestinal homeostasis using FRB supplementation is currently undocumented. In this study, we observed the effects of dietary FRB supplementation on intestinal restoration and the development of fibrosis after DSS-induced colitis. DSS (1.5%) was introduced in the drinking water of mice for 5 days. Eight mice were sacrificed immediately after the DSS treatment ended. The remaining mice were divided into three groups, comprising the following diets: control, 10% rice bran (RB), and 10% FRB-supplemented. Diet treatment was continued for 2 weeks, after which half the population of mice from each group was sacrificed. The experiment was continued for another 3 weeks before the remaining mice were sacrificed. FRB supplementation could reduce the general observation of colitis and production of intestinal pro-inflammatory cytokines. FRB also increased intestinal mRNA levels of anti-inflammatory cytokine, tight junction, and anti-microbial proteins. Furthermore, FRB supplementation suppressed markers of intestinal fibrosis. This effect might have been achieved via the canonical Smad2/3 activation and the non-canonical pathway of Tgf-β activity. These results suggest that FRB may be an alternative therapeutic agent against inflammation-induced intestinal fibrosis.

The pregnane X receptor (PXR) is the key regulator of our defense mechanism against foreign substances such as drugs, dietary nutrients, or environmental pollutants. Because of increased health consciousness, the use of dietary supplements has gradually increased, and most of them can activate PXR. Therefore, an analysis of the interaction between drugs and nutrients is important because altered levels of drug-metabolizing enzymes or transporters can remarkably affect the efficiency of a co-administered drug. In the present study, we analyzed the effect of vitamin K-mediated PXR activation on drug metabolism-related gene expression in intestine-derived LS180 cells via gene expression studies and western blotting analyses. We demonstrated that menaquinone 4 (MK-4), along with other vitamin Ks, including vitamin K1, has the potential to induce MDR1 and CYP3A4 gene expression. We showed that PXR knockdown reversed MK-4-mediated stimulation of these genes, indicating the involvement of PXR in this effect. In addition, we showed that the expression of MDR1 and CYP3A4 genes increased synergistically after 24 h of rifampicin and MK-4 co-treatment. Our study thus elucidates the importance of drug–nutrient interaction mediated via PXR.

Hypogonadism, associated with low levels of testosterone synthesis, has been implicated in several diseases. Recently, the quest for natural alternatives to prevent and treat hypogonadism has gained increasing research interest. To this end, the present study explored the effect of S-allyl cysteine (SAC), a characteristic organosulfur compound in aged-garlic extract, on testosterone production. SAC was administered at 50 mg/kg body weight intraperitoneally into 7-week-old BALB/c male mice in a single-dose experiment. Plasma levels of testosterone and luteinizing hormone (LH) and testis levels of proteins involved in steroidogenesis were measured by enzymatic immunoassay and Western blot, respectively. In addition, mouse testis-derived I-10 cells were also used to investigate the effect of SAC on steroidogenesis. In the animal experiment, SAC significantly elevated testosterone levels in both the plasma and the testis without changing the LH level in plasma and increased phosphorylated protein kinase A (p-PKA) levels. Similar results were also observed in I-10 cells. The findings demonstrating the increasing effect of SAC on p-PKA and mRNA levels of Cyp11a suggest that SAC increases the testosterone level by activating the PKA pathway and could be a potential target for hypogonadism therapeutics.

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.

Pregnane X receptor (PXR) is a nuclear receptor activated by various compounds, including prescribed drugs and dietary ingredients. Ligand-specific activation of PXR alters drug metabolism and affects many other physiological conditions. Species-specific ligand preference is a considerable challenge for studies of PXR function. To increase translational value of the results of mouse studies, humanized mouse model expressing human PXR (hPXR) has been developed. Menaquinone-4 (MK-4), one of vitamin K2 analogs prescribed in osteoporosis, is a PXR ligand. We hypothesized that MK-4 could modulate the physiological conditions endogenously influenced by PXR, including those that have not been yet properly elucidated. In the present study, we investigated the effects of a single oral treatment with MK-4 on hepatic gene expression in wild-type and hPXR mice by using quantitative RT-PCR and DNA microarray. MK-4 administration altered mRNA levels of genes involved in drug metabolism (Abca3, Cyp2s1, Sult1b1), bile acid synthesis (Cyp7a1, Cyp8b1), and energy homeostasis (Aldoc, Slc2a5). Similar mRNA changes of CYP7A1 and CYP8B1 were observed in human hepatocarcinoma HepG2 cells treated with MK-4. These results suggest that MK-4 may modulate bile acid synthesis. To our knowledge, this is the first report showing the effect of MK-4 in hPXR mice.

The objective of this study was to evaluate the effect of rehydrating and ensiling dry ground corn (DGC) with varying concentrations of wet brewers grain (WBG) on fermentation profile and ruminal in vitro starch digestibility (ivSD; 7-h incubations on dried and 4-mm ground samples). Samples of DGC and WBG were weighed separately and mixed into 100% WBG (WBG); mixture of DGC and WBG targeting 60 (RC60), 65 (RC65), or 70% (RC70) of dry matter (DM); and DGC rehydrated with distilled water targeting for 70% of DM (REH). Samples were ensiled in vacuum-sealed bags and allowed to ferment for 0, 1, 3, 7, 14, and 28 d. The experiment consisted of 30 treatments (5 mixtures of DGC and WGB × 6 ensiling time points) and 120 mini-silos (4 silos per treatment). All samples were analyzed for fermentation profile and water-soluble carbohydrates. Except for WBG, samples from 0 and 28 d were analyzed for ivSD. Content of DM was greater for REH (70.0%), followed by RC70 (69.2%), RC65 (63.9%), RC60 (58.4%), and WBG (17.5%) on d 0, with a slight decrease (1 to 2 percentage units) observed for all treatments until 28 d. Measurements of pH were highest for REH (6.19) and lowest for WBG (4.68) on 0 d, but all other treatments were lower than WBG on 14 and 28 d (3.83 vs. 4.14, on average). Except for WBG, all treatments had a gradual increase in lactic acid concentration from 0 to 28 d. In contrast, butyric acid gradually increased from 0 (0.25%) to 28 d (2.16% of DM) in WBG but not the other treatments. Fermentation patterns were related to water-soluble carbohydrates concentration, which was greater for all treatments except WBG from 0 (1.41% on average vs. 0.38% of DM, respectively) to 28 d (0.37% on average vs. 0.19% of DM, respectively). Except for RC60, greater ivSD was observed for all treatments on 28 than 0 d, but magnitude of the difference was greater for REH and RC70 (14.5 percentage units on average). Rehydration and ensiling of DGC with WBG resulted in adequate fermentation and enhanced starch digestibility.