Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa

The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.     

Local immunity of the respiratory mucosal system in chickens and turkeys

This review article presents fundamental mechanisms of the local mucosal immunity in selected regions of the respiratory tract in healthy birds and in some pathological conditions. The respiratory system, whose mucosa come into direct contact with microorganisms contaminating inhaled air, has some associated structures, such as Harderian gland (HG), conjunctive-associated lymphoid tissue (CALT) and paranasal glands (PG), whose participation in local mechanisms of the mucosal immunity has been corroborated by numerous scientific studies. The nasal mucosa, with structured clusters of lymphoid tissue (NALT - nasal-associated lymphoid tissue) is the first to come into contact with microorganisms which contaminate inhaled air. Lymphoid nodules, made up of B cells with frequently developed germinal centres (GC), surrounded by a coat of CD4+ cells, are the major NALT structures in chickens, whereas CD8+ cells are situated in the epithelium and in the lamina propria of the nasal cavity mucosa. Studies into respiratory system infections (e.g. Mycoplasma gallisepticum) have shown the reactivity of the tracheal mucosa to infection, despite a lack of essential lymphoid tissue. Bronchus-associated lymphoid tissue (BALT) takes part in bronchial immune processes and its structure, topography and ability to perform defensive function in birds is largely age-dependent. Mature BALT is covered by a delicate layer of epithelial cells, called follicle-associated epithelium (FAE). Germinal centres (GC), surrounded by CD4+ cells are developed in most mature BALT nodules, while CD8+ lymphocytes are dispersed among lymphoid nodules and in the epithelium, and they are rarely present in GC. Macrophages make up the first line of defence mechanisms through which the host rapidly responds to microorganisms and their products in the respiratory mucosal system. Another very important element are polymorphonuclear cells, with heterophils being the most important of them. Phagocytic cells obtained from lung lavages in birds are referred to as FARM (free avian respiratory macrophages). Their number in chickens and turkeys is estimated to be 20 times lower than that in mice and rats, which indicates a deficit in the first-line of defence in the birds' respiratory system. There are numerous B cells and antibody secreting cells (ASC) present throughout the respiratory system in birds. Their role comes down to perform antigen-specific protection by producing antibodies (IgM, IgY or IgA class) as a result of contact with pathogenic factors.     

The effect of different doses of methisoprinol on the percentage of CD4+ and CD8+ T lymphocyte subpopulation and the antibody titers in pigeons immunised against PPMV-1

As immunosuppression in pigeons is common and results in reduced post-vaccination immunity and lower health status of the birds, studies have been taken up aimed at evaluation of the effect of three doses of methisoprinol on the percentage of CD4+ and CD8+ T lymphocyte subpopulation in peripheral blood and in the spleen and the titre of anti-NDV antibodies in the serum of pigeons in four groups (A, B, C, D), with 20 birds each. Pigeons in each group were immunised against paramyxovirosis at week 6 and 9 of life. Water for injection (group A - control) or methisoprinol at 100 mg/kg of body weight (group B), 200 mg/kg of body weight (group C) and 600 mg/kg of body weight (group D) was administered intramuscularly for 3 days before each vaccination. The immunological analyses were carried out by flow cytometry and the ELISA test. The findings indicate that methisoprinol administered intramuscularly at 100 and 200 mg/kg of body weight for 3 successive days before vaccination against paramyxovirosis mainly stimulates the mechanisms of non-specific humoral and cellular immunity, which is indicated by a higher percentage of the subpopulation of CD4+ T lymphocytes in peripheral blood and in the spleen and a higher titre of anti-NDV antibodies.     

Distribution of Potato virus Y in potato plant organs, tissues, and cells

The distribution of Potato virus Y (PVY) in the systemically infected potato (Solanum tuberosum) plants of the highly susceptible cultivar Igor was investigated. Virus presence and accumulation was analyzed in different plant organs and tissues using real-time polymerase chain reaction and transmission electron microscopy (TEM) negative staining methods. To get a complete insight into the location of viral RNA within the tissue, in situ hybridization was developed and optimized for the detection of PVY RNA at the cellular level. PVY was shown to accumulate in all studied leaf and stem tissues, in shoot tips, roots, and tubers; however, the level of virus accumulation was specific for each organ or tissue. The highest amounts of viral RNA and viral particles were found in symptomatic leaves and stem. By observing cell ultrastructure with TEM, viral cytoplasmic inclusion bodies were localized in close vicinity to the epidermis and in trichomes. Our results show that viral RNA, viral particles, and cytoplasmic inclusion bodies colocalize within the same type of cells or in close vicinity.     

Use of the BCR three-step sequential extraction procedure for the study of the partitioning of Cd, Pb and Zn in various soil samples

A slightly modified three-step sequential extraction procedure proposed by the Community Bureau of Reference (BCR) for analysis of sediments was successfully applied to soil samples. Contaminated soil samples from the lead and zinc mining area in the Mezica valley (Slovenia) and natural soils from a non-industrial area were analysed. The total concentrations of Cd, Pb and Zn and their concentrations in fractions after extraction were determined by flame or electrothermal atomic absorption spectrometry (FAAS, ETAAS). Total metal concentrations in natural soils ranged from 0.3 to 2.6 mg kg^-1 for Cd, from 20 to 45 mg kg^-1 for Pb and from 70 to 140 mg kg^-1 for Zn, while these concentrations ranged from 0.5 to 35 mg kg^-1 for Cd, from 200 to 10000 mg kg^-1 for Pb and from 140 to 1500 mg kg^-1 for Zn in soils from contaminated areas. The results of the partitioning study applying the slightly modified BCR three-step extraction procedure indicate that Cd, Pb and Zn in natural soils prevails mostly in sparingly soluble fractions. Cd in natural soils is bound mainly to Fe and Mn oxides and hydroxides, Pb to organic matter, sulphides and silicates, while Zn is predominantly bound to silicates. In contaminated soils, Cd, Pb and Zn are distributed between the easily and sparingly soluble fractions. Due to the high total Cd, Pb and Zn concentrations in contaminated soil close to the smelter, ! and their high proportions in the easily soluble fraction (80% of Cd, 50% of Pb and 70% of Zn), the soil around smelters represents an environmental hazard.     

Determination of Plant-Available Nutrients in Two Wood Ashes: The Influence of Combustion Conditions

Wood ashes were traditionally used as a fertilizing agent from the very beginning of working land but the fertilizing quality varies according to the combusted biomass and also the industrial burning equipment. The differences between wood ash originated from fluid and classic boiler were investigated by X-ray powder diffraction (XRD), X-ray fluorescence spectrometry (XRF), differential thermal analysis (DTA), thermo-gravimetric analysis (TGA), scanning electron microscopy (SEM), and Mehlich 3 and deionized water extraction procedures in relation to extraction time. The results, especially of the SEM and chemical extractions, have demonstrated that the type of burning boiler determines the portions of the leachable nutrient elements. The silica and alumina ash content conducted by the elevated temperature transforms the main proportion of calcium and potassium into the insoluble feldspars. Due to this effect only a small amount of nutrients could be released and served as a fertilizing matter.     

The effect of “acid rain” and mineral fertilizers on the biometrical features of larix decidua mill. Seedlings

The purpose of the present experiment was to describe the influence of various mineral fertilizers on the development of Larix decidua Mill. seedlings subject to the influence of simulated acid rain of pH 2.5, 3.0, 3.5, 4.0, 4.5. The seedlings were fertilized every month (from April up to October), in order to counteract acid rain. The composition of the fertilizers was diversified in order to select an optimum variant. Fertilizer with NK applied to the needles (Florovit) and NPK applied to the soil (Fruktus 2) as well as dolomite lime applied to the soil. One row per set was left without fertilization as a control for each pH step of acid rain treatment. The analysis of biometric characteristics showed that a two-year period of investigations was too short to estimate effects on Larix decidua Mill. seedlings. It, however, indicated important trends. High correlations were found between particular growth parameters i.e. diameter of root neck, root mass, total mass of: seedlings, needles, main shoots, lateral shoots, ligneous parts, total overground parts. No strong correlations were observed between the above mentioned parameters and the total height of the seedlings. Among the different treatments the best development was achieved at pH 4.0, whereas the weakest development was observed at pH 2.5. The analysis showed a decrease in biometrical features along with an increase in acidity of acid rain solution. The first year of the experiment had the greatest influence upon the growth of Larix decidua Mill. seedlings. Significant improvement in growth, in relation to control plots, was achieved by using NK-fertilizers onto needles — the best results, and NPK onto soil. The best increment of the height of seedling was noticed at pH 3.5. In the second year of the experiment the effects of pH of acid rain and fertilization decreased — no significant effect on biometrical features was observed. Applaying NPK fertilizer onto soil showed a positive effect in relation to dolomite treatment. The best increment was noticed at pH 4.0.     

Saprotrophic fungi transform organic phosphorus from spruce needle litter

Fungal decomposition of and phosphorus transformation from spruce litter needles (Picea abies) were simulated in systems containing litter needles inoculated with individual saprotrophic fungal strains and their mixtures. Fungal strains of Setulipes androsaceus (L.) Antonín, Chalara longipes (Preus) Cooke, Ceuthospora pinastri (Fr.) Höhn., Mollisia minutella (Sacc.) Rehm, Scleroconidioma sphagnicola Tsuneda, Currah & Thormann and an unknown strain NK11 were used as representatives of autochthonous mycoflora. Systems were incubated for 5.5 months in laboratory conditions. Fungal colonization in systems and competition among strains were assessed using the reisolation of fungi from individual needles. After incubation, needles were extracted with NaOH and extracts were analysed using ³¹P nuclear magnetic resonance spectroscopy (NMR). Needle decomposition was determined based on the decrease in C:N ratio. Systems inoculated with the basidiomycete S. androsaceus revealed substantial decrease in C:N ratio (from 25.8 to 11.3) while the effect of ascomycetes on the C:N ratio was negligible. We suppose that tested strains of saprotrophic ascomycetes did not participate substantially in litter decomposition, but were directly involved in phosphorus transformation and together with S. androsaceus could transform orthophosphate monoesters and diesters from spruce litter needles into diphosphates, polyphosphates and phosphonates. These transformations seem to be typical for saprotrophic fungi involved in litter needle decomposition, although the proportion of individual phosphorus forms differed among studied fungal strains. Phosphonate presence in needles after fungal inoculation is of special interest because no previous investigation recorded phosphonate synthesis and accumulation by fungi. Our results confirmed that the ³¹P NMR spectroscopy is an excellent instrumental method for studying transformations of soil organic phosphorus during plant litter decomposition. We suggest that polyphosphate production by S. androsaceus may contribute to the phosphorus cycle in forest ecosystems because this fungus is a frequent litter colonizer that substantially participates in decomposition.     

Macroinvertebrate Community and Chemistry of the Most Atmospherically Acidified Streams in the Czech Republic

We performed a comparative study of strongly acidified mountain streams covering 10 sites in the Czech Republic in the season 1999/2000. The aim of the study was to determine how acidification influenced macroinvertebrate community structure within a relatively narrow pH range. We focused on strongly acidified, non-humic running waters with low ionic content (pH < 4.6, total organic carbon < 10 mg l^?1, specific conductivity < 100 μS cm^?1) and minimum human influence in the catchment. The actual pH values ranged from 3.98 to 4.65, and concentrations of reactive aluminium ranged from 0.2 to 2.0 mg l^?1. Characteristic macroinvertebrates were the stoneflies Leuctra nigra, Nemurella pictetii, and Protonemura spp.; the chironomids Corynoneura spp.; and the caddisfly Plectrocnemia conspersa – the former two stoneflies formed together 46% of total organisms. The sites were divided into three groups with use of divisive classification. Individual groups reflected similarity in water chemistry, catchment characteristics, and geographical proximity. The largest biotic difference detected by PCA was in the abundance of stoneflies, mainly Diura bicaudata and Leuctra major. The strongest correlation with this gradient was shown by pH (and associated heavy metals), followed by the distance from source. The results show that even in such narrow pH range, the number of taxa is determined by the low pH value and related high concentrations of aluminium and heavy metals.