In Vitro Fermentation Characteristics and Rumen Microbial Population of Diet Supplemented with Saccharomyces Cerevisiae and Rumen Microbe Probiotics

The objective of this study was to select three strains of probiotic Saccharomyces cerevisiae and to evaluate the effect of S. cerevisiae and rumen bacteria isolate (MR4) supplementation and their combination on rumen fermentability and rumen microbial population. Experiment 1 was designed in a 4 x 5 factorial randomized block design with 3 replications. The first factor was S. cerevisiae strain consisted of control treatment (without S. cerevisiae supplementation), NBRC 10217, NRRL Y 567 and NRRL 12618, and the second factor was incubation time consisted of 0, 1, 2, 3, and 4 h. Ration was basal ration for feedlot with forage to concentrate ratio (F:C)= 60:40. Dosage of each treatment with S. cerevisiae was 5 x 1010 cfu/kg ration. Experiment 2 was designed in randomized block design with 4 treatments: P0= basal ration of feedlot; P1= P0 + S. cerevisiae; P2= P0 + MR4 isolate (5 x 107 cfu/kg ration); P3= P0 + S. cerevisiae and MR4 isolate. The result of experiment 1 showed that supplementation of S. cerevisiae NRRL 12618 had the highest S. cerevisiae population and increased rumen bacterial population. This strain was selected as probiotic in experiment 2. The result from experiment 2 showed that probiotic supplementation stabilized rumen pH and produced the highest NH3 concentration (P<0.05) and bacterial population (P<0.05). As compared with control, all treatments reduced protozoa population (P<0.05). Combination of S. cerevisiae and MR4 probiotics produced the highest total volatile fatty acids (VFA) and isovalerate (P<0.05). It was concluded that strain S. cerevisiae NRRL 12618 had potential as probiotic yeast. Supplementation with this strain increased fermentability, rumen isoacid and decreased A:P ratio. Those abilities could be improved with MR4 rumen isolate probiotic

Fermentation Quality and in Vitro Nutrient Digestibility of Fresh Rice Straw-Based Silage Treated with Lactic Acid Bacteria

The aim of the experiment was to evaluate fermentation characteristics and in vitro nutrient digestibility of fresh rice straw-based silage ensiled with addition of epiphytic lactic acid bacteria (LAB) inoculant. The experiment was arranged in a completely randomized design, with 2 × 2 factorial arrangement of treatments. The first factor was the ratio of fresh rice straw (FRS), tofu waste (TW) and cassava waste (CW) consisted of two levels i.e., 40 : 20 : 40 and 40 : 25 : 35, on dry matter (DM) basis). The second factor was the level of LAB inoculant with two levels ie., 0 and 20 mL/kg FM. The treatments were (A) FRS + TW + CW with the ratio of 40 : 20 : 40, without LAB inoculant; (B) FRS + TW + CW with the ratio of 40 : 20 : 40 + LAB inoculant; (C) FRS + TW + CW with the ratio of 40 : 25 : 35, without LAB inoculant; (D) FRS + TW + CW with ratio of 40 : 25 : 35 + LAB inoculant. Results showed that addition of LAB inoculant in silage increased lactic acid concentration (P0.05) on chemical composition, fermentation quality of silage and in vitro digestibility. It was concluded that mixture silage with ratio of 40 : 20 : 40 with the addition of LAB inoculant had the best fermentation quality and nutrient digestibility than other silages

In vitro and in vivo inhibition of breast cancer cell growth by targeting the Hedgehog/GLI pathway with SMO (GDC-0449) or GLI (GANT-61) inhibitors.

Aberrant Hedgehog (Hh)/glioma-associated oncogene (GLI) signaling has been implicated in cancer progression. Here, we analyzed GLI1, Sonic Hedgehog (Shh) and NF-κB expression in 51 breast cancer (ductal carcinoma) tissues using immunohistochemistry. We found a positive correlation between nuclear GLI1 expression and tumor grade in ductal carcinoma cases. Cytoplasmic Shh staining significantly correlated with a lower tumor grade. Next, the in vitro effects of two Hh signaling pathway inhibitors on breast cancer cell lines were evaluated using the Smoothened (SMO) antagonist GDC-0449 and the direct GLI1 inhibitor GANT-61. GDC-0449 and GANT-61 exhibited the following effects: a) inhibited breast cancer cell survival; b) induced apoptosis; c) inhibited Hh pathway activity by decreasing the mRNA expression levels of GLI1 and Ptch and inhibiting the nuclear translocation of GLI1; d) increased/decreased EGFR and ErbB2 protein expression, reduced p21- Ras and ERK1/ERK2 MAPK activities and inhibited AKT activation; and e) decreased the nuclear translocation of NF-κB. However, GANT-61 exerted these effects more effectively than GDC-0449. The in vivo antitumor activities of GDC-0449 and GANT- 61 were analyzed in BALB/c mice that were subcutaneously inoculated with mouse breast cancer (TUBO) cells. GDC-0449 and GANT-61 suppressed tumor growth of TUBO cells in BALB/c mice to different extents. These findings suggest that targeting the Hh pathway using antagonists that act downstream of SMO is a more efficient strategy than using antagonists that act upstream of SMO for interrupting Hh signaling in breast cancer

In Vitro Fertility of Post-thawed Epididymal Ram Spermatozoa After Storage at 5 °C Before Cryopreservation

This study addressed the effects of storage duration of epididymides at 5 °C before sperm collection and their fertility after cryopreservation in vitro. Spermatozoa from one of the testes pairs were immediately collected, evaluated and frozen (control group). The remaining epididymides were cooled to 5 °C and stored for 24, 48, 72, and 96 h (experimental groups), after which spermatozoa were collected and frozen as in the control group. Before and after thawing, sperm motility, sperm viability and plasma membrane integrity were assessed. The fertilizing ability of frozen-thawed spermatozoa of each group was evaluated by in vitro fertilization of matured sheep oocytes. Sperm quality (sperm motility, viability, and plasma membrane integrity) at collection and after cryopreservation decreased as the duration of the epididymal storage interval increase (P < 0.05). The motility decreased steadily along the studied time periods. Although, the fertilizing ability of post-thawed epididymal spermatozoa gradually decreased as the storage period was prolonged, the spermatozoa collected from the cauda epididymides stored at 5 °C for up to 96 h were able to fertilize 16%-65% of oocytes in vitro. Results of the present study showed that ram epididymal spermatozoa survive in storage at 5 °C for up to 96 h. These spermatozoa maintain their fertilizing ability and may be suitable for use in IVF and other assisted reproductive procedures

In Vitro Rumen Fermentation and Anti Mastitis Bacterial Activity of Diet Containing Betel Leaf Meal (Piper Betle L.)

The aims of this experiment was to study the inhibition effect of betel leaf meal (BLM) addition into concentrate diet on mastitis causing bacteria and on rumen fermentation condition. The study consisted of five dietary treatments of BLM level in concentrate feed, i.e., 0%, 2%, 4%, 6%, and 8% and four replicates of each treatment. The treatment diets together with napier grass in ratio of 40 : 60 were fermented using rumen liquor. All treatments were examined their antibacterial activity before and after fermentation. After four hours fermentation, supernatant of each samples were analyzed for VFA, NH3, number of bacteria and protozoa. Dry matter (DM) and organic matter (OM) digestibility were analyzed after 48 h fermentation. The results showed that before fermentation, 8% BLM addition caused the bigest (P<0.05) inhibition diameter of Staphylococcus spp. growth compared to other lower levels. However after fermentation there were no significant differences among the addition levels of BLM. Two per cent of BLM addition produced higher VFA (P<0.05) than the other addition levels. Ammoniaconcentration, dry matter (DM) and organic matter (OM) digestibility were not different among the treatments. Addition of BLM significantly (P<0.01) decreased protozoa number, but did not affect bacterial count. It is concluded that the addition of 2% BLM in concentrate feed can be used effectively to inhibit the growth of mastitis causing bacteria (Staphylococcus spp.) and does not disturb rumen fermentation condition

Research Article

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In vitro evaluation of carrier based obturation technique: a CBCT study

AIM: The goal of the study was to compare the ability of two different carrier based obturation (CBO) techniques to reach working length and fill in three-dimensions root canal systems, by using CBCT. MATERIALS AND METHODS: Twenty-six extracted molars were scanned with CBCT and 40 curved canals were selected (between 30° and 90°) and divided in two similar groups (n=20). All canals were prepared up to size 25 taper .06 using nickel-titanium instrumentation. The canals in the Group SC were obturated using Soft-Core obturators (Kerr, Romulus, Mi, USA), while Group TH canals (n= 20) were obturated using Thermafil Endodontic Obturators (Tulsa Dental Products, Tulsa, OK, USA), strictly following manufacturers' instructions for use. The obturations were analyzed by means of CBCT to measure the distance from the apical limit of obturation to the apical foramen and the presence of voids inside root canals. RESULTS: There was no significant difference between the two groups in the mean distance of the apical extent of the obturation (t test, p>0.05). Overfilling occurred in only 3 cases (2 in Group TH and 1 in Group SC). The percentages of voids in both groups were very low with no significant difference (Z test, p>0.05). CONCLUSIONS: The two tested CBO techniques showed similar positive results in terms of performance, even if, after checking with verifiers, in most cases the size of the selected Soft-Core obturator was one size smaller than Thermafil

Analysis of indole derivatives in methanolic extracts from mycelium of Agaricus bisporus cultured in vitro on liquid Oddoux medium

Methanolic extracts obtained from biomass of Agaricus bisporus (J.E. Lange) Imbach cultured in vitro were analyzed for qualitative and quantitative composition of non-hallucinogenic indole compounds in order to compare their amount with fruiting bodies of these species. Extracts demonstrated to contain six indole compounds. Contents of individual compounds ranged from 0.01 to 21.33 mg/100 g d.w. in biomass from in vitro cultures. The quantitatively dominating compounds included: 5-hydroxytryptophan (12.50 mg/100 g d.w.), Ltryptophan (14.00 mg/100 g d.w.) and serotonin (7.00 mg/100 g d.w.). Total content of the remaining indole compounds under analysis in the study was 55.32 mg/100 g d.w.Po raz pierwszy zidentyfikowane i ilościowo oznaczone zostały związki indolowe w kulturach in vitro Agaricus bisporus na płynnym podłożu wg Oddoux. Analiza wykazała, że ekstrakty metanolowe otrzymane z grzybni zawierają sześć związków indolowych: L -tryptofan, 5 - hydroksytryptofan, serotoninę, melatoninę, tryptaminę i 5-metylotryptamię. Zawartości poszczególnych składników w biomasie z kultur in vitro były zróżnicowane w zakresie od 0,01 do 21,33 mg/100 g s. m. Dominującymi ilościowo związkami były: 5-hydroksytryptofan (12,50 mg/100 g s. m.), L-tryptofan (14,00 mg/100 g) i serotonina (7,00 mg/100 g). Całkowita zawartość związków indolowych w badanym materiale wynosiła 55,32 mg/100 g s. m. Biomasa z kultur in vitro badanego gatunku jest dobrym źródłem 5-hydroksytryptofanu i L- tryptofanu. Kultury in vitro A. bisporus mogą być wykorzystane jako model do badań nad akumulacją i metabolizmem związków indolowych

Developing an Analytical Method for Separating and Quantifying RNA Generated in In Vitro Transcription Reactions

The generation of run-off transcripts from in vitro transcription reactions is a useful technique in the study of transcription regulation. There are currently limited ways in which these run-off transcripts can be analyzed, and few that are truly quantitative. Our aim is to establish a method using ion-pair reverse phase high performance liquid chromatography (IP RP HPLC) to analyze RNA transcripts from in vitro transcription reactions. We were able to demonstrate that we could recapitulate the separation of DNA based on size using our IP RP HPLC method. The application of this method was also able to effectively separate RNA based on size in the size range of 281 – 1908 bp. Using a simple in vitro transcription system with T7 RNA polymerase and a linear DNA template with a single promoter, we showed detection of the RNA run-off transcript in the size range demonstrated. We would like to apply this method to more complex in vitro transcription systems and demonstrate the ability to quantify RNA using IP RP HPLC

Effects of preconditioning and extrusion of linseed on the ruminal biohydrogenation of fatty acids. 2. In vitro and in situ studies

The extent and/or intermediates of ruminal biohydrogenation (BH) of fatty acids (FA) were investigated in vitro and in situ using a raw, pre-conditioned or extruded blend of linseed and wheat bran (70:30). The duration of in vitro incubations were 2, 4, 8, 16 and 24 h, with 5 replicates. In situ studies used 3 dry ruminally fistulated Holstein cows in a 3 × 3 Latin square design, with 3 weeks adaptation to the linseed form. The diet contained 20% (DM basis) of the linseed based blend. The duration of in situ incubations were 2, 4, 8, 16, 24 and 48 h. BH was much slower in situ than in vitro, resulting in a much lower effective disappearance of C18:2 and C18:3. Moreover, the in situ technique suggested that the technological pre-treatment of linseed did not affect C18:2 and C18:3 rate of BH, whereas reduced rates were observed in vitro. After 8 h of in vitro incubation and onwards, proportions of cis-9,trans-11C18:2 were the highest with extruded linseed. The BH of FA from linseed resulted in the appearance of great proportions of trans-10+11 to trans-16C18:1 intermediates. Extrusion increased the proportions of trans-10+11C18:1 both in vitro and in situ and proportions or trans-C18:1 were higher in situ than in vitro. Compared to previous in vivo results with the same material, the in situ method provided poor estimates of BH rates and intermediates