385 research outputs found

    A Standardized Ultrasonography Classification for Channel Catfish Ovarian Development

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    The goal of this dissertation was to develop application of ultrasonography as a decision-making tool in genetic improvement programs for channel catfish Ictalurus punctatus. A literature review on the use of ultrasonography in fish reproduction generated a comprehensive reference data set intended to benefit existing and potential users. It exposed the need for reporting of instrument control settings and standardization of fish handling and imaging procedures. These issues were addressed from the onset of this work by assessing more than 6,300 channel catfish ovaries by use of initial fish handling and imaging procedures developed (2004-2005) at the Louisiana State University Agricultural Center Aquaculture Research Station. The development of a standardized and systematic approach to interpretation of ultrasound images emphasized the interplay of technical and biological aspects of ultrasonography assessments. This showed the importance of the control settings and identified disruptive ultrasound artifacts to avoid for observation of the ovary and oocytes. A preliminary ultrasound imaging classification index for assessing ovarian development during the annual reproductive cycle was developed, used and evaluated. This led to the creation of seven well-defined, standardized ultrasound imaging classifications of channel catfish ovarian development based on the annual cycle. Histology of each ultrasound image in the classification index was included as a Reference Guide to provide insight into the processes observed during ultrasonography. Finally, the ultrasound imaging classification index was used for identification and selection of females for hormone-induced spawning in commercial hatchery production of F1 hybrids (channel catfish female x blue catfish male I. furcatus). In sum, this dissertation provides a systematic method of ultrasound imaging assessment of channel catfish ovarian development enabling progress towards standardization in the use of ultrasonography in fish reproduction

    Democratized image analytics by visual programming through integration of deep models and small-scale machine learning

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    Analysis of biomedical images requires computational expertize that are uncommon among biomedical scientists. Deep learning approaches for image analysis provide an opportunity to develop user-friendly tools for exploratory data analysis. Here, we use the visual programming toolbox Orange (http://orange.biolab.si) to simplify image analysis by integrating deep-learning embedding, machine learning procedures, and data visualization. Orange supports the construction of data analysis workflows by assembling components for data preprocessing, visualization, and modeling. We equipped Orange with components that use pre-trained deep convolutional networks to profile images with vectors of features. These vectors are used in image clustering and classification in a framework that enables mining of image sets for both novel and experienced users. We demonstrate the utility of the tool in image analysis of progenitor cells in mouse bone healing, identification of developmental competence in mouse oocytes, subcellular protein localization in yeast, and developmental morphology of social amoebae

    Accessing Nuclear Structure for Field Emission, in Lens, Scanning Electron Microscopy (FEISEM)

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    Scanning electron microscopy (SEM) has had a shorter time course in biology than conventional transmission electron microscopy (TEM) but has nevertheless produced a wealth of images that have significantly complemented our perception of biological structure and function from TEM information. By its nature, SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by the considerably reduced resolution in conventional SEM in comparison to TEM. This restriction has been removed by the recent advent of high-brightness sources used in lensfield emission instruments (FEISEM) which have produced resolution of around 1 nanometre, which is not usually a limiting figure for biological material. This communication reviews our findings in the use of FEISEM in the imaging of nuclear surfaces, then associated structures, such as nuclear pore complexes, and the relationships of these structures with cytoplasmic and nucleoplasmic elements. High resolution SEM allows the structurally orientated cell biologist to visualise, directly and in three dimensions, subcellular structure and its modulation with a view to understanding its functional significance. Clearly, intracellular surfaces require separation from surrounding structural elements in vivo to allow surface imaging, and we review a combination of biochemical and mechanical isolation methods for nuclear surfaces

    Advanced methods in reproductive medicine: Application of optical nanoscopy, artificial intelligence-assisted quantitative phase microscopy and mitochondrial DNA copy numbers to assess human sperm cells

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    Declined fertility rate and population is a matter of serious concern, especially in the developed nations. Assisted Reproductive Technologies (ART), including in vitro fertilization (IVF), have provided great hope for infertility treatment and maintaining population growth and social structure. With the help of ART, more than 8 million babies have already been born so far. Despite the worldwide expansion of ART, there is a number of open questions on the IVF success rates. Male factors for infertility contribute equally as female factors, however, male infertility is primarily focused on the “semen quality”. Therefore, the search of new semen parameters for male fertility evaluation and the exploration of the optimal method of sperm selection in IVF have been included among the top 10 research priorities for male infertility and medically assisted reproduction. The development of imaging systems coupled with image processing by Artificial Intelligence (AI) could be the revolutionary step for semen quality analysis and sperm cell selection in IVF procedures. For this work, we applied optical nanoscopy technology for the analysis of human spermatozoa, i.e., label-based Structured Illumination Microscopy (SIM) and non-invasive Quantitative Phase Microscopy (QPM). The SIM results demonstrated a prominent contrast and resolution enhancement for subcellular structures of living sperm cells, especially for mitochondria-containing midpiece, where features around 100 nm length-scale were resolved. Further, non-labeled QPM combined with machine learning technique revealed the association between gradual progressive motility loss and the morphology changes of the sperm head after external exposure to various concentrations of hydrogen peroxide. Moreover, to recognize healthy and stress-affected sperm cells, we applied Deep Neural Networks (DNNs) to QPM images achieving an accuracy of 85.6% on a dataset of 10,163 interferometric images of sperm cells. Additionally, we summarized the evidence from published literature regarding the association between mitochondrial DNA copy numbers (mtDNAcn) and semen quality. To conclude, we set up the high-resolution imaging of living human sperm cells with a remarkable level of subcellular structural details provided by SIM. Next, the morphological changes of sperm heads resulting from peroxidation have been revealed by QPM, which may not be explored by microscopy currently used in IVF settings. Besides, the implementation of DNNs for QPM image processing appears to be a promising tool in the automated classification and selection of sperm cells during IVF procedures. Moreover, the results of our meta-analysis showed an association of mtDNAcn in human sperm cells and semen quality, which seems to be a relevant sperm parameter for routine clinical practice in male fertility assessment

    Automated Glaucoma Detection Using Hybrid Feature Extraction in Retinal Fundus Images

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    Glaucoma is one of the most common causes of blindness. Robust mass screening may help to extend the symptom-free life for affected patients. To realize mass screening requires a cost-effective glaucoma detection method which integrates well with digital medical and administrative processes. To address these requirements, we propose a novel low cost automated glaucoma diagnosis system based on hybrid feature extraction from digital fundus images. The paper discusses a system for the automated identification of normal and glaucoma classes using higher order spectra (HOS), trace transform (TT), and discrete wavelet transform (DWT) features. The extracted features are fed to a support vector machine (SVM) classifier with linear, polynomial order 1, 2, 3 and radial basis function (RBF) in order to select the best kernel for automated decision making. In this work, the SVM classifier, with a polynomial order 2 kernel function, was able to identify glaucoma and normal images with an accuracy of 91.67%, and sensitivity and specificity of 90% and 93.33%, respectively. Furthermore, we propose a novel integrated index called Glaucoma Risk Index (GRI) which is composed from HOS, TT, and DWT features, to diagnose the unknown class using a single feature. We hope that this GRI will aid clinicians to make a faster glaucoma diagnosis during the mass screening of normal/glaucoma images

    Life history parameters and valorisation of a low commercial value fish species: the case study of piper gurnard, Trigla lyra Linnaeus 1758

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    Many fishing activities involve high discarding rates constituting, a major problem for the marine ecosystem. Trigla lyra presents low commercial value in Portuguese landings and is mainly caught as accessory species in trawls, trammel nets, traps and pots. Published information on this species in Portuguese waters is scarce. So, the main objectives of the present study are to contribute to the knowledge of biological, namely age and growth and reproduction, and chemical parameters of T. lyra. Approximately 30 specimens were collected monthly from fishing vessels operating off Peniche (West coast of Portugal). The study of age and growth involved age estimation using whole sagitta otoliths, marginal increment ratio (MIR) and otolith edge type analysis, determination of length-weight relationship and estimation of parameters of the von Bertalanffy growth model. Regarding reproduction, the structure of the gonads was studied, and reproductive strategy was characterized, including the definition and duration of spawning season and determination of fecundity type. Chemical composition of the muscle was determined in terms of moisture, ash content, protein content, total fat and profile of fatty acids. MIR and otolith edge type analysis suggest that a single set of translucent and opaque increments is formed every year in T. lyra otoliths. There was a lack of small individuals in the sample, leading to the application of three adjustment methods for estimating growth parameters, with back-calculation being the best method. Sex ratio analysis indicated higher abundance of females in relation to males. Spawning season occurred between November and February and fecundity type was found to be determinate. The species proved to be beneficial regarding protein content but especially regarding fatty acid content, with the results indicating high EPA and DHA concentrations. The present study contributed to improve the knowledge on biological and chemical parameters of T. lyra in Portuguese waters, essential for its exploration and future valorisation.Em geral, a pesca está associada a taxas de rejeições mais ou menos elevadas. A rejeição corresponde à parte da captura que é trazida para bordo da embarcação de pesca, mas que é posteriormente devolvida ao mar morta ou viva. As razões associadas a rejeição prendem-se com motivos pessoais, económicos ou legais, sendo as causas mais comuns o baixo ou nenhum valor comercial da espécie capturada, a má condição do pescado (rápida deterioração ou corpo danificado), restrições em termos de tamanho mínimo, existência de quotas, TACs ou proibições de capturas acessórias, ou ainda a falta de espaço de armazenamento a bordo da embarcação de pesca. Os ruivos são espécies que ocorrem ao longo da costa Portuguesa e que são capturadas como espécies acessórias em redes de arrasto, redes de emalhar e de tresmalho e armadilhas. Podem ser rejeitadas no mar ou desembarcadas em lota, apresentando um baixo valor comercial. Entre elas, Trigla lyra, conhecida como cabra-lira, é uma das espécies de ruivos mais desembarcada. É uma espécie bentónica, pouco estudada em termos biológicos em Portugal, sendo que a maior parte da informação existente se refere ao Mar Mediterrâneo. O presente estudo pretende contribuir para um maior conhecimento de alguns parâmetros biológicos e químicos de T. lyra, nomeadamente a idade e crescimento, a reprodução e a composição química. Aproximadamente 30 indivíduos foram recolhidos mensalmente entre Maio de 2018 e Maio de 2019, por embarcações de pesca de arrasto, de redes de emalhar e tresmalho e de armadilhas, a operar ao largo de Peniche (costa Oeste de Portugal). No laboratório, foram recolhidos dados individuais relativos ao comprimento e peso total, removidas as gónadas e restantes órgãos da cavidade abdominal, registado o peso eviscerado, o peso da gónada e o peso do fígado. O estudo da idade e crescimento envolveu a estimativa de idades através da análise de otólitos sagitta inteiros e a sua validação recorrendo à análise da evolução do bordo do otólito e do incremento marginal ao longo dos meses de amostragem. Foi determinada a relação comprimento-peso e os parâmetros de crescimento da espécie foram estimados utilizando o modelo de crescimento de von Bertallanffy. Devido à sub-representação na amostra de indivíduos com comprimento total inferior a 23 cm, foram utilizados três métodos de ajuste ao modelo de crescimento: otólito inteiro, retro cálculo e método combinado. O estudo da estratégia reprodutiva envolveu a determinação da proporção dos sexos, a caracterização do aspecto macroscópico e histológico das gónadas incluindo a identificação dos tipos celulares sexuais e medição do seu diâmetro, a definição da época de reprodução e sua duração tendo por base a evolução mensal da frequência das fases de maturação do ciclo sexual bem como dos índices gonadossomático, hepatossomático e factor de condição. O tipo de fecundidade da espécie foi avaliado tendo em conta os quatro critérios estabelecidos em literatura relativamente à distribuição ao longo dos meses dos ovócitos em vitelogénese avançada no ovário, variação do seu número e diâmetro e percentagem de atrésia ao longo dos meses. A composição química no músculo foi determinada em termos de humidade e teor de cinza, com base nas normas portuguesas, de teor proteico através do método de Kjeldahl, de teor de gordura através do método de Folch e de perfil de ácidos gordos através de cromatografia em fase gasosa. Os resultados demonstraram diferenças nas distribuições frequência-comprimento entre machos e fêmeas, alcançando as fêmeas maiores comprimentos que os machos. O comprimento e idade máximos das fêmeas foram de 45.1 cm e 12 anos, respectivamente, enquanto que o comprimento e idade máximos dos machos foram de 41.0 cm e 10 anos, respectivamente. No estudo da idade e crescimento, a análise da evolução mensal do incremento marginal bem como do bordo do otólito indicaram que os incrementos são depositados anualmente. A relação comprimento-peso revelou a inexistência de diferenças significativas no crescimento entre os dois sexos e que a espécie possui um crescimento isométrico. Quanto aos parâmetros de crescimento, o retro cálculo revelou melhor ajuste ao conjunto dos dados, tendo-se obtido valores de L∞ de 42.579 cm, de k de 0.204 e de t0 de -1.455. Relativamente à reprodução da cabra-lira, a estrutura das gónadas revelou a presença de todos os estados de desenvolvimento de ovócitos e de células sexuais masculinas. A proporção dos sexos indicou que as fêmeas foram mais abundantes do que os machos em quase todos os meses de amostragem, incluindo os meses da época de reprodução, que foi definida como o período entre Novembro e Fevereiro. A evolução do índice gonadossomático ao longo dos meses corroborou a definição da época de postura, uma vez que os valores mais altos se verificaram no período acima indicado. O tipo de fecundidade de T. lyra foi classificado como determinado, uma vez que se verificou a presença de hiato entre a população de ovócitos não vitelados e vitelados, a diminuição do número de ovócitos em vitelogénese avançada e um aumento do seu diâmetro ao longo dos meses, assim como a inexistência de atrésia massiva no final da época de postura. Em termos de estratégia reprodutiva, os resultados sugerem que a espécie apresenta fertilização externa, é gonocorística, apresenta um desenvolvimento dos ovócitos do tipo grupo-síncrono e a desova é seriada ao longo da época de postura. No que diz respeito à composição química, o componente principal do músculo de T. lyra foi a água, seguido pelas proteínas, cinzas e lípidos. A composição em lípidos sugere que a espécie é magra. A análise do perfil de ácidos gordos permitiu identificar 21 tipos, com uma maior concentração de ácidos gordos polinsaturados. Entre os ácidos gordos saturados, o mais abundante foi o ácido palmítico, e entre os ácidos monoinsaturados o mais comum foi o ácido oleico. Os ácidos DHA e EPA destacaram-se entre os ácidos polinsaturados. Os resultados permitiram concluir que a cabra-lira constitui uma boa fonte de proteínas e ácidos gordos sendo benéfica para consumo humano. Em resumo, o presente estudo contribuiu para um melhor conhecimento dos parâmetros de história de vida de T. lyra, nomeadamente da idade, do crescimento e da reprodução, informação fundamental para uma correta gestão da exploração da espécie em Portugal. A análise da composição química permitiu conhecer as suas características nutricionais e, assim, avançar na valorização futura da espécie enquanto recurso pesqueiro, passando por exemplo por um melhor aproveitamento do recurso e criação de novos produtos ao consumo, o que pode ser importante, já que T. lyra é uma espécie, presentemente, com baixo valor comercial em Portugal

    Using adaptive thresholding and skewness correction to detect gray areas in melanoma \u3ci\u3ein situ\u3c/i\u3e images

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    The incidence of melanoma in situ (MIS) is growing significantly. Detection at the MIS stage provides the highest cure rate for melanoma, but reliable detection of MIS with dermoscopy alone is not yet possible. Adjunct dermoscopic instrumentation using digital image analysis may allow more accurate detection of MIS. Gray areas are a critical component of MIS diagnosis, but automatic detection of these areas remains difficult because similar gray areas are also found in benign lesions. This paper proposes a novel adaptive thresholding technique for automatically detecting gray areas specific to MIS. The proposed model uses only MIS dermoscopic images to precisely determine gray area characteristics specific to MIS. To this aim, statistical histogram analysis is employed in multiple color spaces. It is demonstrated that skew deviation due to an asymmetric histogram distorts the color detection process. We introduce a skew estimation technique that enables histogram asymmetry correction facilitating improved adaptive thresholding results. These histogram statistical methods may be extended to detect any local image area defined by histograms --Abstract, page iv

    Generation and molecular analysis of in vitro germ cells from mouse and human pluripotent stem cells

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    Zusammengefasst zeigen unsere Daten, dass ESC-abgeleitete humane PGCs und in vitro generierte Oozyten der Maus ihren natürlichen Gegenstücken stark ähneln. Zusätzlich weisen unsere Studien auf bisher unbekannte Mechanismen während der humanen Keimzellspezifikation hin. Weitere Untersuchungen werden zeigen wie vorteilhaft solche in vitro Differenzierungsmodelle als alternative Ansätze für Untersuchungen in der reproduktiven Entwicklung tatsächlich sind.<br

    Morphological reflectors and molecular predictors of preimplantation developmental competence in bovine oocytes and embryos

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    The efficiency of in vitro production (IVP) of bovine embryos still remains low, reaching blastocyst rates between 25 and 35 %. Accordingly, the necessity to define reliable predictors for developmental competence is indispensible. Up to now most morphological and molecular markers are subjective and partly invasive and thus relatively inaccurately. Therefore, the aim of the first experiment of the present approach was to evaluate the effect of structural changes in zona pellucidas’ (ZP) morphology during in maturation on developmental competence using polarized light microscopy. Structural changes in terms of number and size of pores were displayed by the Scanning electron microscopy and determined quantitatively in zona pellucida birefringence (ZPB). Immature oocytes classified as high presumble quality oocytes exhibited highest values for birefringence, and in vivo matured oocytes showed the lowest values for birefringence compared to their in vitro matured counterparts. These results implicate, that decreasing values for ZPB during maturation are typical for high developmental competence. In the second study of the present thesis, the transcriptome profile of one blastomere of a bovine 2-cell stage embryo was directly correlated with the developmental potential of the corresponding sister blastomere. Analyses using microarray technology revealed specific molecular fingerprints for blastomeres whose sister blastomere developed to the blastocyst stage in contrast to those blastomeres whose sister blastomeres stopped cleaving after separation or were blocked before embryonic genome activation (EGA). Pathway analyses uncovered, that differentially regulated genes (DEG) were mostly involved in oxidative phosphorilation, oxidative stress response and antioxidant activity. Five candidate genes, namely, NDUFS1, MAPK14, CAT, PRDX1 and PRDX6, which are known to act as either direct or indirect scavengers of reactive oxygen species (ROS), were selected and their expression levels were further characterized using two independent models for developmental competence. Furthermore, ROS staining revealed high ROS accumulations in late cleaved or rather developmentally incompetent 2-cell stage embryos, compared to low ROS levels in early cleaved and therefore presumable highly competent 2-cell stage embryos. Taken together, noninvasive morphological criteria predicting subsequent developmental competence of matured bovine oocytes were identified in this work. Moreover, a direct correlation between molecular mechanisms and the individual developmental competence, which was found to be already determined in the 2-cell stage, could be detected.Untersuchungen boviner Eizellen und Embryonen auf morphologischer und molekularer Ebene zur Identifizierung von entwicklungsrelevanten Markern In Anbetracht der relativ geringen Effizienz der in vitro Produktion (IVP) boviner Embryonen mit Blastozystenraten von 25 bis 35 %, ist es unverzichtbar, zuverlässige Prädiktoren für das Entwicklungspotential von Eizellen und Embryonen zu definieren. Bis heute sind die meisten morphologischen und molekularen Qualitätsparameter subjektiv und von invasiver Natur. Daher war es das Ziel in der ersten Studie der vorliegenden Arbeit, strukturelle Veränderungen in der Zona pellucida (ZP) während der in vitro Maturation (IVM) mit Hilfe der Polarisationslichtmikroskopie quantitativ zu erfassen und mit dem Entwicklungspotential zu korrelieren. Strukturelle Änderungen bezogen auf Anzahl und Größe der Poren in der ZP zeigten sich bei der Analyse mittels eines Rasterelektronenmikroskops und spiegelten sich in der Doppellichtbrechungsintensität der Zona pellucida (ZPB) quantitativ wieder. Immature Eizellen, die als qualitativ sehr gut eingestuft wurden, zeigten die höchsten Werte für die ZPB, wohingegen die in vivo maturierten Eizellen im Vergleich zu den in vitro maturierten die niedrigsten Werte aufwiesen. Diese Ergebnisse implizieren, dass abnehmende Werte für die ZPB während der Maturation als Pärdiktor für hohe Eizellqualität genutzt werden können. Zusammenfassend konnte gezeigt werden, dass die Polarisationslichtmikroskopie als nicht invasisver Prädiktor für das Entwicklungspotential genutzt werden kann. In der zweiten Studie der vorliegenden Arbeit wurde eine direkte Korrelation zwischen dem Transkriptionsprofil einer Blastomere eines bovinen Zweizellers zum Entwicklungspotential der korrespondierenden Schwesterblastomere hergestellt. Beim Vergleich mittels Mikroarraytechnologie wurden spezifische molekulare Fingerabdrücke für Blastomeren ermittelt, deren Schwesterblastomeren sich zur Blastozyste entwickelten im Gegensatz zu denen, deren Schwesterblastomeren sich nicht weiter teilten oder vor der embryonalen Genomaktivierung (EGA) geblockt wurden. Nach Analyse der durch die differentiell regulierten Transkripte betroffenen Stoffwechselwege zeigte sich, dass diese Gene hauptsächlich in oxidative Phosphorilierung, oxidative Stressantwort und antioxidative Aktivität involviert waren. Die Expremierung der daraus selektierten Kandidatengene NDUFS1, MAPK14, CAT, PRDX1 und PRDX6, deren direkte und indirekte Funktionen bei der Eliminierung von freien Radikalen bekannt ist, wurde an Hand von zwei unabhängigen Kompetenzmodellen weiter charakterisiert. Des Weiteren zeigte Färbung, dass spät geteilte Zweizeller mit einem geringeren Entwicklungspotential sehr hohe Anreicherungen an freien Radikalen aufwiesen im Gegensatz zu früh geteilten Zweizellern mit einem hohen Entwicklungspotential, die nur niedrige Anreicherungen zeigten. Schlussfolgernd konnte gezeigt werden, dass spezifische molekulare Mechanismen direkt mit dem Entwicklungspotential korrelieren und es bereits im Zweizeller determinieren
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