Blood pressure lowering effects of Lactobacillus helveticus fermented milk containing bioactive peptides Ile-Pro-Pro and Val-Pro-Pro; mechanistic, kinetic and clinical studies

The purpose of the present study was to evaluate the effects of Lactobacillus helveticus fermented milk (peptide milk) containing the casein-derived tripeptides Isoleucyl-prolyl-proline (Ile-Pro-Pro) and Valyl-prolyl-proline (Val-Pro-Pro) on blood pressure and vascular function in hypertensive subjects. The peptide milk lowered systolic and diastolic blood pressure in long-term use in hypertensive subjects when blood pressure was measured by using 24-hour ambulatory blood pressure measurement (ABPM). The blood pressure lowering effect was seen with the dose of 50 mg of tripeptides, and a tendency for lowering blood pressure was also observed when the dose was 5 mg. No adverse effects compared to the placebo group were reported or detected in laboratory analysis. The effect of the peptide milk on arterial stiffness was shown using two different methods, the ambulatory arterial stiffness index (AASI) and pulse wave analysis (PWA). According to the AASI, arterial stiffness was significantly reduced in the peptide milk group compared to the baseline level, but the difference was not significant compared to the placebo group. PWA showed that the peptide milk reduced arterial stiffness significantly compared to the placebo group. Endothelium-independent relaxation (nitroglycerin) and endothelium-dependent relaxation (salbutamol) did not differ between the groups. The blood pressure lowering mechanisms of the tripeptides and the kinetics of Ile-Pro-Pro were investigated using spontaneously hypertensive rats (SHR) and Sprague-Dawley rats. Previous studies have suggested that the blood pressure lowering effect of the tripeptides Ile-Pro-Pro and Val-Pro-Pro is based on angiotensin-converting enzyme inhibition, but the present findings did not agree with these previous studies. It was shown in SHR that calcium, potassium and magnesium may also have an important role in attenuating the development of hypertension as part of the peptide milk effect. In addition, the present study suggests indirectly that improved endothelial nitric oxide release capacity is not the mechanism by which peptide milk mediates its favourable circulatory effects. The kinetics of Ile-Pro-Pro were studied using adult Sprague-Dawley rats. The results showed that orally administered Ile-Pro-Pro is absorbed at least partly intact from the gastrointestinal tract. Radiolabelled Ile-Pro-Pro was distributed in different tissues and considerable radioactivity levels were found in tissues related to the renin-angiotensin system (RAS), adrenals, aorta and kidneys. Ile-Pro-Pro does not bind to plasma proteins, and therefore it is possible that its blood pressure lowering effect is mediated by free Ile-Pro-Pro. In conclusion, consumption of the peptide milk lowers blood pressure and reduces arterial stiffness in hypertensive subjects. Ile-Pro-Pro can be absorbed partly intact from the gastrointestinal tract and might accumulate in tissues related to the RAS. The precise blood pressure lowering mechanism of peptide milk remains to be studied.

Dietary elecrolytes and cyclosporine toxicity. Experimental studies in spontaneuosly hypertensive rats

Cyclosporine-A (CsA) is widely used after organ transplantation to prevent rejection and in the treatment of autoimmune diseases. Hypertension and nephrotoxicity are common side-effects of CsA. Studies in patients on the prevention of the side-effects of CsA are difficult to conduct because the patients often receive a combination of different drugs thus making study of the side-effects of a single drug impossible. A challenge in experimental studies has been the lack of an animal model in which the side-effects concomitantly occur. Epidemiological data show an association between sodium (Na) intake and blood pressure. There is also evidence on low dietary intake of magnesium (Mg) and potassium (K) and high blood pressure. Our study was designed to develop an experimental model to study the side-effects of CsA in spontaneously hypertensive rats (SHR). On high dietary sodium, CsA caused hypertension, left ventricular hypertrophy (LVH), narrowing of the coronary arteries, small myocardial infarctions, and proteinuria, reduced creatinine clearance and histopathological renal injury in SHR. Loss of Mg into the urine caused by CsA resulted in Mg depletion in the tissues. Renal excretion of dopamine was reduced and the renin-angiotensin-aldosterone system was activated. We investigated the effects of dietary Mg and/or K and the calcium antagonist drug, isradipine, on the prevention of CsA toxicity. Dietary supplementation of Mg alone or in combination with K prevented from the deleterious pathophysiological and histopathological changes in the kidneys and the heart. K alone had little effect. Isradipine protected better than Mg from LVH, but the combination of isradipine and Mg was the most effective. Isradipine did not, however, protect against Mg loss. In our animal model, the combination of high dietary Na and treatment with CsA accelerated the development of the cardiovascular and renal changes clinically known as the side-effects of CsA. Dietary supplementation of Mg and K and reduction of Na intake and the calcium antagonist drug isradipine prevent from the deleterious effects of CsA.

Effects of living crown reduction on needle element status of Scots pine

A wide range of biotic and abiotic factors, operating over different time perspectives and intensities, cause defoliation and a rapid decrease in the crown size of trees. Scleroderris canker disease [Gremmeniella abietina (Lagerb.) Morelet] has caused widespread crown reduction and tree mortality in Scots pine (Pinus sylvestris L) in forests in Scandinavia during the last three decades. In the 1980's, attempts were made to show, on the basis of the higher foliar N and S concentrations of affected pines in the diseased area, that sulphur and nitrogen deposition predispose trees to G. abietina. Unfortunately, in many studies on defoliated trees, exceptionally high or low needle mineral nutrient concentrations are still often interpreted as one of the causes of tree injury and not, conversely, as the result. In this thesis, three different field experiments, with foliar analysis as the main study method, were conducted in order to asses the possible long-term effects of living crown reduction on the needle nutrient concentrations of Scots pine trees in southern Finland. The crown ratio and length of the living crown were used to estimate the amount of defoliation in the reduced canopies. The material for the partial studies was collected and a total of 968 foliar samples were analysed individually (15-17 elements/sample) on a total of 488 sample trees (140 diseased, 116 pruned and 232 control trees) during the years 1987-1996 in 13 Scots pine stands. All the three experiments of this thesis provided significant evidence that severe, disease-induced defoliation or artificial pruning of the living branches can induce long-lasting nutritional changes in the foliage of the recovering trees under the typical growing conditions for Scots pine. The foliar concentrations of all the 17 mineral nutrients/elements analysed were affected, to a varying degree, by artificial pruning during the following three years. Although Scots pine, as an evergreen conifer, is considered to have low induced chemical responses to defoliation, this study proved experimentally under natural forest conditions that severe artificial pruning or disease-induced defoliation of Scots pine trees may induce biologically significant changes in the concentrations of most of the important macro- and micronutrients, as well as of carbon, in refoliated needles. Concerning the studies in this thesis, I find the results significant in providing new information about the long-term effects of rapid living crown reduction on the foliar nutrient and element status of Scots pine trees. Key words: Foliar analysis, defoliation, needle loss, pruning, nutrients, Pinus sylvestris, Gremmeniella abietina

Enzyme molecular choreography : studies on soluble inorganic pyrophosphatases

Inorganic pyrophosphatases (PPases, EC 3.6.1.1) hydrolyse pyrophosphate in a reaction that provides the thermodynamic 'push' for many reactions in the cell, including DNA and protein synthesis. Soluble PPases can be classified into two families that differ completely in both sequence and structure. While Family I PPases are found in all kingdoms, family II PPases occur only in certain prokaryotes. The enzyme from baker's yeast (Saccharomyces cerevisiae) is very well characterised both kinetically and structurally, but the exact mechanism has remained elusive. The enzyme uses divalent cations as cofactors; in vivo the metal is magnesium. Two metals are permanently bound to the enzyme, while two come with the substrate. The reaction cycle involves the activation of the nucleophilic oxygen and allows different pathways for product release. In this thesis I have solved the crystal structures of wild type yeast PPase and seven active site variants in the presence of the native cofactor magnesium. These structures explain the effects of the mutations and have allowed me to describe each intermediate along the catalytic pathway with a structure. Although establishing the ʻchoreographyʼ of the heavy atoms is an important step in understanding the mechanism, hydrogen atoms are crucial for the mechanism. The most unambiguous method to determine the positions of these hydrogen atoms is neutron crystallography. In order to determine the neutron structure of yeast PPase I perdeuterated the enzyme and grew large crystals of it. Since the crystals were not stable at ambient temperature, a cooling device was developed to allow neutron data collection. In order to investigate the structural changes during the reaction in real time by time-resolved crystallography a photolysable substrate precursor is needed. I synthesised a candidate molecule and characterised its photolysis kinetics, but unfortunately it is hydrolysed by both yeast and Thermotoga maritima PPases. The mechanism of Family II PPases is subtly different from Family I. The native metal cofactor is manganese instead of magnesium, but the metal activation is more complex because the metal ions that arrive with the substrate are magnesium different from those permanently bound to the enzyme. I determined the crystal structures of wild type Bacillus subtilis PPase with the inhibitor imidodiphosphate and an inactive H98Q variant with the substrate pyrophosphate. These structures revealed a new trimetal site that activates the nucleophile. I also determined that the metal ion sites were partially occupied by manganese and iron using anomalous X- ray scattering.

Properties of Ammonium Nitrate based fertilisers

The text is divided into three parts; Properties, Application and Safety of Ammonium Nitrate (AN) based fertilisers. In Properties, the structures and phase transitions of ammonium and potassium nitrate are reviewed. The consequences of phase transitions affect the proper use of fertilisers. Therefore the products must be stabilised against the volume changes and consequent loss of bulk density and hardness, formation of dust and finally caking of fertilisers. The effect of different stabilisers is discussed. Magnesium nitrate, ammonium sulphate and potassium nitrate are presented as a good compromise. In the Application part, the solid solutions in the systems (K+,NH4+)NO3- and (NH4+,K+)(Cl-,NO3-) are presented based on studies made with DSC and XRD. As there are clear limits for solute content in the solvent lattice, a number of disproportionation transitions exist in these process phases, e.g., N3 (solid solution isomorphous to NH4NO3-III) disproportionates to phases K3 (solid solution isomorphous to KNO3-III) and K2 (solid solution isomorphous to KNO3-II). In the crystallisation experiments, the formation of K3 depends upon temperature and the ratio K/(K+NH4). The formation of phases K3, N3, and K2 was modelled as a function of temperature and the mole ratios. In introducing chlorides, two distinct maxima for K3 were found. Confirmed with commercial potash samples, the variables affecting the reaction of potassium chloride with AN are the particle size, time, temperature, moisture content and amount of organic coating. The phase diagrams obtained by crystallisation studies were compared with a number of commercial fertilisers and, with regard to phase composition, the temperature and moisture content are critical when the formation and stability of solid solutions are considered. The temperature where the AN-based fertiliser is solidified affects the amount of compounds crystallised at that point. In addition, the temperature where the final moisture is evaporated affects the amount and type of solid solution formed at this temperature. The amount of remaining moisture affects the stability of the K3 phase. The K3 phase is dissolved by the moisture and recrystallised into the quantities of K3, which is stable at the temperature where the sample is kept. The remaining moisture should not be free; it should be bound as water in the final product. The temperatures during storage also affect the quantity of K3 phase. As presented in the figures, K3 phase is not stable at temperatu¬res below 30 °C. If the temperature is about 40 °C, the K3 phase can be formed due to the remaining moisture. In the Safety part, self-sustaining decomposition (SSD), oxidising and energetic properties of fertilisers are discussed. Based on the consequence analysis of SSD, early detection of decomposition in warehouses and proper temperature control in the manufacturing process is important. SSD and oxidising properties were found in compositions where K3 exists. It is assumed that potassium nitrate forms a solid matrix in which AN can decompose. The oxidising properties can be affected by the form of the product. Granular products are inherently less oxidising. Finally energetic properties are reviewed. The composition of the fertiliser has an importance based on theoretical calculations supported by experimental studies. Materials such as carbonates and sulphates act as diluents. An excess of ammonium ions acts as a fuel although this is debatable. Based on the experimental work, the physical properties have a major importance over the composition. A high bulk density is of key importance for detonation resistance.

Studies on metal complex formation of environmentally friendly aminopolycarboxylate chelating agents

Aminopolykarboksyylaatteja, kuten etyleenidiamiinitetraetikkahappoa (EDTA), on käytetty useiden vuosikymmenien ajan erinomaisen metalli-ionien sitomiskyvyn vuoksi kelatointiaineena lukuisissa sovelluksissa sekä analytiikassa että monilla teollisisuuden aloilla. Näiden yhdisteiden biohajoamattomuus on kuitenkin herättänyt huolta viime aikoina, sillä niiden on havaittu olevan hyvin pysyviä luonnossa. Tämä työ on osa laajempaa tutkimushanketta, jossa on tavoitteena löytää korvaavia kelatointiaineita EDTA:lle. Tutkimuksen aiheena on kuuden kelatointiaineen metalli-ionien sitomiskyvyn kartoitus. EDTA:a paremmin luonnossa hajoavina nämä ovat ympäristöystävällisiä ehdokkaita korvaaviksi kelatointiaineiksi useisiin sovelluksiin. Työssä tutkittiin niiden kompleksinmuodostusta useiden metalli-ionien kanssa potentiometrisella titrauksella. Metalli-ionivalikoima vaihteli hieman kelatointiaineesta riippuen sisältäen magnesium-, kalsium-, mangaani-, rauta-, kupari-, sinkki-, kadmium-, elohopea-, lyijy- ja lantaani-ionit. Tutkittavat metallit oli valittu tähtäimessä olevien sovellusten, synteesissä ilmenneiden ongelmien tai ympäristönäkökohtien perusteella. Tulokset osoittavat näiden yhdisteiden metallinsitomiskyvyn olevan jonkin verran heikompi kuin EDTA:lla, mutta kuitenkin riittävän useisiin sovelluksiin kuten sellunvalkaisuprosessiin. Myrkyllisten raskasmetallien, kadmiumin, elohopen ja lyijyn kohdalla EDTA:a heikompi sitoutuminen on eduksikin, koska se yhdistettynä parempaan biohajoavuuteen saattaa alentaa tutkittujen yhdisteiden kykyä mobilisoida kyseisiä metalleja sedimenteistä. Useimmilla tutkituista yhdisteistä on ympäristönäkökulmasta etuna myös EDTA:a pienempi typpipitoisuus.

Stabilization of Phospholipid Coatings in Capillary Electrophoresis

The development of a simple method of coating a semi-permanent phospholipid layer onto a capillary for electrochromatography use was the focus of this study. The work involved finding good coating conditions, stabilizing the phospholipid coating, and examining the effect of adding divalent cations, cetyltrimethylammonium bromide, and polyethylene glycol (PEG)-lipids on the stability of the coating. Since a further purpose was to move toward more biological membrane coatings, the capillaries were also coated with cholesterol-containing liposomes and liposomes of red blood cell ghost lipids. Liposomes were prepared by extrusion, and large unilamellar vesicles with a diameter of about 100 nm were obtained. Zwitterionic phosphatidylcholine (PC) was used as a basic component, mainly 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) but also eggPC and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Different amounts of sphingomyelin, bovine brain phosphatidylserine, and cholesterol were added to the PC. The stability of the coating in 40 mM N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) solution at pH 7.4 was studied by measuring the electroosmotic flow and by separating neutral steroids, basic proteins, and low-molar-mass drugs. The presence of PC in the coating solution was found to be essential to achieving a coating. The stability of the coating was improved by the addition of negative phosphatidylserine, cholesterol, divalent cations, or PEGylated lipids, and by working in the gel-state region of the phospholipid. Study of the effect on the PC coating of divalent metal ions calcium, magnesium, and zinc showed a molar ratio of 1:3 PC/Ca2+ or PC/Mg2+ to give increased rigidity to the membrane and the best coating stability. The PEGylated lipids used in the study were sterically stabilized commercial lipids with covalently attached PEG chains. The vesicle size generally decreased when PEGylated lipids of higher molar mass were present in the vesicle. The predominance of discoidal micelles over liposomes increased PEG chain length and the average size of the vesicles thus decreased. In the capillary electrophoresis (CE) measurements a highly stable electroosmotic flow was achieved with 20% PEGylated lipid in the POPC coating dispersion, the best results being obtained for disteroyl PEG (3000) conjugates. The results suggest that smaller particles (discoidal micelles) result in tighter packing and better shielding of silanol groups on the silica wall. The effect of temperature on the coating stability was investigated by using DPPC liposomes at temperatures above (45 C) and below (25 C) the main phase transition temperature. Better results were obtained with DPPC in the more rigid gel state than in the fluid state: the electroosmotic flow was heavily suppressed and the PC coating was stabilized. Also dispersions of DPPC with 0−30 mol% of cholesterol and sphingomyelin in different ratios, which more closely resemble natural membranes, resulted in stable coatings. Finally, the CE measurements revealed that a stable coating is formed when capillaries are coated with liposomes of red blood cell ghost lipids.

The effects of human induced disturbances on the sediment geochemistry of Baltic Sea embayments with a focus on eutrophication history

Historical sediment nutrient concentrations and heavy-metal distributions were studied in five embayments in the Gulf of Finland and an adjacent lake. The main objective of the study was to examine the response of these water bodies to temporal changes in human activities. Sediment cores were collected from the sites and dated using 210Pb and 137Cs. The cores were analyzed for total carbon (TC), total nitrogen (TN), total phosphorus (TP), organic phosphorus (OP), inorganic phosphorus (IP), biogenic silica (BSi), loss on ignition (LOI), grain size, Cu, Zn, Al, Fe, Mn, K, Ca, Mg and Na. Principal component analysis (PCA) was used to summarize the trends in the geochemical variables and to compare trends between the different sites. The links between the catchment land use and sediment geochemical data were studied using a multivariate technique of redundancy analysis (RDA). Human activities produce marked geochemical variations in coastal sediments. These variations and signals are often challenging to interpret due to various sedimentological and post-depositional factors affecting the sediment profiles. In general, the sites studied here show significant upcore increases in sedimentation rates, TP and TN concentrations. Also Cu, which is considered to be a good indicator of anthropogenic influence, showed clear increases from 1850 towards the top part of the cores. Based on the RDA-analysis, in the least disturbed embayments with high forest cover, the sediments are dominated by lithogenic indicators Fe, K, Al and Mg. In embayments close to urban settlement, the sediments have high Cu concentrations and a high sediment Fe/Mn ratio. This study suggests that sediment accumulation rates vary significantly from site to site and that the overall sedimentation can be linked to the geomorphology and basin bathymetry, which appear to be the major factors governing sedimentation rates; i.e. a high sediment accumulation rate is not characteristic either to urban or to rural sites. The geochemical trends are strongly site specific and depend on the local geochemical background, basin characteristics and anthropogenic metal and nutrient loading. Of the studied geochemical indicators, OP shows the least monotonic trends in all studied sites. When compared to other available data, OP seems to be the most reliable geochemical indicator describing the trophic development of the study sites, whereas Cu and Zn appear to be good indicators for anthropogenic influence. As sedimentation environments, estuarine and marine sites are more complex than lacustrine basins with multiple sources of sediment input and more energetic conditions in the former. The crucial differences between lacustrine and estuarine/coastal sedimentation environments are mostly related to Fe. P sedimentation is largely governed by Fe redox-reactions in estuarine environments. In freshwaters, presence of Fe is clearly linked to the sedimentation of other lithogenic metals, and therefore P sedimentation and preservation has a more direct linkage to organic matter sedimentation.

Geological factors affecting on after-use of Finnish cut-over peatlands : with implications on the carbon accumulation

In Finland, peat harvesting sites are utilized down almost to the mineral soil. In this situation the properties of mineral subsoil are likely to have considerable influence on the suitability for the various after-use forms. The aims of this study were to recognize the chemical and physical properties of mineral subsoils possibly limiting the after-use of cut-over peatlands, to define a minimum practice for mineral subsoil studies and to describe the role of different geological areas. The future percentages of the different after-use forms were predicted, which made it possible to predict also carbon accumulation in this future situation. Mineral subsoils of 54 different peat production areas were studied. Their general features and grain size distribution was analysed. Other general items studied were pH, electrical conductivity, organic matter, water soluble nutrients (P, NO3-N, NH4-N, S and Fe) and exchangeable nutrients (Ca, Mg and K). In some cases also other elements were analysed. In an additional case study carbon accumulation effectiveness before the intervention was evaluated on three sites in Oulu area (representing sites typically considered for peat production). Areas with relatively sulphur rich mineral subsoil and pool-forming areas with very fine and compact mineral subsoil together covered approximately 1/5 of all areas. These areas were unsuitable for commercial use. They were recommended for example for mire regeneration. Another approximate 1/5 of the areas included very coarse or very fine sediments. Commercial use of these areas would demand special techniques - like using the remaining peat layer for compensating properties missing from the mineral subsoil. One after-use form was seldom suitable for one whole released peat production area. Three typical distribution patterns (models) of different mineral subsoils within individual peatlands were found. 57 % of studied cut-over peatlands were well suited for forestry. In a conservative calculation 26% of the areas were clearly suitable for agriculture, horticulture or energy crop production. If till without large boulders was included, the percentage of areas suitable to field crop production would be 42 %. 9-14 % of all areas were well suitable for mire regeneration or bird sanctuaries, but all areas were considered possible for mire regeneration with correct techniques. Also another 11 % was recommended for mire regeneration to avoid disturbing the mineral subsoil, so total 20-25 % of the areas would be used for rewetting. High sulphur concentrations and acidity were typical to the areas below the highest shoreline of the ancient Litorina sea and Lake Ladoga Bothnian Bay zone. Also differences related to nutrition were detected. In coarse sediments natural nutrient concentration was clearly higher in Lake Ladoga Bothnian Bay zone and in the areas of Svecokarelian schists and gneisses, than in Granitoid area of central Finland and in Archaean gneiss areas. Based on this study the recommended minimum analysis for after-use planning was for pH, sulphur content and fine material (<0.06 mm) percentage. Nutrition capacity could be analysed using the natural concentrations of calcium, magnesium and potassium. Carbon accumulation scenarios were developed based on the land-use predictions. These scenarios were calculated for areas in peat production and the areas released from peat production (59300 ha + 15 671 ha). Carbon accumulation of the scenarios varied between 0.074 and 0.152 million t C a-1. In the three peatlands considered for peat production the long term carbon accumulation rates varied between 13 and 24 g C m-2 a-1. The natural annual carbon accumulation had been decreasing towards the time of possible intervention.

Snow characteristics in Dronning Maud Land, Antarctica

Snow cover is very sensitive to climate change and has a large feedback effect on the climate system due to the high albedo. Snow covers almost all surfaces in Antarctica and small changes in snow properties can mean large changes in absorbed radiation. In the ongoing discussion of climatic change, the mass balance of Antarctica has received increasing focus during recent decades, since its reaction to global warming strongly influences sea-level change. The aim of the present work was to examine the spatial and temporal variations in the physical and chemical characteristics of surface snow and annual accumulation rates in western Dronning Maud Land, Antarctica. The data were collected along a 350-km-long transect from the coast to the plateau during the years 1999-2004 as a part of the Finnish Antarctic Research Programme (FINNARP). The research focused on the most recent annual accumulation in the coastal area. The results show that the distance from the sea, and the moisture source, was the most predominant factor controlling the variations in both physical (conductivity, grain size, oxygen isotope ratio and accumulation) and chemical snow properties. The sea-salt and sulphur-containing components predominated in the coastal region. The local influences of nunataks and topographic highs were also visible on snow. The variations in all measured properties were wide within single sites mostly due to redistribution by winds and sastrugi topography, which reveals the importance of the spatially representative measurements. The mean accumulations occurred on the ice shelf, in the coastal region and on the plateau: 312 ± 28, 215 ± 43 and 92 ± 25 mm w.e., respectively. Depth hoar layers were usually found under the thin ice crust and were associated with a low dielectric constant and high concentrations of nitrate. Taking into account the vast size of the Antarctic ice sheet and its geographic characteristics, it is important to extend investigation of the distribution of surface snow properties and accumulation to provide well-documented data.

Components of fractionated stallion seminal plasma and the effects of seminal plasma on sperm longevity

Seminal plasma (SP) is the fluid portion of semen, secreted by the epididymides and the accessory glands before and during ejaculation. The stallion s ejaculate is a series of jets that differ in sperm concentration, semen volume and biochemical composition. Before the actual ejaculation, a clear and watery pre-sperm fluid is secreted. The first three jets form the sperm-rich fractions, and contain ¾ of the total number of sperm. The semen volume and sperm concentration in each of the jets decrease towards the end of the ejaculation, and the last jets are sperm-poor fractions with a low sperm concentration. The aims of these studies were to examine the effects of the different SP fractions, and the presence of SP, on sperm survival during storage. Pre-sperm fluid, and semen fractions with a high (sperm-rich) and low (sperm-poor) sperm concentration were collected in five experiments. The levels of selected enzymes, electrolytes and proteins in different SP fractions were determined. These studies also aimed at assessing the individual variation in the levels of the selected SP components and in the effects of SP on spermatozoa. The association between the components of SP and semen quality, sperm longevity, and fertility was examined with a stepwise linear regression analysis. Compared to samples containing SP during storage, centrifugation and the subsequent removal of SP reduced sperm motility parameters during 24 h of cooled storage in all SP fractions, but sperm membrane integrity was not affected. Some of the measured post-thaw motility parameters were also higher in samples containing SP compared to samples stored without SP. In contrast, the proportion of DNA-damaged spermatozoa was greater in the samples stored with SP than those without SP, and this effect was seen in both sperm-rich and sperm-poor fractions. There were no differences in DNA integrity between fractions stored with SP, but the sperm-rich fraction showed less DNA damage than the sperm-poor fraction after SP removal. The differences between fractions in sperm motility after cooled storage were non-significant. The levels of alkaline phosphatase, acid phosphatase and β-glucuronidase were higher in the sperm-rich fractions compared to the sperm-poor fractions, while the concentrations of calcium and magnesium were higher in sperm-poor fractions than in sperm-rich fractions. The concentrations of sodium and chloride were highest in pre-sperm fluid. In the sperm-poor fraction, the level of potassium was associated with the maintenance of sperm motility during storage. The levels of alkaline and acid phosphatase were associated with sperm concentration and the total number of spermatozoa in the ejaculates. None of the measured SP components were correlated to the first cycle pregnancy rate. In summary, the removal of SP improved DNA integrity after cooled storage compared with samples containing SP. There were no differences in the maintenance of sperm motility between the sperm-rich and sperm-poor fractions and whole ejaculates during cooled storage, irrespective of the presence of SP. The lowest rate of DNA damage was found in the sperm-rich fractions stored without SP. In practice, the results presented in this thesis support the use of individual modifications of semen processing techniques for cooled transported semen from subfertile stallions.

Veden ja järvisedimenttien laatu Vihtamonjoen valuma-alueella Sotkamossa

The study area, Vihtamonjoki catchment area, is 55 square kilometres and a third of it has been ditched. The largest ditchings have been done in years 1959-1970. The water system in the catchment area builds up of several lake basins, brooks and rivers. This study tries to discover the water quality at present. It also tries to determine the sedimentation rate and the changes on the sediment quality during the past decades. The water samples were collected in August 2003 and in March 2004 from several places in the catchment area. On March 2004 the sediment samples were collected from four lake basins. Organic matter, total phosphorus, iron, manganese, Fe/Mn-ratio, zinc and copper were determined from sediment samples. The water quality was determined by electric conductivity, alkalinity, pH, oxygen content and the content of sodium, potassium, magnesium, calcium, sulphate, chlorine and fluoride. Also the nutrients, nitrate, ammonium and phosphate, were determined. Chemical analyses and loss on ignition analyses showed clear changes in sediment quality in samples taken from 15-25 cm depth, thus showing the time of the ditching. In most cases the forest ditching had caused increase in mineral matter, iron, zinc and copper and decrease in total phosphorus and organic matter. Sedimentation rates vary between 4,1 to 6,7 mm/year in lakes after the forest ditching. Sedimentation rates have probably increased due to the forest ditching. The Fe/Mn-ratio shows that there has been a lack of oxygen in the lakes for some years after the forest ditching. The water quality proved to be normal in the Vihtamonjoki catchment area. Oxygen content in March 2004 pointed to the conclusion that there might be lack of oxygen in winter. Other analysis showed the water quality to be typical for the Kainuu area.