Efficiency of three boar sperm enrichment techniques

The objective of the study was to investigate the efficiency of three enrichment methods to separate boar spermatozoa. Twenty-four ejaculates from 12 boars (2 ejaculates/boar) were extended (30 × 10⁶ spermatozoa/mL) in commercial Beltsville Thawing Solution. Each semen sample was processed with glass wool column (GW) and glass beads (GB) filtration and with the single-layer centrifugation (SLC) technique. Semen samples before (control; C) and after treatment were evaluated for sperm CASA motility/kinetics and concentration, viability, morphology and chromatin integrity. Data were analysed with mixed models. The concentration of total and motile spermatozoa was significantly decreased after treatment in groups GW and SLC, but not in group GB. Group GW showed increased values of WOB compared with both groups C and GB. Group GB showed greater values of rapid movement spermatozoa and lower values of slow movement spermatozoa compared with group C. In group SLC, higher values of VSL, LIN and STR were observed compared with group C. In conclusion, all techniques under examination enhanced various CASA variables. Based on our results, the GB method is a promising alternative separation technique for boar sperm and deserves further research regarding swine in vitro fertilization.     

Genetic diversity,structure and fruit trait associations in Greek sweet cherry cultivars using microsatellite based (SSR/ISSR) and morpho-physiological markers

It is important to couple phenotypic analysis with genetic diversity for germplasm conservation in gene bank collections. The use of molecular markers supports the study of genetic marker-trait associations of biological and agronomic interest on diverse genetic material. In this report, 19 Greek traditional sweet cherry cultivars and two international cultivars, which were used as controls, were grown in Greece and characterized for 17 morpho-physiological traits, 15 simple sequence repeat (SSR) loci and 10 inter simple sequence repeat (ISSR) markers. To our knowledge, this is the first report on molecular genetic diversity studies in sweet cherry in Greece. Principal component analysis (PCA) of nine qualitative and eight quantitative morphological parameters explain over 77.33% of total variability in the first five axes. The SSR markers yielded a combined matching probability ratio (MPR) of 9.569 × e−12. The 15 SSR loci produced a total of 92 alleles. Ten ISSR primers generated 91 bands, with an average of 9.1 bands per primer. Expected heterozygosity (gene diversity) values of 15 SSR loci and 10 ISSR markers averaged at 0.683 and 0.369, respectively. Based on stepwise multiple regression analysis (MRA), SSR alleles were found associated with harvest time and fruit polar diameter. Furthermore, three ISSR markers were correlated with fruit harvest and soluble solids and four ISSR markers were correlated with fruit skin color. Stepwise MRA identified six SSR alleles associated with harvest time with a high correlation (P < 0.001), with linear associations with high F values. Hence, data analyzed by the use of MRA could be useful in marker-assisted breeding programs when no other genetic information is available.     

Characterizing NAD-dependent alcohol dehydrogenase enzymes of Methylobacterium extorquens and strawberry (Fragaria x ananassa cv. Elsanta)

The methylotroph Methylobacterium extorquens (strain with CABI registration number IMI 369321), which has been isolated from strawberry (Fragaria x ananassa cv. Elsanta) callus cultures, was grown on a mixture of methanol (0.25% v/v) and 1,2-propanediol (0.75% v/v). The microbial biotransformation of 1,2-propanediol to 2-hydroxypropanal (lactaldehyde) was studied. The bacterial alcohol dehydrogenase (ADH) enzymatic activities were assessed, and the optimum pH for ADH activity was found to be pH 6.0. Enzyme assays were carried out for both the bacterial and the strawberry extracts to define the best substrate specificity. For Methylobacterium extorquens, the best substrates were found to be methanol (Km = 0.78 mM) and 1,2-propanediol (Km = 15.84 mM), whereas for strawberries, 1-propanol (Km = 3.54 mM) and ethanol (Km = 6.66 mM) were the best substrates. A wide variety of metals as well as EDTA were shown to decrease the enzymatic activity. Furthermore, SDS-PAGE experiments showed molecular weights of 45.0 and 24.6 kDa for the alcohol dehydrogenases of Methylobacterium extorquens and Fragaria x ananassa, respectively.     

Rapid and direct low micromolar NMR method for the simultaneous detection of hydrogen peroxide and phenolics in plant extracts

A rapid and direct low micromolar 1H NMR method for the simultaneous identification and quantification of hydrogen peroxide and phenolic compounds in plant extracts was developed. The method is based on the highly deshielded 1H NMR signal of H?O? at ～10.30 ppm in DMSO-d? and the combined use of picric acid and low temperature, near the freezing point of the solution, in order to achieve the minimum proton exchange rate. Line widths of H?O? below 3.8 Hz were obtained for several Greek oregano extracts which resulted in a detection limit of 0.7 μmol L?1. Application of an array of NMR experiments, including 2D 1H-13C HMBC, spiking of the samples with H?O?, and variable temperature experiments, resulted in the unequivocal assignment of H?O? precluding any confusion with interferences from intrinsic phenolics in the extract.     

Irrigating Onions and Potatoes with Chromium and Nickel: Its Effects on Catalase and Peroxidase Activities and the Cross-Contamination of Plants

Tropospheric ozone (O₃) has long been documented to cause an injury to plants, but a plants’ protectant, widely applicable in agronomical practice, does not exist. We evaluated the potential antiozonate efficacy of the antitranspirant di-1-p-menthene (Vapor Gard) compared with ethylenediurea (EDU) on Bel-W3 tobacco plants. Plants were treated either with water, or by EDU (10, 100, and 500 mg dm^?3), or by vapor (1, 5, 10, and 50 ml dm^?3) and were exposed either to O₃-enriched (90 ppb) or O₃-free air, for 12 days and 8 h day^?1. EDU when applied at 10 mg dm^?3 did not protect the plants against O₃, but when applied at 100 and 500 mg dm^?3 offered a significant protection to the plants. Vapor, when applied at 1 ml dm^?3 did not protect the plants against O₃, neither by terms of foliar visible injury nor by terms of aboveground biomass. In addition, when applied at 10 and 50 ml dm^?3 caused phytotoxicity to all the plants, which it was expressed as necrotic spots on the leaves’ surface, misshaping of the leaves, or short plants' height_. It is obvious that vapor does not protect Bel-W3 tobacco plants against O₃. The antiozonate role of di-1-p-menthene is species-specific and probably occurs only under short-term exposures.     

Reduction of Pb,Zn and Cd Availability from Tailings and Contaminated Soils by the Application of Lignite Fly Ash

The effectiveness of lignite fly ash for stabilization of Pb,Zn and Cd in tailings and contaminated soils was examined. Flyash was mixed with the contaminated soil or oxidic tailingssamples at various doses and pot experiments were performed.The effectiveness of stabilization was mainly evaluated by thestandard US EPA TCLP toxicity test. The 5-stage sequentialextraction procedure was also applied to determine the form ofcontaminants in the fly ash amended soil or tailings samples.Complementary EDTA extraction tests were also carried out. Thefly ash treatment resulted in the reduction of TCLP solubilityof Pb, Zn and Cd to below the respective regulatory limits at5 and 10% w/w fly ash addition rates in tailings and soil,respectively. Speciation of lead, in the treated soil andtailings samples indicated that there was a significanttransition of the heavy metals form from the exchangeable andcarbonate fractions to reducible and residual, suggesting thatthe potential mechanisms of heavy metals retention are, apartfrom increase of pH, sorption on the oxides and hydroxidessurfaces and binding with the hydrated fly ash compounds.     

Local management and landscape drivers of pollination and biological control services in a Kenyan agro-ecosystem

Arthropods that have a direct impact on crop production (i.e. pests, natural enemies and pollinators) can be influenced by both local farm management and the context within which the fields occur in the wider landscape. However, the contributions and spatial scales at which these drivers operate and interact are not fully understood, particularly in the developing world. The impact of both local management and landscape context on insect pollinators and natural enemy communities and on their capacity to deliver related ecosystem services to an economically important tropical crop, pigeonpea was investigated. The study was conducted in nine paired farms across a gradient of increasing distance to semi-native vegetation in Kibwezi, Kenya. Results show that proximity of fields to semi-native habitats negatively affected pollinator and chewing insect abundance. Within fields, pesticide use was a key negative predictor of pollinator, pest and foliar active predator abundance. On the contrary, fertilizer application significantly enhanced pollinator and both chewing and sucking insect pest abundance. At a 1 km spatial scale of fields, there were significant negative effects of the number of semi-native habitat patches within fields dominated by mass flowering pigeonpea on pollinators abundance. For service provision, a significant decline in fruit set when insects were excluded from flowers was recorded. This study reveals the interconnections of pollinators, predators and pests with pigeonpea crop. For sustainable yields and to conserve high densities of both pollinators and predators of pests within pigeonpea landscapes, it is crucial to target the adoption of less disruptive farm management practices such as reducing pesticide and fertilizer inputs.     

The Impact of Cl ?? Cd Interrelationship on Planning Wastewater Reuse in Cabbage

Two greenhouse pot experiments were conducted in Agrinion, Greece, using a randomized block design in four replications, respectively, as follows: The first one included five levels of treated municipal wastewater (TMWW), being used as an irrigation water source. The second one, five levels of applied Cl at a constant soil applied Cd level of 10.36?mg/kg soil, the plants being irrigated with fresh well water. The purpose of these experiments was to study the impact of the Cl ?? Cd interrelationship on planning TMWW reuse, for the irrigation of Brassica oleracea var. Capitata (cabbage) cv F1 Gloria, ehich was used as test plant, in both of these experiments. It was found that the TMWW Cl content, originating mainly from the procedure of wastewater chlorination, was synergistically interrelated with the toxic heavy metal Cd, increasing its soil availability and cabbage plant leaf uptake (edible plant part). As this increase is directly associated with the consumer??s health, it was suggested that the TMWW be subjected to dechlorination process or the disinfection be made by ozonation or UV, which do not include Cl.     

A new process for the management of olive oil mill waste water and recovery of natural antioxidants

The high polyphenol content of the wastewater is the major environmental problem caused by the olive mills. A pilot scale system for the treatment of the olive oil mills wastewater was developed aiming at the recovery of high added value-contained polyphenols and the reduction of the environmental problems. The treatment system consists of three main successive sections: The first one includes successive filtration stages aiming at the gradual reduction of the wastewater suspended solids up to a limit of 25 microm. The second section includes passing of the filtered wastewater through a series of adsorbent resins (XAD16 and XAD7HP) in order to achieve the de-odoring and decolorization of the wastewater and the removal/ recovery of the polyphenol and lactone content. The third section of the procedure includes the thermal evaporation and recovery of the organic solvents mixture, which has been used in the resin regeneration process, and finally the separation of the polyphenols and other organic substance contents using fast centrifuge partition chromatography. The final outcome of the whole procedure is (i) an odorless yellowish wastewater with a 99.99% reduced content in polyphenols and 98% reduced COD, (ii) an extract rich in polyphenols and lactones with high antioxidant activity and high added value, (iii) an extract containing the coloring substances of the olive fruit, and (iv) pure hydroxytyrosol.     

Quantitative bioluminometric method for DNA-based species/varietal identification in food authenticity assessment

A method is reported for species quantification by exploiting single-nucleotide polymorphisms (SNPs). These single-base changes in DNA are particularly useful because they enable discrimination of closely related species and/or varieties. As a model, quantitative authentication studies were performed on coffee. These involved the determination of the percentage of Arabica and Robusta species based on a SNP in the chloroplastic trnL(UAA)-trnF(GAA) intraspacer region. Following polymerase chain reaction (PCR), the Robusta-specific and Arabica-specific fragments were subjected to 15 min extension reactions by DNA polymerase using species-specific primers carrying oligo(dA) tags. Biotin was incorporated into the extended strands. The products were captured in streptavidin-coated microtiter wells and quantified by using oligo(dT)-conjugated photoprotein aequorin. Aequorin was measured within 3 s via its characteristic flash-type bioluminescent reaction that was triggered by the addition of Ca(2+). Because of the close resemblance between the two DNA fragments, during PCR one species serves as an internal standard for the other. The percentage of the total luminescence signal obtained from a certain species was linearly related to the percent content of the sample with respect to this species. The method is accurate and reproducible. The microtiter well-based assay configuration allows high sample throughput and facilitates greatly the automation.