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.

Efeito da idade sobre a fertilidade de éguas inseminadas com sêmen diluído, resfriado a 14ºC e transportado

Para estudar o efeito da idade sobre características ovulatórias e de fertilidade, 147 ciclos estrais de 99 éguas foram agrupados em classes por idade (1 - 3-6 anos; 2 - 7-10 anos; 3 - 11-14 anos; e 4 - 15-19 anos), de acordo com o ano do nascimento. As éguas foram inseminadas com sêmen diluído, resfriado e transportado de apenas um garanhão, três vezes por semana (segundas, quartas e sextas-feiras).O controle folicular, por meio da palpação retal, e a rufiação foram realizados diariamente, durante todo o período experimental. Utilizou-se, para o transporte, o sêmen diluído no diluidor de leite desnatado-glicose no conteiner Celle modificado, sendo a dose inseminante de 400 x 10(6) espermatozóides móveis, no momento da diluição final, pré-resfriamento. O tempo médio da coleta do sêmen à inseminação artificial foi de 3,5 horas e a temperatura final do sêmen, no momento da inseminação, de 14ºC. Não houve influência da idade sobre a velocidade de crescimento folicular e o tamanho do folículo ovulatório. A fertilidade decaiu após os 15 anos de idade, traduzida pela diminuição da taxa de concepção/ciclo e eficiência de prenhez, entretanto, não foram observadas características indicativas de senescência até os 19 anos de idade.

Efeito do número de inseminações artificiais sobre a fertilidade de éguas inseminadas com sêmen diluído, resfriado a 14ºC e transportado

Estudou-se o efeito do número de inseminações sobre a fertilidade de éguas inseminadas, três vezes/semana (segundas, quartas e sextas-feiras), com sêmen diluído, resfriado e transportado, de apenas um garanhão. As éguas foram inseminadas a partir da detecção, pela palpação retal, de um folículo de 3,0 a 3,5 cm de diâmetro, em um dos ovários, até a ovulação. Utilizaram-se para o transporte o sêmen diluído no diluidor leite desnatado-glicose e o conteiner Celle modificado, sendo a dose inseminante de 400 x 10(6) espermatozóides móveis, no momento da diluição final, pré-resfriamento. de acordo com o número de inseminações artificiais (IA) utilizadas/ciclo, os resultados de 148 ciclos, de 100 éguas, foram agrupados em: 1 IA, 2IA, 3 IA e 4 ou mais IA. A eficiência de prenhez foi de 4,29; 5,04; 5,67; e 3,43, para 1, 2, 3 e 4 ou mais IA, respectivamente. As características foliculares diferiram em relação à freqüência de inseminações com os maiores valores observados no grupo de éguas inseminadas quatro ou mais vezes. Concluiu-se que menor velocidade de crescimento folicular e maior diâmetro do folículo ovulatório estiveram associados a maior número de inseminações artificiais/ciclo. Na presença de uma concentração espermática adequada, o número de inseminações/ciclo não exerceu influência sobre a fertilidade.

Fertilidade do sêmen eqüino diluído, resfriado e transportado

Objetivou-se neste experimento estudar o efeito do transporte de sêmen equino, entre haras, sobre a fertilidade de éguas inseminadas. Setenta e seis fêmeas da raça Mangalarga Marchador foram distribuídas, ao acaso, com número desigual de repetições, para se estudarem os efeitos do sêmen in natura, transportado ou não, e do sêmen diluído, resfriado e transportado, sobre a fertilidade de éguas e diferentes características reprodutivas. O sêmen foi diluído no diluidor lactose-gema de ovo sem glicerina. As fêmeas foram inseminadas, em dias alternados, com sêmen de apenas um garanhão, com volumes/dose inseminante variáveis dependendo do número de fêmeas a serem inseminadas por dia com determinado ejaculado. Utilizou-se para o transporte do sêmen um contêiner especialmente projetado para este fim (MSP-1). As taxas de concepção ao primeiro ciclo e totais, a eficiência de prenhez, o número de ciclos/égua, ciclos/égua gestante, ciclo/prenhez e prenhez/ciclo, o número de IA/égua, o número de IA/égua gestante e o número de IA/égua vazia não foram influenciados pelos tratamentos. O diluidor de lactose gema, modificado pela retirada do glicerol de sua formulação, constitui uma opção para a diluição do sêmen a ser resfriado e transportado. O suceso do transporte do sêmen, diluído e resfriado, a pequenas distâncias, por período de tempo curto, depende mais de um bom diluídor que de um sofisticado contêiner de transporte.

Avaliação de uma Técnica de Diluição e Transporte de Sêmen Eqüino, para as Condições Brasileiras

Ninety-seven Mangalarga Marchador mares were allocated in a randomized experimental design, with different number of replicates per treatments. The objective of this experiment was to study the effect of raw (T3) or diluted, cooled semen transported to the breeding farms 1 (T1) and 3 (T2), on mare fertility and different aspects of reproductive efficiency. The mares were inseminated, with semen of only one stallion (Farm 2), on alternate days using different inseminating volumes, according to number of inseminated mares per day using only one ejaculate. For transportation, semen diluted in a lactose-egg yolk modified extender and the MSP-2 container were used. There was no difference among treatments in relation to conception rates of mares inseminated with raw or diluted, cooled and transported semen. Regardless of the farm, the total fertility obtained with cooled semen was 80% (52/65). The following variables were not affected by the treatments: cycles/mare, cycles/pregnant mare, cycles/pregnancy, pregnancy/cycle, number of inseminations/mare, number of inseminations/pregnant mare, number of inseminations/open mare and efficiency of pregnancy. However, the breeding farm had an effect on water and semen temperature in the container, time between collection and insemination, according to time and distance covered. Based on the results, cooling and transporting equine semen, using an extender, lactose-egg yolk without glycerine and the MSP-2 container should be recommended for short distances. The use of raw semen immediately after collection is also recommended.

Efeito de Diferentes Concentrações Espermáticas sobre a Fertilidade de Éguas Inseminadas com Sêmen Eqüino Diluído, Resfriado a 20oC e Transportado

One hundred and one estral cycles of 92 Mangalarga Marchador breed mares were analyzed to study the effect of different spermatic concentrations on different reproductive characteristics. The mares were inseminated with diluted equine semen, cooled at 20 degrees C and transported: T1-concentrations below 250 x 10(6) (177.58 +41.74 x 10(6) viable spermatozoa per inseminate dose), T2-concentrations between 250 and 350 10(6) sptz (315.51 +/- 20.78 x 10(6)), and T3-concentrations above 350 x 10(6) sptz (477.71 +/- 136.83 x 10(6)). The conception rates in the first cycle were 43.75 (7/16), 57.89 (11/19)and 54.55% (18/33) for treatments 1, 2 and 3,respectively. After five cycles, the conception rates were 36.00 (9/25), 44.12 (15/34) and 54.76% (23/42) for treatments 1,2 and 3, respectively. Concentrations of 177.58 +/- 41,74 x 10(6) viable spermatozoa perinseminate dose had moderate fertility index in a commercial program using cooled and transported semen.

Efeito de Diferentes Diluídores na Fertilidade de Éguas de Diferentes Haras Inseminadas com Sêmen Diluído, Resfriado e Transportado em Conteiner Especial

The effects of extender and farms on the fertility of sixty-five Mangalarga Marchador breed mares were studied. The mares were randomly assigned to a 2 x 2 factorial experiment, with two breeding farms (H1 and H2) and two extenders (glycine-egg yolk-T1 and lactose-egg yolk modified-T2), from only one stallion, with different number of replicates. The mares were inseminated, on alternated days, with diluted, cooled, transported semen, in a special container (MSP-2). There were no differences in the conception rate at first cycle, regardless of the breeding farm or extender used. Also. there was no difference among breeding farm or extender concerning the conception rate/cycle and the following reproductive characteristics: number of cycles/pregnancy, number of inseminations/mare, inseminations/pregnant mare, inseminations/open mare, water temperature in the container, temperature of semen in the container, time between collection and insemination and efficiency of pregnancy. The use of lactose-egg yolk modified extender for the dilution, cooling and transport of stallion semen between farms is recommended. The new container MSP-2 showed to be a option for the transportation between farms, for the same purpose.

Efeito de diferentes concentrações espermáticas sobre a fertilidade de éguas inseminadas com sêmen eqüino diluído, resfriado a 20°C e transportado

One hundred and one estral cycles of 92 Mangalarga Marchador breed mares were analyzed to study the effect of different spermatic concentrations on different reproductive characteristics. The mares were inseminated with diluted equine semen, cooled at 20°C and transported: T1 - concentrations below 250×10 6 (177.58±41.74×10 6 viable spermatozoa per inseminate dose), T2 - concentrations between 250 and 350 10 6 sptz (315.51±20.78×106), and T3 - concentrations above 350×10 6 sptz (477.71±136.83×10 6). The conception rates in the first cycle were 43.75 (7/16), 57.89 (11/19) and 54.55% (18/33) for treatments 1, 2 and 3, respectively. After five cycles, the conception rates were 36.00 (9/25), 44.12 (15/34) and 54.76% (23/42) for treatments 1, 2 and 3, respectively. Concentrations of 177.58±41,74×10 6 viable spermatozoa per inseminate dose had moderate fertility index in a commercial program using cooled and transported semen.

Ureaplasma parvum and ureaplasma urealyticum detected in semen after washing prior to assisted reproductive technology procedures

Objective: This study investigated: (i) the prevalence of ureaplasmas in semen and washed semen and (ii) the effect of ureaplasmas on semen andrology parameters. Design: Prospective study. Setting: IVF unit -private hospital, Brisbane, Australia. Patient(s): Three hundred and forty three men participating in an assisted reproductive technology (ART) treatment cycle. Intervention(s): Semen and washed semen tested by culture, PCR assays and indirect immunofluorescent antibody assays. Statistical differences were determined by a t-test, Wilcoxon or Pearson’s Chi- square test where appropriate. Main Outcome Measure(s): The prevalence of ureaplasmas in semen and washed semen and the effect of these microorganisms on semen andrology parameters. Result(s): Ureaplasmas were detected in 73/343 (22%) semen samples and 29/343 (8.5%) washed semen samples. Ureaplasmas adherent to the surface of spermatozoa were demonstrated by indirect immunofluorescent antibody testing. U. parvum serovar 6 (36.6%) and U. urealyticum (30%) were the most prevalent isolates in washed semen. A comparison of the semen andrology parameters of washed semen ureaplasma positive and negative groups demonstrated a lower proportion of non-motile sperm in the washed semen ureaplasma positive group. Conclusion(s): Ureaplasmas are not always removed from semen by a standard ART washing procedure and can remain adherent to the surface of spermatozoa.

Isolation of bovine herpesvirus type 5 from the semen of a healthy bull in Australia.

Artificial insemination is widely used in the cattle industry and a major challenge is to ensure that semen is free of infectious agents. A healthy donor bull was tested for freedom from infectious agents. A bovine herpesvirus was isolated in testis cells and identified as bovine herpesvirus type 5 (BoHV-5) by polymerase chain reaction and by direct amplicon sequencing. The amplicon sequence shared 100% similarity with the published sequence of BoHV-5. This is the first report in Australia of BoHV-5 in semen. The implications of this finding are discussed.

Characterisation and source identification of pollution episodes caused by long-range transported aerosols

Aerosol particles can cause detrimental environmental and health effects. The particles and their precursor gases are emitted from various anthropogenic and natural sources. It is important to know the origin and properties of aerosols to efficiently reduce their harmful effects. The diameter of aerosol particles (Dp) varies between ~0.001 and ~100 μm. Fine particles (PM2.5: Dp < 2.5 μm) are especially interesting because they are the most harmful and can be transported over long distances. The aim of this thesis is to study the impact on air quality by pollution episodes of long-range transported aerosols affecting the composition of the boundary-layer atmosphere in remote and relatively unpolluted regions of the world. The sources and physicochemical properties of aerosols were investigated in detail, based on various measurements (1) in southern Finland during selected long-range transport (LRT) pollution episodes and unpolluted periods and (2) over the Atlantic Ocean between Europe and Antarctica during a voyage. Furthermore, the frequency of LRT pollution episodes of fine particles in southern Finland was investigated over a period of 8 years, using long-term air quality monitoring data. In southern Finland, the annual mean PM2.5 mass concentrations were low but LRT caused high peaks of daily mean concentrations every year. At an urban background site in Helsinki, the updated WHO guideline value (24-h PM2.5 mean 25 μg/m3) was exceeded during 1-7 LRT episodes each year during 1999-2006. The daily mean concentrations varied between 25 and 49 μg/m3 during the episodes, which was 3-6 times higher than the mean concentration in the long term. The in-depth studies of selected LRT episodes in southern Finland revealed that biomass burning in agricultural fields and wildfires, occurring mainly in Eastern Europe, deteriorated air quality on a continental scale. The strongest LRT episodes of fine particles resulted from open biomass-burning fires but the emissions from other anthropogenic sources in Eastern Europe also caused significant LRT episodes. Particle mass and number concentrations increased strongly in the accumulation mode (Dp ~ 0.09-1 μm) during the LRT episodes. However, the concentrations of smaller particles (Dp < 0.09 μm) remained low or even decreased due to the uptake of vapours and molecular clusters by LRT particles. The chemical analysis of individual particles showed that the proportions of several anthropogenic particle types increased (e.g. tar balls, metal oxides/hydroxides, spherical silicate fly ash particles and various calcium-rich particles) in southern Finland during an LRT episode, when aerosols originated from the polluted regions of Eastern Europe and some open biomass-burning smoke was also brought in by LRT. During unpolluted periods when air masses arrived from the north, the proportions of marine aerosols increased. In unpolluted rural regions of southern Finland, both accumulation mode particles and small-sized (Dp ~ 1-3 μm) coarse mode particles originated mostly from LRT. However, the composition of particles was totally different in these size fractions. In both size fractions, strong internal mixing of chemical components was typical for LRT particles. Thus, the aging of particles has significant impacts on their chemical, hygroscopic and optical properties, which can largely alter the environmental and health effects of LRT aerosols. Over the Atlantic Ocean, the individual particle composition of small-sized (Dp ~ 1-3 μm) coarse mode particles was affected by continental aerosol plumes to distances of at least 100-1000 km from the coast (e.g. pollutants from industrialized Europe, desert dust from the Sahara and biomass-burning aerosols near the Gulf of Guinea). The rate of chloride depletion from sea-salt particles was high near the coasts of Europe and Africa when air masses arrived from polluted continental regions. Thus, the LRT of continental aerosols had significant impacts on the composition of the marine boundary-layer atmosphere and seawater. In conclusion, integration of the results obtained using different measurement techniques captured the large spatial and temporal variability of aerosols as observed at terrestrial and marine sites, and assisted in establishing the causal link between land-bound emissions, LRT and air quality.

Maximizing transported data in a wireless sensor network: how much can the network transport before partition?

We consider a scenario where the communication nodes in a sensor network have limited energy, and the objective is to maximize the aggregate bits transported from sources to respective destinations before network partition due to node deaths. This performance metric is novel, and captures the useful information that a network can provide over its lifetime. The optimization problem that results from our approach is nonlinear; however, we show that it can be converted to a Multicommodity Flow (MCF) problem that yields the optimal value of the metric. Subsequently, we compare the performance of a practical routing strategy, based on Node Disjoint Paths (NDPs), with the ideal corresponding to the MCF formulation. Our results indicate that the performance of NDP-based routing is within 7.5% of the optimal.