Selected plant essential oils inhibit biofilm formation and luxS- and pfs-mediated quorum sensing by Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) causes food-borne outbreaks worldwide and the bacteria form antimicrobial-tolerant biofilm. We investigated the abilities of Thymus daenensis and Satureja hortensis essential oils (EOs) to inhibit bacterial growth, biofilm formation and quorum sensing (QS) by E. coli O157:H7. The tested EOs were isolated from plant material by hydrodistillation and analysed under chromatography-mass spectrometry. The antibacterial and anti-biofilm activities of the EOs were determined by microdilution broth and microtitre-plate (MtP) tests, respectively. The QS inhibitory (QSI) potential was examined by inhibition of swimming and swarming motility at sub-MIC levels. Real-time quantitative reverse transcription PCR (RT-qPCR) was used to determine the expression of QS-system-related genes. The MICs for T. daenensis and S. hortensis EOs against EHEC were 3·12 and 6·25 µg ml-1 , respectively and the MBCs were 6·25 and 12·5 µg ml-1 , respectively. The MtP test showed a significant (P < 0·05) inhibitory and disruptive effect on both EOs for EHEC biofilm formation at MIC/2 (1·56 µg ml-1 for T. daenensis; 3·12 µg ml-1 for S. hortensis) and MIC/4 (0·78 µg ml-1 for T. daenensis; 1·56 µg ml-1 for S. hortensis) concentrations. Gene expression analysis revealed significant down-regulation of luxS and pfs following treatment with MIC/2 concentrations. The results of the present research point to the promising antibacterial, anti-biofilm and anti-QS potential of T. daenensis and S. hortensis EOs against E. coli O157:H7.

Development of a subacute ruminal acidosis risk score and its prediction using milk mid-infrared spectra in early-lactation cows.

A routine monitoring for subacute ruminal acidosis (SARA) on the individual level could support the minimization of economic losses and the ensuring of animal welfare in dairy cows. The objectives of this study were (1) to develop a SARA risk score (SRS) by combining information from different data acquisition systems to generate an integrative indicator trait, (2) the investigation of associations of the SRS with feed analysis data, blood characteristics, performance data, and milk composition, including the fatty acid (FA) profile, (3) the development of a milk mid-infrared (MIR) spectra-based prediction equation for this novel reference trait SRS, and (4) its application to an external data set consisting of MIR data of test day records to investigate the association between the MIR-based predictions of the SRS and the milk FA profile. The primary data set, which was used for the objectives (1) to (3), consisted of data collected from 10 commercial farms with a total of 100 Holstein cows in early lactation. The data comprised barn climate parameters, pH and temperature logging from intrareticular measurement boluses, as well as jaw movement and locomotion behavior recordings of noseband-sensor halters and pedometers. Further sampling and data collection included feed samples, blood samples, milk performance, and milk samples, whereof the latter were used to get the milk MIR spectra and to estimate the main milk components, the milk FA profile, and the lactoferrin content. Because all measurements were characterized by different temporal resolutions, the data preparation consisted of an aggregation into values on a daily basis and merging it into one data set. For the development of the SRS, a total of 7 traits were selected, which were derived from measurements of pH and temperature in the reticulum, chewing behavior, and milk yield. After adjustment for fixed effects and standardization, these 7 traits were combined into the SRS using a linear combination and directional weights based on current knowledge derived from literature studies. The secondary data set was used for objective (4) and consisted of test day records of the entire herds, including performance data, milk MIR spectra and MIR-predicted FA. At farm level, it could be shown that diets with higher proportions of concentrated feed resulted in both lower daily mean pH and higher SRS values. On the individual level, an increased SRS could be associated with a modified FA profile (e.g., lower levels of short- and medium-chain FA, higher levels of C17:0, odd- and branched-chain FA). Furthermore, a milk MIR-based partial least squares regression model with a moderate predictability was established for the SRS. This work provides the basis for the development of routine SARA monitoring and demonstrates the high potential of milk composition-based assessment of the health status of lactating cows.

Statistical modeling of ruminal pH parameters from dairy cows based on a meta-analysis.

Adequate feeding of high-performance dairy cows is extremely important to avoid the digestive disorder subacute ruminal acidosis. Subacute ruminal acidosis is defined as a status with a below-average ruminal pH that does not cause direct clinical symptoms at the individual level but is relevant for animal welfare due to a higher risk of secondary health problems at the herd level. The main objective of this study was to apply meta-analytical methods in an exploratory approach to investigate the association between pH parameters of the ventral rumen with milk and diet parameters. Data from 32 studies using continuous pH measurement in the ventral rumen of lactating cows were included in the meta-analysis. Available information extracted from all studies was categorized into parameters associated with management, cow, diet, milk, and pH. The statistical analysis was divided into 4 sections. First, a multiple imputation procedure based on a principal component model was applied, since approximately 19% of the data set consisted of missing values due to heterogeneity in provided information between the studies included in the analysis. In a second step, all potential predictors for the pH parameters, including the daily mean pH, the time with a pH below 5.8, and the pH range, were examined for their prediction suitability using multi-level mixed effects meta-regression models. These analyses were performed on the raw and the imputed data. Because the results of both approaches were consistent, the imputing procedure was considered to be appropriate. Third, automated variable selection was applied to all 3 pH parameters separately for the predictor groups milk and diet using the imputed data set. Thereby, multi-model inference was used to estimate the relative importance of the selected variables. Finally, a functional relationship between the 3 pH parameters was established. The fat to protein ratio of milk, milk fat, and milk protein showed significant associations in meta-regression analysis for all 3 pH parameters when used as a single predictor. Out of the group of diet-specific variables, the acid detergent fiber, neutral detergent fiber, nonfiber carbohydrate, starch content, as well as the forage to concentrate ratio, showed the highest significance in the models. In particular, the multi-model inference showed that the protein, fat, and lactose content of the milk can best quantify the association to the daily mean pH and the time with a pH below 5.8 in a multiple regression model.

Modeling reticular and ventral ruminal pH of lactating dairy cows using ingestion and rumination behavior.

The prevention and control of metabolic and digestive diseases is an enormous challenge in dairy farming. Subacute ruminal acidosis (SARA) is assumed to be the most severe feed-related disorder and it impairs both animal health and economic efficiency. Currently, ruminal pH as well as variables derived from the daily pH curve are the main indicators for SARA. The objective of this study was to explain the daily pH course in the ventral rumen and reticulum of dairy cows using ingestion pattern and rumination behavior data gathered by automated data recording systems. The data of 13 ruminally fistulated lactating cows were collected at the experimental station of the Friedrich-Loeffler-Institut (Brunswick, Germany). The data included continuous pH measurements, which were recorded simultaneously in the reticulum by pH-measuring boluses and in the ventral rumen by a separate data logger. In addition, rumination behavior was measured using jaw movement sensors, and feed and water intakes were recorded by transponder-assisted systems. Milk yield and body weight were determined during and after each milking, respectively. For statistical evaluation, the data were analyzed using time-series modeling with multiple linear mixed regressions. Before applying the developed mathematical statistical modeling, we performed a plausibility assessment to ensure data quality. The major part of the mathematical statistical modeling consisted of data preparation, where all variables were transformed into a uniform 1-min resolution. Signal transformations were used to model individual feed and water intakes as well as rumination behavior events over time. Our results indicated that diurnal pH curves of both the reticulum and ventral rumen could be predicted by the transformed feed and water intake rates. Rumination events were associated with a marginal temporal increase in pH. We observed that the pH of the ventral rumen was delayed by approximately 37 min compared with that of the reticulum, which was therefore considered in the modeling. With the models developed in this study, 67.0% of the variance of the reticular pH curves and 37.8% of the variance of the ruminal pH curves could be explained by fixed effects. We deduced that the diurnal pH course is, to a large extent, associated with the animal's individual feed intake and rumination behavior.

Analysis of porcine body size variation using re-sequencing data of miniature and large pigs.

BACKGROUND: Domestication has led to substantial phenotypic and genetic variation in domestic animals. In pigs, the size of so called minipigs differs by one order of magnitude compared to breeds of large body size. We used biallelic SNPs identified from re-sequencing data to compare various publicly available wild and domestic populations against two minipig breeds to gain better understanding of the genetic background of the extensive body size variation. We combined two complementary measures, expected heterozygosity and the composite likelihood ratio test implemented in "SweepFinder", to identify signatures of selection in Minipigs. We intersected these sweep regions with a measure of differentiation, namely FST, to remove regions of low variation across pigs. An extraordinary large sweep between 52 and 61 Mb on chromosome X was separately analyzed based on SNP-array data of F2 individuals from a cross of Goettingen Minipigs and large pigs. RESULTS: Selective sweep analysis identified putative sweep regions for growth and subsequent gene annotation provided a comprehensive set of putative candidate genes. A long swept haplotype on chromosome X, descending from the Goettingen Minipig founders was associated with a reduction of adult body length by 3% in F2 cross-breds. CONCLUSION: The resulting set of genes in putative sweep regions implies that the genetic background of body size variation in pigs is polygenic rather than mono- or oligogenic. Identified genes suggest alterations in metabolic functions and a possible insulin resistance to contribute to miniaturization. A size QTL located within the sweep on chromosome X, with an estimated effect of 3% on body length, is comparable to the largest known in pigs or other species. The androgen receptor AR, previously known to influence pig performance and carcass traits, is the most obvious potential candidate gene within this region.

Characterization of Enterococcus faecalis isolates by chicken embryo lethality assay and ERIC-PCR.

Enterococcus faecalis is the major causative agent of amyloid arthropathy in chickens. Given the difficulty of estimating the risk from field strains, the embryo lethality assay (ELA) is proposed in this study as a model to predict the virulence of 68 avian E. faecalis strains. Additionally, Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) was used to characterize the genetic diversity of the E. faecalis strains. The ELA was performed 10 times with subsets of 7-8 E. faecalis strains each on a sample of 9987 eggs, including control groups. An estimated 3-24 colony-forming units were inoculated into the allantoic cavity of 10-day-old embryos. The embryonic mortality rate (EMR) was determined by means of candling the eggs over a period of seven days. The ELA was able to distinguish the virulence of the E. faecalis strains. Twenty-six strains were considered as avirulent strains with an EMR of below 40%. Five strains were highly virulent with an EMR above 80%. The remaining 37 strains were classified as strains of moderate virulence, causing an EMR between 40% and 80%. The highest EMR occurred three and four days post-inoculation (p.i.). From the fourth day p.i., almost no embryonic mortality was observed. Therefore, the ELA could be optimized by reducing experiment duration to four days p.i. ERIC-PCR did not cluster the strains according to its virulence, although ERIC banding patterns revealed a considerable genetic diversity. In conclusion, the ELA can be considered a reliable and useful tool to predict the virulence of avian E. faecalis strains.

Antibacterial, antibiofilm and antiquorum sensing effects of Thymus daenensis and Satureja hortensis essential oils against Staphylococcus aureus isolates.

AIMS: The present study was conducted to investigate the effects of Thymus daenensis and Satureja hortensis essential oils (EOs) on the planktonic growth, biofilm formation and quorum sensing (QS) of some Staphylococcus aureus isolates (strong biofilm producers). METHODS AND RESULTS: Minimum inhibitory concentration (MIC) of the EOs, inhibition of biofilm formation as well as disruption of preformed Staph. aureus biofilms were assessed. The antibiofilm activity of the EOs was determined using microtitre plate test (MtP) and scanning electron microscope (SEM). The QS inhibitory activity was also examined on the pregrown biofilms by gene expression analysis using quantitative real-time RT-polymerase chain reaction (PCR) of hld gene (RNAIII transcript). Moreover, tetrazolium-based colorimetric assay (MTT) was performed to detect cytotoxic effects of these EOs on the Vero cell line. Finally, the major components of the tested EOs were determined using Gas Chromatography-Mass Spectrometry (GC-MS). The MICs of T. daenensis and S. hortensis EOs against planktonic cells of the isolates were 0·0625 and 0·125 µl ml-1 respectively. The minimum bactericidal concentrations for both of the EOs was 0·125 µl ml-1 . The MtP test showed a significant inhibitory effect of the EOs on the biofilm formation and disruption at sub-MIC concentrations. These results were confirmed by SEM. Real-time PCR revealed a significant down-regulation of hld gene following treatment with MIC/2 concentration of S. hortensis EO. GC-MS analysis showed that carvacrol, terpinene and thymol were the major components of the applied EOs. CONCLUSIONS: As selected EOs did not show significant cytotoxic effects even up to tenfold of MIC concentration, the applied EOs seem to be good candidates for preventing of biofilm formation of Staph. aureus cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study introduced T. daenensis and S. hortensis EOs as new antibiofilm, and S. hortensis EO as anti-QS herbal agents with natural origin against Staph. aureus.

Antibacterial and anti-PmrA activity of plant essential oils against fluoroquinolone-resistant Streptococcus pneumoniae clinical isolates.

Streptococcus pneumoniae (pneumococcus) is recognized as one of the major cause of infections in communities and hospitals. In this study, anti-pneumococcal and anti-efflux pump activity of two medicinal plants (Thymus daenensis and Origanum vulgare) essential oils were evaluated. Checkerboard assay test was performed for investigation of the effects of selected EOs on ciprofloxacin (CIP) and ethidium bromide (EtBr) uptake in pmrA-overexpressed fluoroquinolone-resistant pneumococcus. Using quantitative real-time RT-PCR the PmrA efflux pump gene (pmrA) expression was evaluated following treatment with selected EOs. Gas chromatography-mass spectrometry analysis was performed for identifying the major components of the tested EOs. The minimum inhibitory concentration (MIC) values for pneumococcus isolates were 0·625-2·5 µl ml-1 for T. daenensis and 1·25-5 µl ml-1 for O. vulgare EOs. We confirmed that in all strains T. daenensis and O. vulgare have a total or partial synergistic effects with CIP and EtBr (FICI from 0·14 to 0·75). In other hand MIC/2 concentration of T. daenensis and O. vulgare EOs caused a significant downregulation of pmrA gene (P < 0·05) in seven of eight strains. This study showed that T. daenensis and O. vulgare EOs have strong antimicrobial and anti-efflux pump activity against clinical isolates of S. pneumoniae and might be useful in controlling pneumococcal infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This study introduced Thymus daenensis and Origanum vulgare essential oil as new antibacterial and anti-efflux pump agents against fluoroquinolone-resistant Streptococcus pneumoniae clinical isolates. These findings indicate that combination of these two essential oils with fluoroquinolone antibiotics may provide alternative methods to overcome the fluoroquinolone-resistant S. pneumoniae.

SNP g.1007A>G within the porcine DNAL4 gene affects sperm motility traits and percentage of midpiece abnormalities.

The flagellar beating of a spermatozoa's axoneme is caused by the varying activation and inactivation of dynein molecules. Dynein, axonemal, light chain 4 (DNAL4) is a functional candidate gene for sperm motility as it encodes a small subunit of the dyneins. We resequenced the porcine DNAL4 using three artificial insemination (AI) boars each with high (>68%) or low (<60%) motility, and detected 23 SNP. These were then genotyped for 82 AI boars. Using spermatological records, significantly negative genetic correlations between ejaculate volume (VOL) and the further spermatological parameters concentration (CONC) (r = -.43), motility of undiluted semen (MOTUD) (r = -.09), motility after 24 h (MOT1) (r = -.17) and after 48 hr (MOT2) (r = -.23) were estimated. Significantly positive correlations existed between CONC and MOT1 (r = .07) as well as MOT2 (r = .10), between MOTUD and MOT1 (r = .33), between MOTUD and MOT2 (r = .36), and finally between MOT1 and MOT2 (r = .70). Significantly negatively correlated were all motility traits with the parameters abnormal acrosome (AA) (MOTUD r = -.06; MOT1 r = -.08, and MOT2 r = -.1) and presence of cytoplasmic droplet (CD) (MOTUD r = -.07; MOT1 r = -.08; MOT2 r = -.07). Association analyses (single marker regression model; SMR) propose that SNP g.1007A>G, located in the second intron, reduces motility significantly (MOTUD -4.59%; MOT1 -10.33%; MOT2 -19.37%). According to the dominant-recessive model (DRM), genotype AA is always superior compared to genotypes AG and GG (i.e. MOTUD 67.67%, 64.16% and 53.91%; MOT1 54.17%, 43.75% and 28.44%; MOT2 44.12%, 24.91% and 4.97%). The average effect of gene substitution (g.1007A>G) on abnormal midpiece (AM) was 0.71%, the genotypic values-as expressed by LSmeans-were 0.1 (AA) and 0.81 (AG).

Chicken embryo lethality assay for determining the lethal dose and virulence of Enterococcus faecalis.

Enterococcus faecalis is the major pathogen found in field cases of amyloid arthropathy in chickens. Given the need for a better understanding of the virulence mechanisms of the causative strains, the embryo lethality assay (ELA) is proposed in the present study as a model to evaluate the virulence of E. faecalis strains, specifically the pathogenic avian strain K923/96, which was previously related with amyloid arthropathy. Hence, 0.2 ml of five doses of the cited strain (from 2.5 to 2500 colony-forming units (CFU) per ml) were inoculated into the allantoic cavity of 10-day-old embryos. The embryo mortality rate (EMR) was determined by daily candling of the eggs over a period of seven days and based on this information the median lethal dose (LD50) was calculated. The ELA was repeated four times on a sample of 3443 eggs. The infectious dose showed a significant effect on the EMR. The EMR with the doses of 2.5, 5, 25, 250 and 2500 CFU/ml was 43%, 45%, 63%, 90% and 93%, respectively. The estimated dose at LD50 was 6.6 CFU/ml. As expected, the higher the infectious dose, the greater the EMR and the lower the embryo survival time. The highest EMR was recorded after three and four days post-inoculation in all doses. In conclusion, these results can be used as a basis for further researches on the E. faecalis virulence. In order to corroborate its model capacity to predict the virulence of this bacterium, more ELAs with different E. faecalis strains are required.

A reaction norm sire model to study the effect of metabolic challenge in early lactation on the functional longevity of dairy cows.

Due to the discrepancy of the high energy demand for rapidly increasing milk production and limited feed intake in the transition period around parturition, dairy cows require considerable metabolic adaptations. We hypothesize that some cows are genetically less suited to cope with these metabolic needs than others, leading to adverse follow-up effects on longevity. To test this, we designed a reaction norm model in which functional lifetime was linked to the metabolic challenge in the beginning of the first lactation. As challenge variables, we used either the sum of milk yield or the accumulated fat-to-protein ratio of the first 3 test-days (<120 d in milk), pre-adjusted for herd-test-day variance. We defined a random regression sire model, in which a random slope was estimated for each sire to assess whether a bull had robust (neutral or positive slopes) or non-robust (negative slopes) daughters. We fitted the model to data of ∼580,000 daughters of ∼5,000 Brown Swiss bulls with suitable observations available (≥10 daughters per bull). To validate our proposed model and assess the reliability of the estimated (co)variance components, we conducted an extensive bootstrap approach. For both challenge variables, we found the sire variance for the slope of the random regression to be significantly different from zero, suggesting a genetic component for metabolic adaptability. The results of the study show that the ability to cope with metabolic stress in the transition period has a genetic component, which can be used to breed metabolically robust dairy cows.

Modeling Nitrogen and Carbon dynamics in wetland soils and water using a mechanistic wetland model.

In this article, extension and application to variably-saturated wetland conditions of a process-based wetland model, namely WetQual is demonstrated. The new model described in this article is an improved version of an earlier model, which was only capable of capturing nutrient dynamics in continuously ponded wetlands. The upgraded model is capable of simulating nutrient cycling and biogeochemical reactions in both ponded and unsaturated zones of the wetland. To accomplish this goal, a comprehensive module for tracking water content in wetland soil was implemented in the model, and biogeochemical relationships were added to explain cycling of nitrogen (N) and carbon (C) in variably saturated zones of wetlands. The developed model was applied to a small, restored wetland receiving agricultural runoff, located on Kent Island, Maryland. On average, during the two year study period, the ponded compartment of the study wetland covered 65% of the total 1.2 ha area. Through mass balance analysis, it was revealed that the mass of nitrogen lost to denitrification at the variably saturated compartment of the study wetland was about 3 times higher than that of the ponded compartment (32.7 ± 29.3 kg vs. 9.5 ± 5.5 kg) whereas ammonia volatilization at the variably saturated compartment was a fraction of that of ponded compartment (1.2 ± 1.9 kg vs. 11.3 ± 11.8 kg). Sensitivity analysis showed that cycling of carbon related constituents in variably saturated compartment had high sensitivity to temperature and available soil moisture.

Capturing Spatial Variability of Biogeochemical Mass Exchanges and Reaction Rates in Wetland Water and Soil through Model Compartmentalization.

A common phenomenon observed in natural and constructed wetlands is short-circuiting of flow and formation of stagnant zones that are only indirectly connected with the incoming water. Biogeochemistry of passive areas is potentially much different than that of active zones. In the research reported in this paper, the spatial resolution of a previously developed wetland nutrient cycling model was improved in order to capture the spatial variability of concentrations and reaction rates regarding nitrogen and carbon cycles throughout active and passive zones of wetlands. The upgraded model allows for several compartments in the horizontal domain, with all neighboring compartments connected through advective and dispersive/diffusive mass transport. The model was applied to data collected from a restored wetland in California that was characterized by the formation of a large stagnant zone at the southern end of the wetland due to close vicinity of the inlet and outlet structures in the northern end. Mass balance analysis revealed that over the course of the research period, about 23.4±3.9% of the incoming total nitrogen load was removed or retained by the wetland. It was observed that mass of all exchanges (physical and biogeochemical) regarding nitrogen cycling decreased along the activity gradient from active to passive zones. Model results also revealed that anaerobic processes become more significant along the activity gradient towards passive areas.

RNAi-induced silencing in floral tissues of Petunia hybrida by agroinfiltration: a rapid assay for chalcone isomerase gene function analysis.

Variegation in flower color is commonly observed in many plant species and also occurs on Petunia (Petunia hybrida) as an ornamental plant. Variegated plants are of highly valuable in the floricultural market. Agroinfiltration is an Agrobacterium-mediated transient assay for the analysis of gene function and genetic modification in leaves, flowers and fruit tissues of various plants. Transient RNAi-induced silencing by agroinfiltration has been developed in leaves and fruits of several plant species. Here we report the establishment of a transient hairpin RNAi-induced silencing system for color modification assay in floral tissues of Petunia with different colors. chiRNAi construct was cloned into the pBI121 vector under the control of 35S promoter. Transient RNA silencing of chi in the floral tissues of Petunia was induced by delivering 530 bp chi hairpin RNAs (hpRNAs) into the petals of flowers using agroinfiltration. Impaired anthocyanin accumulation and reduction of endogenous mRNAs of the corresponding targets were observed in the infiltrated areas of the petals of four colors of Petunia. Silencing of the endogenous chi mRNAs was highly effective in reduction of chi gene and anthocyanin accumulation. This transient silencing system is a prototype for modification of the anthocyanin biosynthetic pathway in Petunia through chi gene suppression.

Modified Spiral Intestinal Lengthening and Tailoring for Short Bowel Syndrome.

BACKGROUND: The spiral intestinal lengthening and tailoring (SILT) procedure is a new surgical technique for autologous intestinal reconstruction in patients with short bowel syndrome. The aim of this work is to present a first description of a modified SILT technique by which the mucosal layer is left intact to possibly reduce the severe postoperative complications of intestinal leakage and abdominal abscess formation. MATERIALS AND METHODS: The modified SILT technique was performed on a 10-cm-long intestinal segment in 2 pigs to determine the technical feasibility. Thereafter, the short-term clinical feasibility was monitored clinically in 2 dogs by gastrointestinal X-ray series at postoperative day 4 and by relaparotomy postoperative day 10. RESULTS: It was technically feasible to lengthen the intestinal segment from 10 cm to 20 cm and tailoring it from 3 cm to 1.7 cm in diameter, while leaving the intestinal mucosal layer intact. The postoperative course was uneventful for both dogs. The gastrointestinal X-ray series showed an inconspicuous intestinal transit time without any signs of stricture, perforation, or leakage. In the relaparotomy, the initially achieved lengthening and tailoring extents were preserved and the operated intestinal segment was well perfused with no early signs of necrosis, stenosis, or leakage. CONCLUSIONS: Leaving the mucosal layer intact during SILT is technically and clinically feasible in the short term in a large animal model. Further studies are needed to fully assess the impact of this technical modification on the long-term outcome of larger series.

Effects of feeding different levels of corn steep liquor on the performance of fattening lambs.

This study was conducted to assess the effect of feeding corn steep liquor (CSL) on in vivo digestibility, ruminal pH, ammonia and hydrolytic enzyme activities, blood metabolites, feed intake (FI) and growth performance in fattening lambs. The CSL is a by-product of wet milling process of maize starch industry. The crude protein (CP), rumen-degradable protein (RDP), lactic acid and metabolisable energy contents of this by-product were 420, 324, 200 g/kg dry matter (DM) and 12.6 MJ/kg DM respectively. Twenty-seven male Moghani lambs were assigned randomly into three groups of nine lambs each in a completely randomised design. Three iso-energetic and iso-nitrogenous diets containing different levels (0, 50 or 100 g/kg dry matter) of CSL were offered ad libitum three times a day. Forage to concentrate ratio of the diets was 30:70. With inclusion of CSL in diet, the contents of canola meal, fish meal, wheat bran, corn grain and sugar beet pulp were decreased. The contents of DM, ash-free neutral detergent fibre (NDFom), ether extract, starch, Ca and S were numerically lower, but soluble protein, RDP and non-fibre carbohydrates were greater in the diets containing CSL in comparison with the control diet. The lambs fed with the diets containing CSL had lower [linear (L), p < 0.06] digestibility coefficients of DM and NDFom as compared to those fed with the diet free of CSL. Ruminal ammonia-N concentration increased (L, p < 0.05), but pH decreased (L, p < 0.05) with raising CSL level in diet. Carboxymethyl cellulase and filter paper-degrading activities decreased (L, p < 0.05), while proteases activity increased (L, p < 0.05) as dietary rates of CSL increased. Microcrystalline cellulase and ɑ-amylase activities were similar among the treatments. Within blood metabolites, only urea-N concentration increased (L, p < 0.05) in the lambs receiving CSL as compared to those fed with diet without CSL. Dietary inclusion of CSL resulted in linear decreases (L, p < 0.05) in the intakes of DM, organic matter, CP, NDFom and ash-free acid detergent fibre, and average daily gain. However, the feed conversion ratio was similar among the experimental animals. Overall, feeding CSL up to 100 g/kg diet DM in lamb resulted in reductions of rumen fibrolytic microbial enzyme activities, in vivo digestibility, FI and growth performance, but rumen proteases activity increased.

Polymorphisms in EGFR and IL28B are associated with spontaneous clearance in an HCV-infected Iranian population.

Although most hepatitis C virus (HCV)-infected individuals develop chronic infection, about 25% of them are able to clear the virus spontaneously without any therapeutic intervention. The aim of the present study was to identify genes associated with spontaneous HCV clearance in a population of Iranian patients. We genotyped 110 single-nucleotide polymorphisms (SNPs) in 59 selected--candidate--genes in a cohort of 107 HCV-infected participants who spontaneously cleared the infection and 176 participants whose infection persisted. Three out of the 110 SNPs were found to be associated with HCV outcome (P-values<0.03). rs11506105 in EGFR (epidermal growth factor receptor gene), and rs11881222 and rs12979860 in IL28B (interferon-λ3 gene). Multivariate logistic regression of the three markers showed that the A/A genotypes in both rs11506105 (EFGR) and rs11881222 (IL28B), and the C/C genotype in rs12979860 (IL28B) are associated with HCV clearance (recessive model: odds ratio (OR)=2.06, 95% confidence interval (95% CI)=1.09-3.88, P=0.025; OR=2.09, 95% CI=1.23-3.60, P=0.007; and OR=1.95, 95% CI=1.15-3.35, P=0.014 for rs11506105, rs12979860 and rs11881222, respectively). In conclusion, EGFR and IL28B SNPs are strong independent predictive markers of spontaneous viral clearance.

Changing the incubation temperature during embryonic myogenesis influences the weight performance and meat quality of male and female broilers.

Eggs of broilers were initially incubated at 37.8°C, then at 38.8°C (group H) and 36.8°C (group L) between embryonic days 7 to 10 (time A) and days 10 to 13 (time B) and further incubated at 37.8°C until hatching. The chicks were fattened until day 35 and then slaughtered. The effect of treatment, time, and sex and their interactions on carcass and meat quality traits were determined. No significant impact of the "treatment time" was analysed, but "treatment" and "sex" and their interactions influenced (P < 0.05) the different parameters. After hatching, group L broilers were heavier (P < 0.05), followed by slower growth compared to group H chicken. At slaughter day, body, carcass, and leg weights of group H broiler were higher (P < 0.05) and yield results lower (P < 0.05) compared to group L animals. pH, drip loss, shear force, and lightness values of group H broiler breast muscles were higher (P < 0.05) and grill loss and redness values lower (P < 0.05) compared to group L Musculus pectoralis superficialis (MPS). Body, carcass, MPS, and leg weights, leg yields and grill loss and lightness values of the male broiler were higher (P < 0.05) and drip loss results lower (P < 0.05) than the female birds. Male group H broiler showed lower (P < 0.05) carcass and MPS yields compared to the control and group L males, whereas females group H birds had higher (P < 0.05) carcass and MPS weights and lower leg yields (P < 0.05) than the female group L animals. The study shows that an increasing incubation temperature during early embryogenesis positively influences the growth and carcass traits of the broilers, accompanied with a partly negative impact on meat quality (drip loss, shear force, lightness). The growth effects were sex-dependent, as significant weight differences could be only found in female broilers. The results indicate that incubation temperature alteration influences molecular mechanisms in the muscle and other tissues with an impact on growth after hatch.

Investigating the protective effect of lithium against high glucose-induced neurotoxicity in PC12 cells: involvements of ROS, JNK and P38 MAPKs, and apoptotic mitochondria pathway.

Hyperglycemia that occurs under the diabetic condition is a major cause of diabetic complications such as diabetic neuropathy, one of the most common diabetes-related complications. It is well known that hyperglycemia could result in generation of reactive oxygen species (ROS). Over production of ROS recommended as an important mediator for apoptotic signaling pathway as well as a key early event in the development of diabetic neuropathy. Recently, many studies have indicated that lithium has robust neuroprotective effect in relation to several neurodegenerative diseases. The present study aimed to examine effects of lithium on high glucose (HG)-induced neurotoxicity and to determine some of the underlying molecular mechanisms involved in this response in PC12 cells as a neuronal culture model for diabetic neuropathy. PC12 cells were pretreated with different concentrations of lithium for 7 days, exposed to HG for 24 h. Cell viability was measured by MTT assay. ROS and lipid peroxidation levels as well as superoxide dismutase activity were measured. In order to examine the underlying molecular mechanisms, the expressions of Bax, Bcl-2, Caspase-3, total and phosphorylated JNK and P38 MAPK were also analyzed by Western blotting. The present results indicated that pretreatment with 1 mM lithium has protected PC12 cells against HG-induced apoptotic cell death. It could reduce ROS generation, Bax/Bcl-2 ratio, Caspase-3 activation, and JNK and P38 MAPK phosphorylation. It may be concluded that in HG condition, lithium pretreatment could prevent mitochondrial apoptosis as well as JNK and P38 MAPK pathway in PC12 cells.

Footprints of recent selection and variability in breed composition in the Göttingen Minipig genome.

The Göttingen Minipig (GMP) developed at the University of Göttingen is a synthetic breed that is widely used in medical research and toxicology. It combines the high fertility of the Vietnamese potbellied pig, the low body weight of the Minnesota Minipig and the white coat colour of the German Landrace pig. The aim of this study was to find genomic regions that may have undergone selection since the creation of the breed in the 1960s. Therefore, the whole genome was screened for footprints of recent selection based on single nucleotide polymorphism (SNP) genotypes from the Illumina Porcine SNP60 BeadChip using two methods: the extended haplotype homozygosity (EHH) test and the estimation of the genomic proportion of the three original breeds at each SNP using a Bayesian approach. Local deviations from the average genome-wide breed composition were tested with a permutation-based empirical test. Results for a comprehensive whole-genome scan for both methods are presented. Several regions showing the highest P-values in the EHH test are related to breeding goals relevant in the GMP, such as growth (SOCS2, TXN, DDR2 and GRB10 genes) and white colour (PRLR gene). Additionally, the calculated proportion of the founder breeds diverged significantly in many regions from the pedigree-based expectations and the genome average. The results provide a genome-wide map of selection signatures in the GMP, which leads to a better understanding of selection that took place over the last decades in GMP breed development.