Submitted by (edna.saturno@ufrpe.br) on 2016-08-01T15:31:24Z No. of bitstreams: 1 Andre Mariano Batista.pdf: 715032 bytes, checksum: 92e93d004a4a86c672f3ad4c9e8e4af1 (MD5) / Made available in DSpace on 2016-08-01T15:31:24Z (GMT). No. of bitstreams: 1 Andre Mariano Batista.pdf: 715032 bytes, checksum: 92e93d004a4a86c672f3ad4c9e8e4af1 (MD5) Previous issue date: 2009-02-12 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Sperm processing methods are routinely applied on the in vitro fertilization (IVF) systems for various species with the objective to remove seminal plasma/crioprotectant and to increase sperm quality. Aiming at study the sperm viability after use of the Swim-up or density gradient methods (Percoll® and CapriPure®) in frozen-thawed goat semen samples for use in assisted reproduction, six Boer goat semen samples were collected. Following, the samples were evaluated for concentration, progressive motility and morphology, diluted, packaged in straws (0.25 mL), frozen using machine and stored in liquid nitrogen. After thawing (37 oC for 30 s), sperm of all six goat were mixed-up making a pool and then were evaluated for progressive motility, membrane integrity (PI/DCF), acrossomal integrity (FITC/PNA) and mitochondrial membrane potential (JC-1). After that, the pool was split into two aliquots for the accomplishment of the sperm selection using the methods Swim-up and Percoll® (Experiment 1) and centrifugation in Percoll® and CapriPure® density gradient (Experiment 2). After use of each method of selection, the analyses was proceeded it from progressive motility, membrane integrity, acrossomal integrity and of mitochondrial integrity. Exp. 1 evidenced lower percentage of progressive motility spermatozoa (P<0.05) with intact plasma membrane (P<0.01) after use of Swim-up method, when compared with samples submitted to sperm preparation with Percoll®. In the Exp. 2, Percoll® gradient determined reduction (P<0.05) in percentage of acrosome intact spermatozoa when compared with percentage of the sample immediately after thawing, not having significant difference (P>0.05) if comparing submitted samples with the CapriPure® gradient. There were significant differences in the percentage of spermatozoa with high mitochondrial membrane potential inthe samples of semen submitted the selection using Percoll® (P<0.05) and CapriPure® (P<0.01), density gradients, when compared with the values of samples evaluated immediately after thawing. However, there were no significant difference (P<0.05) in the high mitochondrial membrane potential values between Percoll® and CapriPure® density gradients. On the basis the results of mobile spermatozoa and with the intact plasma membrane, are possible to conclude that to select viable sperm of frozen goat semen samples should use density gradient methods and CapriPure® is a good alternative to Percoll®. / Métodos de processamento espermático são rotineiramente utilizados nos sistemas de fertilização in vitro (FIV) de várias espécies, com o objetivo de remover o plasma seminal/crioprotetores e aumentar a qualidade espermática. Visando estudar a viabilidade espermática após utilização dos métodos Swim-up ou gradiente de densidade (Percoll® e CapriPure®) em amostras criopreservadas de sêmen caprino para uso em reprodução assistida, foram colhidas amostras de sêmen de seis reprodutores Boer. A seguir, as amostras foram avaliadas (motilidade progressiva, concentração, e morfologia), submetidas a duas lavagens em solução Tris, diluídas, envasadas em palhetas (0,25 mL), criopreservadas em máquina e armazenadas em botijão criobiológico (-196 oC). Após descongelação (37 oC; 30 segundos),procedeu-se à formação do pool das amostras dos reprodutores de cada dia de congelação e, a seguir, a avaliação de integridade de membrana (IP/DCF), integridade acrossomal (FITC/PNA) e potencial de membrana mitocondrial (JC-1). Em seguida, o pool foi dividido em duas alíquotas para a realização da seleção espermática utilizando os métodos Swim-up e Percoll® (Experimento 1) e centrifugação em gradiente de densidade Percoll® e CapriPure® (Experimento 2). Após utilização de cada método de seleção, procederam-se às análises de motilidade progressiva, concentração, integridade de membrana, integridade acrossomal e potencial de membrana mitocondrial. No Exp. 1 foram constatados menores porcentuais de espermatozóides portadores de motilidade progressiva (P<0,05) e de espermatozóides com membranas intactas (P<0,01) após utilização do método Swim-up, quando comparado as amostras submetidas à preparação espermática com Percoll®. No Exp. 2, o gradiente de Percoll® determinou redução (P<0,05) no porcentual de espermatozóides portadores de acrossoma intacto quando comparado ao porcentual da amostra imediatamente após a descongelação, não havendo diferença significativa (P>0,05) ao se comparar às amostras submetidas ao gradiente CapriPure®. Houve diferença significativa nos porcentuais de espermatozóides com alto potencial de membrana mitocondrial nas amostras de sêmen submetidas à seleção utilizando os gradientes de densidade Percoll® (P<0,05) e CapriPure® (P<0,01), quando comparado aos valores das amostras avaliadas imediatamente após a descongelação. No entanto, nenhuma diferença (P>0,05) foi observada entre os porcentuais de espermatozóides com alto potencial de membrana mitocondrial das amostras submetidas à seleção utilizando os gradientes de densidade Percoll® e CapriPure®. Com base nos resultados de espermatozóides móveis e com membranas intactas, é possível concluir que, para selecionar espermatozóides viáveis de amostras congeladas de sêmen caprino, deve-se usar a centrifugação em gradiente de densidade, e que o CapriPure® é uma boa alternativa ao uso doPercoll®.

Mittels Glaswolle (Microfibre code 112) in einer speziellen Filtrations-Anordnung steht ein effektives Spermienpräparationsverfahren zur Verfügung. Durch Ausnutzung eines gefäßforminduzierten Konzentrierungseffektes kann auf samenzellschädigende Zentrifugation zur Spermatozoen-Anreicherung verzichtet werden. Folgend aufgeführte Ergebnisse sind Mittelwerte aus 30 Messungen. Die Computer-Assistierte Spermienmotilitäts-Analyse (CASA) zeigt eine gegenüber dem nativen Ejakulat signifikante Erhöhung der Spermienkonzentration im Filtrat (27,45 vs. 69,56 Mio/ml). Das Verteilungsverhältnis der Samenzell-Populationen nach Glaswolle- Filtration bietet gute Voraussetzungen für assistierte Reproduktion. Unter Abnahme des immotilen Anteils auf 1/5 (54,53 vs. 11,66 %), erhöht sich konsekutiv der motile Spermien-Anteil (39,53 vs. 82,06 %). In der weiteren Spezifisierung motiler Spermatozoen, kann eine Verschiebung zugunsten linear motiler Spermien registriert werden. Während die Verringerung der nicht linear motilen Subpopulation nur zufälligen Charakters ist, weist der Kreisläuferanteil post filtrationem einen signifikanten Rückgang auf (34,96 vs. 31,1 %). Der Zuwachs linear motiler Samenzellen ist jedoch von besonderem Interesse (7,36 vs. 12,43 %). Bei Zugrundelegung von Absolut- zahlenwerten, läßt sich (auch für oligozoosperme Ejakulate) ein nahezu 9-facher Anstieg linear motiler Spermatozoen von nativ 0,8 auf 7,1 Mio/ml nach Filtration demonstrieren. Ihre Spurgeschwindigkeit (curvilinear velocity-VCL) nimmt dabei überproportional zu (41,26 vs. 62,66 µm/s), während sich über alle motilen Spermien im Mittel nur eine filtrationsbedingte VCL-Erhöhung von 10 µm/s abzeichnet. Weiterhin kam ein Cell Counter + Analyser System (CASY®) zum Einsatz, das ein Verfahren der Partikelmeßtechnik nutzt. So gelang der Nachweis, daß die Glaswolle-Filtration unter Reduktion klein- und großkorpuskulärer Bestandteile (zelluläre und nichtzelluläre) eine deutliche Anreicherung einer Samenzell-Population mit medianen Membrandurchmesser von 3,47 µm bewirkt. Die Ergebnisse der morphologischen Differenzierung (strict criteria) stehen damit im Einklang. Nach Filtration konnte eine signifikante Zunahme der Normalform (13,33 vs. 19,16 %) bei adäquater Verminderung von Makro- und Mikroköpfen (6,5 vs. 3,03 %) sowie Vorstufen (8,33 vs. 5,96 %) registriert werden. Auf den raster-elektronen-mikroskopischen Aufnahmen wird deutlich, wie besonders membrandefekte Spermatozoen und muköses Eiweiß der Glaswolle-Faseroberfläche verhaften. / An efficient procedure for sperm preparation is presented using glass wool (microfibre code 112) in a special filter arrangement. Centrifugation as a means of spermatozoa concentration, a process which is extremely harmful to the sperm cells, can be dispensed with by making use of a concentration effect induced by the form of the vessel. The results that follow are mean values from 30 measurements. Computer-Assisted Sperm-motility Analysis (CASA) shows a significant rise of the sperm cell contents in the filtrate (27,45 vs. 69,56 million/ml) as compared with the native ejaculate. The distribution ratio of sperm populations observed after glass wool filtration provides favourable conditions for assisted reproduction. Reduction of the immotile portion to one fifth (54,53 vs. 11,66 %) yields a consecutive rise of the portion of motile sperms (39,53 vs. 82,06 %). Further specification of motile spermatozoa shows a shift in favour of sperms moving in a straight line. The decrease of the portion moving in circles is significant (34,96 vs. 31,1 %) after filtration, whereas the reduction in the subpopulation of sperms not moving in a straight line is of a rather incidental nature. Of particular interest, however, is the increase in sperm cells moving in straight lines (7,36 vs. 12,43 %). Basing considerations on absolute figures , a nine-fold increase in spermatozoa moving in straight lines, from the native value of 0,8 to 7,1 million/ml, can be demonstrated (even for oligozoospermial ejaculates) after filtration. Their curvilinear velocity (VCL) rises, in this connection, to a disproportionately high degree (41,26 vs. 62,66 µm/sec), whereas a filtration-induced rise of the VCL by only 10 µm/sec is observed for all motile spermatozoa on the average. Furthermore, a Cell Counter + Analyser System (CASY®) was employed making use of a particle test procedure. In this way, it could be proved that glass wool filtration produces a marked enrichment in a population of sperm cells with a mean membrane diameter of 3,47 µm with simultaneous reduction of the small- and large-corpuscular (cellular and non-cellular) fractions. In keeping with this are the results of the morphological differentiation (strict criteria). A significant increase in the standard form (13,33 vs. 19,16 %) with an adequate reduction in macro and micro heads (6,5 vs. 3,03 %) as well as in prestages (8,33 vs. 5,96 %) could be observed after filtration. Pictures taken with the electron-scan microscope clearly show that spermatozoa presenting membrane defects as well as mucous proteins, in particular, attach to the glass wool fibre surface.