Made available in DSpace on 2014-07-29T15:16:30Z (GMT). No. of bitstreams: 1 pre textuais aline franca.pdf: 261338 bytes, checksum: 26489ba3dd2eb948a58b73b531aee0b2 (MD5) Previous issue date: 2010-10-05 / The evaluation of the reproductive capacity of stallions and semen cooled and / or frozen is of fundamental importance in practical breeding of horses. Whereas predicting the fertility of a stallion is still a subjective decision, the present study was conducted to evaluate different staining techniques, as well as tests that assess the viability of semen in horses, studying the effects of the addition of cysteine and glutathione in plasma membrane integrity of sperm DNA, and to evaluate the effects of the addition of these antioxidants in preserving the viability of sperm undergoing incubation and refrigerated for short periods. We used semen from six stallions, wich were split into seven samples, one kept as control (Control Group) and other antioxidants cysteine was added at a ratio of 1.0 mM (Group C1, 0), 1.5 mM (Group C1, 5), 2.5 mM (Group C 2.5) and glutathione also at 1.0 mM (Group G1, 0), 1.5 mM (Group G1, 5) and 2.5 mM (Group G 2.5 ). All tests were performed every six hours. The three treatments were: Treatment I cooled to 12 &#8304;C for 12 hours (zero hour; 6h resf.; 12h resf.) Chilled in boxes; Treatment II incubated in a water bath at 37 &#8304;C for 12 hours (6h 37 ° C, 12h 37 ° C) and Treatment III - cooled and subsequently incubated, according to the treatments I and II. The evaluation of plasma membrane integrity was performed by staining eosine-nigrosin; acrosomal membrane by FITC-PSA and DNA by acridine orange. For each analysis were numbered 200 to 500 cells. The evaluation of the viability of sperm was performed by MTT assay according to Mosmann (1983). The results showed that antioxidants were effective (p <0.05) in keeping the DNA intact chromatin, especially glutathione. In the acrosome of antioxidants were protective, at times 18 and 24 hours, and in other treatments, no significant difference (p> 0.05) between control and treated groups. The MTT test showed that groups treated with antioxidants had absorbance values similar to those of control, showing positive effect (p <0.05) only when cooled by six o'clock in the cysteine group 2.5. In relation to the plasma membrane of spermatozoa stained with eosin-nigrosin, no protective effect of antioxidants in the samples. The values of their averages were close to the control group (p> 0.05). One factor was estimated that cooling per se, independent of the addition of antioxidants used, has been effective in protecting the sperm. And the incubation at 37 &#8304; C causes these cells, and the addition of cysteine and glutathione were efficient, if not protect, but to maintain the integrity of the factors evaluated, not causing more damage to sperm. / A avaliação da eficácia reprodutiva do garanhão e do sêmen resfriado e&#8260;ou congelado é de fundamental importância na prática reprodutiva dos eqüinos. Predizer a fertilidade de um garanhão constitui uma decisão subjetiva. O presente estudo foi realizado com o objetivo de testar diferentes técnicas de coloração, assim como testes que avaliem a viabilidade do sêmen de eqüinos, estudando os efeitos da adição de cisteína e glutationa na integridade da membrana plasmática, do DNA espermático, além de avaliar os efeitos da adição desses antioxidantes na preservação da viabilidade dos espermatozóides submetidos à incubação e refrigeração por curtos períodos. Foi utilizado sêmen de seis garanhões, que foram fracionados em sete amostras, sendo uma mantida como controle (Grupo Controle) e às outras foi adicionado os antioxidantes cisteína, na proporção de 1,0mM (Grupo C1,0); 1,5mM (Grupo C1,5); 2,5mM (Grupo C 2,5) e glutationa também na proporção de 1,0mM (Grupo G1,0); 1,5mM (Grupo G1,5) e 2,5mM (Grupo G 2,5). Todas as análises foram realizadas a cada seis horas. Os três tratamentos foram: Tratamento I resfriado a 12 °C, por 12 horas (zero hora; 6h resf.; 12h resf.), em caixas refrigeradas; Tratamento II incubado em banho-maria a 37 °C, por 12 horas (6h 37° C; 12h 37° C) e Tratamento III resfriado e posteriormente incubado, conforme os Tratamentos I e II. A avaliação da integridade da membrana plasmática foi feita pela coloração eosina-nigrosina; da membrana acrossomal pelo FITC-PSA e do DNA pela laranja de acridina. A avaliação da viabilidade do sêmen foi realizada pelo ensaio do MTT, de acordo com Mosmann(1983). Os resultados obtidos mostraram que os antioxidantes foram eficientes (p<0,05) em manter a cromatina do DNA intacta, especialmente a glutationa. Na membrana acrossomal houve proteção dos antioxidantes, nos momentos 18 e 24 horas, sendo que nos demais tratamentos, não houve diferença significativa (p>0,05) entre os grupos tratados e o grupo controle. O teste do MTT mostrou que os grupos tratados com antioxidantes tiveram valores de absorbância próximos aos do controle, mostrando efeito positivo (p<0,05) apenas quando resfriados por seis horas, no grupo cisteína 2,5. Em relação à membrana plasmática dos espermatozóides, corados por eosina-nigrosina, não houve efeito protetor dos antioxidantes nas amostras avaliadas. Um fator avaliado foi que o resfriamento, por si só, independente da adição dos antioxidantes utilizados, já foi eficaz em proteger os espermatozóides. E a incubação a 37&#8304; C causa danos a essas células, e a adição de cisteína e glutationa foi eficiente em, senão proteger, mas manter a integridade dos fatores avaliados, não causando mais danos aos espermatozóides.

Beurteilung von nativen und aufgetauten Spermatozoen fertiler und subfertiler Hengste mit Hilfe der Phasenkontrast- und Transmissionselektronenmikroskopie. Institut für Veterinär-Pathologie der Veterinärmedizinischen Fakultät, Universität Leipzig Reproduktionsmedizinische Einheit der Kliniken der Tierärztlichen Hochschule Hannover In dieser Arbeit wurde die Ultrastruktur von nativen und tiefgefrorenen Spermien mittels Phasenkontrast- und Transmissionselektronenmikroskopie (TEM) untersucht. Für die Beurteilung der Spermienmotilität und der Morphologie von in Formolzitrat fixierten Spermien standen jeweils drei Ejakulate von 50 Hannoveraner Hengsten des Niedersächsischen Landgestüts Celle zur Verfügung. Aus dieser Gruppe wurden drei fertile, drei subfertile Hengste und 6 Hengste mittlerer Fertilität ausgewählt, von denen sowohl die Nativ-Proben als auch eine Tiefgefrierprobe (TG-Probe) für die TEM im Institut für Veterinär-Pathologie der Universität Leipzig gemäß des Standardprotokolls des Institutes aufbereitet wurden. Die Spermien wurden gewaschen und das Seminalplasma der nativen Proben oder der Verdünner der TG-Proben abpipettiert und durch eine 5%-ige Glutaraldehydlösung in einem 0,1 M Kakodylatpuffer (pH 7,2) ersetzt. Die Fixierungslösung wurde anschließend entfernt und das Pellet gewaschen und danach mit Gelatine gemischt. Die spermienreichen Stellen wurden aus der Gelatine herausgeschnitten und in Glutaraldehyd aufbewahrt. Nach einer Nachfixierung in OsO4 und einer Entwässerung in Ethanollösungen erfolgte eine Einbettung in einer Eponmischung. Nach einer Polymerisation von 5 Tagen wurden die eingebetteten Eponblöckchen angetrimmt und die Semi- und Ultradünnschnitte angefertigt. Die Ultradünnschnitte wurden auf ein Kupfergrid gelegt, mit Uranylazetat und Bleizitrat kontrastiert und mit dem Transmissionselektronenmikroskop (Zeiss EM 900, Oberkochem) bei 80 kV analysiert. In den nativen Proben wurden insgesamt 360 Spermien pro Hengst beurteilt, in den TG-Proben 120 Spermien pro Hengst. Die Qualität der elektronenmikroskopischen Aufnahmen war sehr gut, doch die Plasmamembran zeigte fixierungsbedingte Artefakte. Nach dem Auftauen waren die Bilder heller und der Kontrast etwas geringer. Es gab eine Zunahme an Akrosomdefekten, akrosomreagierten Spermien und Beschädigungen der Plasmamembran, der Mitochondrien, sowie der Mantel- und Ringfasern. Durch die Membranbeschädigungen trat auch eine Verringerung der Anzahl proximaler und distaler Zytoplasmatropfen auf. Sowohl geschwollene Akrosome mit einer niedrigeren Dichte der akrosomalen Matrix als auch Mitochondrien mit einer zu hellen mitochondrialen Matrix waren typische Befunde in den TG-Proben. Die Studie der Ultrastruktur und die wahrgenommenen Defekte führten zur Erstellung eines Standardprotokolls für die transmissionselektronenmikroskopische Beurteilung von Hengstspermien. Die Beurteilung mittels TEM sollte aber nicht zu einer quantitativen, sondern zu einer qualitativen Aussage führen. Sie ermöglicht die Diagnose von Kern- (Kerndeformationen und Taschenbildung im Kern) und Akrosomabweichungen (deformierte Akrosome mit oder ohne Vakuolenbildung, abgehobene Akrosome und akrosomreagierte Spermien), Anomalien der Mitochondrien (Unterbrechung der Mitochondrienscheide, zu viele Mitochondrien, anormale Dichte der mitochondrialen Matrix), Defekten des Axonemas (Ordnung oder Anzahl der Mikrotubuli, Mantel- und Ringfasern) und der Anwesenheit immaturer Spermienvorstufen. Diese Methode eignet sich für die Diagnostik subfertiler Hengste mit normalen Spermienparametern bei der routinemäßige Spermienbeurteilung und kann sowohl in nativen als auch in TG-Proben angewendet werden. Im Vergleich zur Phasenkontrastmikroskopie waren die elektronenmikroskopischen Bilder wegen ihrer stärkeren Vergrößerung und der Darstellung innerer Spermienstrukturen viel aussagekräftiger. Für die Beurteilung von Halsansatzdefekten, abweichende Geißelformen und Mehrfachmißbildungen ist die Phasenkontrastmikroskopie die am besten geeignete Methode. / Evaluation of fresh and frozen-thawed semen samples of fertile and subfertile stallions by light microscopy and transmission electron microscopy. Institut of Pathology of the Faculty of Veterinary Medicine, University of Leipzig Reproduktionsmedizinische Einheit der Kliniken der Tierärztlichen Hochschule Hannover In this study the ultrastructure of fresh and frozen-thawed semen samples of 50 stallions from the National Stud of Lower Saxony (Celle, Germany) were evaluated by light microscopy and transmission electron microscopy (TEM). Three ejaculates of each stallion were available for the motility analysis and the morphological analysis by lightmicroscopy after fixation in formol citrate. Based on the fertility data, the ejaculates of 12 stallions (3 fertile stallions, 3 subfertile stallions and 6 stallions of average fertility) were selected for the morphological analysis by TEM. The native samples and one frozen-thawed sample from these stallions were prepared for the TEM at the Institute of Pathology of the Faculty of Veterinary Medicine, Uni-versity of Leipzig. The sperm cells were washed and the seminal plasma from the native samples and the diluents of the frozen-thawed samples were replaced by a 5%-glutaraldehyde solution in a 0,1 M cacodylate buffer pH 7,2. The fixative was removed, the pellet was washed again and mixed with gelatin. The sperm rich fraction in the gelatin mass was excised and stored in glutaraldehyde. A second fixation in OsO4 was followed by a dehydratation in ethanol and a polymerization phase in epon. After 5 days of polymerization the starred samples were used for semi- and ultratight cuts. The latter were placed on a copper grid, contrasted with uranyl acetate and lead citrate and analyzed with the transmission electron micro-scope (EM 900) by 80 kV. In the fresh samples, 360 sperm cells were examined per stallion, whereas in the frozen-thawed samples only 120 sperm cells per stallion were evaluated. The microscopic pictures were of a high quality. However, the sperm plasma membrane showed some fixation artifacts. In the thawed samples a lower contrast was noticed than in the fresh samples. The sperm cells in the frozen-thawed samples showed an increase in acrosome defects, acrosome reactions, damage of the cell plasma membrane, mitochondria, fibrous sheet and outer dense fibers. The latter defect was associated with a decrease in proximal and distal cytoplasmatic droplets. Swollen acrosomes with a lower matrix density and a bright mitochondrial matrix were typically present in the cryopreserved samples. The ultrastructural defects in these samples, examined by TEM, have led to the development of a standard evaluation protocol with the most common sperm defects in stallion semen. TEM is an expensive and time consuming technique, which cannot be used to obtain quantitative results, but is considered as an accurate method for the qualitative examination of semen samples in cases of unexplained subfertility. TEM can especially be recommended for the diagnosis of nuclear (nuclear malformations and pouches) and acrosomal defects (acrosome deformations, acrosome vacuoles, detached acrosomes and acrosome reactions), mitochondrial (mitochondrial sheet defects, mitochondrial proliferation, decrease in mitochondrial matrix density) and axonema malformations (anormal position or quantity of microtubules and fibrous sheet or outer dense fibers defects) and the detection of immature sperm cells in ejaculates. The results of this study state that TEM can be useful for the evaluation of both fresh and frozen-thawed semen samples. Compared to the light microscopic evaluation of stallion sperm, the TEM images give more precise information because of their higher magnification rate and the ability to reveal internal sperm structures. However, light microscopy remains the best method to detect sperm neck defects, deformed tailes and sperm cells with multiple heads or tails.

Software radio is a radio that is substantially defined in software and whose physical layer behavior can be significantly altered through changes to its software. As a primary goal of software radio is the ability to support existing and future wireless protocols, software radio necessitates the use of a rapidly reprogrammable baseband processing solution. However third generation wireless protocols represent a significant increase in complexity over second generation protocols. Due to the natural performance sacrifices that must be made when moving an application from an Application Specific Integrated Circuit (ASIC) to a general purpose processor or a digital signal processor, it is feared that reprogrammable processing solutions may not suffice for the emerging wireless protocols, which would significantly hinder the realization of software radio, particularly in the handheld domain where power consumption and chip area are critical. Recently, the Configurable Computing Lab at Virginia Tech developed a new breed of reprogrammable processor which they called "custom computing machine" (CCM). Representing a dramatic departure from traditional architectures used for baseband processing solutions, CCMs utilize a large number of optimized and programmable processing cores connected through a programmable mesh. Due to this architectural approach, CCMs have been promoted as supplying a level of processing power and power efficiency similar to ASICs while providing a level of reconfigurability similar to that of a DSP. Subsequently, Dr. Srikathyayani Srikanteswara proposed a new software radio architecture, known as the Layered Radio Architecture, which is intended to facilitate the inclusion of CCMs into a software radio. The primary goal of the research presented in this thesis is to demonstrate how a particular CCM, Stallion, can be used within the Layered Radio Architecture to provide sufficient processing performance, power efficiency, and reconfigurability to meet the constraints of the handheld domain through implementations of a single user adaptive receiver with adaptive complex filtering and a W-CDMA downlink rake receiver. These metrics are measured from a detailed simulation of Stallion and the Configuration Layer of the Layered Radio Architecture using advanced object oriented programming techniques that facilitate the inclusion of statistics gathering routines into normal operation. To provide perspective, these statistics are compared to the performance that could be expected from an implementation on a top-of-the-line DSP. / Master of Science

Equine arteritis virus (EAV) has a global impact on the equine industry being the causative agent of equine viral arteritis (EVA), a reproductive, respiratory, and systemic disease of equids. A distinctive feature of EAV infection is that it establishes long-term persistent infection in the reproductive tract of stallions and is continuously shed in the semen (carrier state). Recent studies showed that long-term persistence is associated with a specific allele of the CXCL16 gene (CXCL16S). However, the cellular and molecular mechanisms underlying the establishment and maintenance of persistent infection are yet to be determined. The studies were undertaken herein unequivocally demonstrated that the ampulla is the main EAV tissue reservoir rather than immunologically privileged tissues (i.e., testes) and that EAV has specific tropism for stromal cells and CD8+ T and CD21+ B lymphocytes but not glandular epithelium in the reproductive tract. Furthermore, persistent EAV infection is associated with a significant humoral, mucosal antibody and inflammatory response at the site of persistence, characterized by induction of high levels of neutralizing antibodies (IgG1), mucosal anti-EAV-specific IgA, IgG1, IgG3/5, and IgG4/7 with variable neutralizing efficacy; and moderate, multifocal lymphoplasmacytic ampullitis, with significant infiltration of T lymphocytes (mainly CD8+ and low numbers of FOXP3+ lymphocytes), CD21+ B lymphocytes, diverse Ig-secreting plasma cells, and Iba-1+ and CD83+ tissue macrophages/dendritic cells. Moreover, EAV long-term persistent infection is associated with a CD8+ T lymphocyte transcriptional profile with upregulation of T-cell exhaustion-related transcripts and homing chemokines/chemokine receptors (CXCL9-11/CXCR3 and CXCL16/CXCR6), orchestrated by a specific subset of transcription factors (EOMES, PRDM1, BATF, NFATC2, STAT1, IRF1, TBX21), which are associated with the presence of the susceptibility allele (CXCL16S). Finally, these studies have determined that long-term EAV persistence is associated with the downregulation of a specific seminal exosome-associated miRNA (eca-mir-128) along with an enhanced expression of CXCL16 in the reproductive tract, a putative target of eca-mir-128. These findings provide evidence that this miRNA plays a crucial role in the regulation of the CXCL16/CXCR6 axis in the reproductive tract of persistently infected stallions, a chemokine axis strongly implicated in EAV persistence. The findings presented herein suggest that complex host-pathogen interactions shape the outcome of EAV infection in the stallion and that EAV employs complex immune evasion mechanisms favoring persistence in the reproductive tract. Further studies to identify specific mechanisms mediating the modulation of the CXCL16/CXCR6 axis and viral immune evasion in the reproductive tract of the EAV long-term carrier stallion are warranted.