A coleta de sêmen da cauda do epidídimo é a última oportunidade de obter espermatozoides de garanhões valiosos, sendo que durante a criopreservação, a etapa de centrifugação é considerada um ponto crítico. A principal hipótese é que a centrifugação com coloides pode melhorar a qualidade dos espermatozoides coletados da cauda do epidídimo de garanhões. Para avaliação da hipótese foram realizados dois experimentos. O experimento um teve por objetivo avaliar o efeito da centrifugação com cushion e com coloide em camada única (SLC) na motilidade de sêmen do epidídimo de garanhões após a etapa de centrifugação. O experimento dois teve o objetivo de determinar o efeito da SLC prévio ao congelamento e após o descongelamento. Experimento 1) Oito garanhões foram submetidos à orquiectomia bilateral e o sêmen foi coletado da cauda dos epidídimos (n=16). Após a coleta, as amostras foram submetidas a três protocolos de centrifugação: Convencional (20 minutos a 600xg), cushioned (20 minutos a 900xg) e SLC (20 minutos a 300xg). Os pellets foram ressuspendidos e as amostras foram submetidas à avaliação laboratorial de motilidade e morfologia espermática. Experimento 2) Dez garanhões foram submetidos a orquiectomia bilateral e o sêmen foi coletado da cauda dos epidídimos (n=20). Para criopreservação, as amostras foram submetidas a: centrifugação convencional (20 minutos a 600xg), SLC prévio a criopreservação (SLC-Pre) (20 minutos a 300xg) e SLC após a criopreservação (SLC+) (20 minutos a 600xg seguidos de uma segunda centrifugação descrita após descongelamento). Os pellets foram ressuspendidos em diluente de congelamento, submetidos ao processo de congelamento em nitrogênio líquido e descongelamento. Os grupos de 6 centrifugação convencional e SLC-Pre foram avaliados imediatamente após descongelamento. O grupo SLC+ foi descongelado e submetido à SLC (20 minutos a 300xg) e ressupendido em diluente de congelamento (SLC+F) ou resfriamento (SLC+C). A motilidade total e a motilidade progressiva das amostras foram avaliadas com análise computadorizada do movimento espermático. A morfologia foi avaliada com auxílio de microscópio com contraste de fase. Funcionalidade de mitocôndria, integridade de membrana e DNA foram avaliados com auxílio de microscópio de fluorescência. Os dados foram analisados por estatística descritiva, simple one-way ANOVA e Teste de Tukey. Experimento 1) a motilidade de espermatozoide submetidos à SLC (p<0,05) e cushion (p>0,05) foi superior do que os submetidos a centrifugação convencional. Experimento 2) SLC-Pre e SLC+F apresentaram maior motilidade total, enquanto SLC+F apresentou maior motilidade progressiva. O percentual de espermatozoides com morfologia normal foi maior em SLC-Pre e SLC+F. A funcionalidade de mitocôndria foi maior em todos grupos com SLC, enquanto a integridade de membrana foi maior em SLC-Pre. A centrifugação com coloides melhorou a qualidade de espermatozoides coletados da cauda do epidídimo de garanhões, tanto no momento prévio ao congelamento como após o descongelamento. / Epididymis cauda sperm recovery and cryopreservation are last opportunity to obtain spermatozoa from a valuable animal, even though during cryopreservation centrifugation step is considered as a critical point. It is hypothesized that colloidal centrifugation could enhance epididymal stallion sperm parameters. To evaluate this hypothesis two experiments were performed. In experiment one, the objective was to evaluate the effect of cushioned and Single Layer Centrifugation (SLC) on epididymal stallion sperm motility postcentrifugation. In experiment two, the objective was to determine the effect of SLC on epididymal stallion sperm quality pre-freezing and post-thawing. Experiment 1) Eight stallions were submitted to bilateral orchiectomy and the resulting epididymal cauda (n = 16) were flushed with semen extender. After harvesting, samples were submitted to three centrifugation protocols: conventional (20 minutes at 600xg), cushioned (20 minutes at 900xg), and SLC (20 minutes at 300xg). Pellets were resuspended, motility and morphology were evaluated. Experiment 2) Ten stallions were submitted to bilateral orchiectomy and epididymal cauda (n=20) were harvested. For cryopreservation, epididymal sperm were submitted to: conventional centrifugation (20 minutes at 600xg), Single Layer Centrifugation prior cryopreservation (SLC-Pre) (20 minutes at 300xg) and Single Layer Centrifugation after cryopreservation (SLC+) (20 minutes at 600xg followed by a second centrifugation described after thawing). Pellets were resuspended in freezing extender, submitted to cryopreservation process in liquid nitrogen and thawed. Conventional and SLC-Pre were evaluated immediately after thawing. SLC+ samples were thawed, submitted to SLC (20 minutes at 300xg) and the pellets were resuspended with freezing (SLC+F) and cooling extender (SLC+C). Total motility (TM) and progressive 8 motility (PM) were evaluated with computer-assisted semen analyses. Sperm morphology was evaluated under a phase-contrast microscope. Mitochondrial functionality, membrane e DNA integrity were evaluated with an epifluorescence microscope. Data was evaluated by descriptive statistics, simple one-way ANOVA and comparison between means by Tukey test. Significance was assigned to all values p<0.05. Experiment 1) Motility of spermatozoa recovered by SLC (p<0.05) and cushioned centrifugation (p>0.05) were higher than those recovered by conventional centrifugation. Experiment 2) SLC-Pre and SLC+F yielded the highest TM, while SLC+F yielded the highest PM. Higher morphological normal sperm was observed in SLC-Pre and SLC+F. Mitochondrial functionality was significantly higher in all treatments with SLC, while membrane integrity was higher in SLC-Pre. Colloidal centrifugation improved epididymal sperm quality before freezing and after thawing.

O sêmen eqüino é muito utilizado na forma refrigerada, porém, para alguns garanhões, a longevidade espermática é baixa. Durante a refrigeração o espermatozóide pode ser afetado por vários fatores, que podem causar a perda da integridade da membrana plasmática, danos no acrossomo e disfunções mitocondriais, resultando em perda irreversível de motilidade e capacidade fecundante. A composição e concentração do plasma seminal podem causar efeitos deletérios ao espermatozóide, sendo a centrifugação do sêmen uma alternativa para minimizar estes efeitos. Este experimento foi realizado para verificar o efeito da centrifugação nas características de movimento, integridade e peroxidação lipídica das membranas do espermatozóide eqüino refrigerado a 5°C por até 72h. Foram realizadas cinco colheitas de sêmen de quatro garanhões. O sêmen foi avaliado quanto ao volume e concentração, e em seguida dividido em dois tratamentos, sem centrifugação (S) e centrifugado (C) (300g/10min). Ambos foram diluídos na concentração final de 30 x106 eptz/mL em diluidor a base de leite desnatado e então refrigerados. Foram analisadas as características do movimento espermático (CASA), morfologia espermática (DIC), integridade das membranas plasmática e acrossomal (PI/FITC-PSA), potencial de membrana mitocondrial (JC-1) e peroxidação lipídica das membranas (C11-BODIPY581/591), sendo estas três últimas análises por citometria de fluxo. As análises foram realizadas no tempo 0 e 24, 48 e 72 horas após a refrigeração. O delineamento experimental foi em blocos generalizados adicionado do fator medidas repetidas no tempo. Os dados foram analisados pelo programa SAS, submetidos à análise de variância e às interações determinadas pelo teste de Greenhouse-Geisser ao nível de 5% de significância. O espermatozóide do sêmen centrifugado apresentou valores médios maiores de motilidade total e progressiva, ALH, STR e LIN (P<0,05), porém os valores das velocidades e BCF foram menores (P<0,05). Houve efeito do tempo nas características de movimento espermático (P<0,05), exceto para STR. Não houve efeito da centrifugação e nem do tempo quanto às características morfológicas dos espermatozóides refrigerados, esta só diferiu entre os garanhões (P<0,05). Por sua vez, a integridade das membranas plasmática e acrossomal e o potencial de membrana mitocondrial espermáticas foram maiores nos sêmen centrifugado (P<0,05) e suas porcentagens decresceram com o tempo (P<0,05). A peroxidação lipídica da membrana do espermatozóide refrigerado aumentou somente nas primeiras 24h (P<0,05) e não foi influenciada pela centrifugação. Conclui-se que a centrifugação e a retirada do plasma seminal melhoram a qualidade do sêmen refrigerado eqüino, principalmente em garanhões com baixa longevidade, sendo esta influenciada pelas características iniciais da morfologia espermática. Ainda, tempo de refrigeração induz a um declínio da qualidade seminal. E, por fim, a peroxidação lipídica da membrana do espermatozóide parece não ser a principal causa de alterações da integridade das membranas e do potencial de membrana mitocondrial durante a refrigeração do sêmen eqüino. / Equine cooled semen is frequently used, although the longevity for some stallions is low. During the cooling process the spermatozoon can be affected by several factors that can cause loss of plasma membrane integrity, acrosomal damage and mitochondrial dysfunctions, resulting in irreversible loss of motility and fertilizing capacity. The composition and concentration of seminal plasma could cause harmful effects to the sperm, being semen centrifugation an alternative to minimize these effects. This experiment was designed to verify the effects of the centrifugation in motion characteristics, membranes integrity and lipid peroxidation of cooled equine sperm to 5°C until 72 hours. Five ejaculates from four stallions were collected. The semen was evaluated for volume and concentration, then split in two treatments, no centrifugation (N) and with centrifugation (W) (300g/10min). Both were diluted in the final concentration 30 x106 spermatozoa/mL in a skin milk extender and then cooled. The following analyses were performed: the sperm motion characteristics (CASA), morphology (DIC), plasma and acrosomal membranes integrity (PI/FITC-PSA), mitochondrial membrane potential (JC-1) and membrane lipid peroxidation (C11-BODIPY581/591), the last three analyses were assessed in flow citometry. The analyses were performed in time 0 and 24, 48 and 72 hours after cooling. The experimental design was random blocks with repeated-measures. The data were analyzed by the SAS program, submitted to analysis of variance and the interactions by the test of Greenhouse-Geisser at the level of 5% of significance. The values were larger in W semen for total and progressive motility, ALH, STR and LIN (P<0,05), however the values of the three types of speed and BCF were smaller (P<0,05). There was effect of time in the motion characteristics of sperm (P<0,05), except for STR. There was not any effect of centrifugation and time for the morphologic characteristics of cooled spermatozoa, that only differed among stallions (P<0,05). On the other hand, the sperm integrity of plasma and acrosomal membranes and the potential of mitochondrial membrane were larger in W semen (P<0,05) and their percentages decreased within time (P<0,05). The membrane lipidic peroxidation of the cooled sperm only increased in the first 24 hours (P<0,05) and it was not influenced by centrifugation. In conclusion, the centrifugation and withdrawal of the seminal plasma improve semen quality of the cooled equine sperm, mainly in stallions with low longevity, which is influenced by the initials characteristics of sperm morphology. Moreover, time of cooling induces a decline of seminal quality. Finally, the membrane lipidic peroxidation of the cooled sperm seems not to be the main cause of alterations in the sperm integrity of plasma and acrosomal membranes and the potential of mitochondrial membrane during the cooling of equine semen.

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-03-08T18:29:37Z No. of bitstreams: 1 117110033.pdf: 882213 bytes, checksum: 12c52b9fad305c8e13e54eeab7fca3ac (MD5) / Made available in DSpace on 2015-03-08T18:29:38Z (GMT). No. of bitstreams: 1 117110033.pdf: 882213 bytes, checksum: 12c52b9fad305c8e13e54eeab7fca3ac (MD5) Previous issue date: 2013-04-26 / A centrifugação por gradiente de Percoll ® é o método mais utilizado para a seleção de sêmen bovino para a fecundação in vitro (FIV), no entanto, até o momento não existe nenhum estudo que indique uma força de centrifugação capaz de remover com sucesso o plasma seminal e outros constituintes, sem causar perdas na recuperação espermática ou danos funcionais aos espermatozoides obtidos no pellet. O presente estudo foi desenvolvido para verificar o efeito de diferentes forças de centrifugação na seleção espermática por gradientes de Percoll ® , através de avaliação de parâmetros de qualidade espermática e posterior desenvolvimento e qualidade de embriões bovinos produzidos in vitro (PIV). Parâmetros de estresse oxidativo também foram avaliados para verificar uma possível relação com os achados na avaliação de esperma. No experimento 1, amostras de sêmen de quatro touros foram homogeneizadas e submetidas a centrifugação em gradientes descontínuo de Percoll ® (30,60 e 90%) em diferentes forças: F1 (9000 X g), F2 (6500 X g), F3 (4500 X g) e F4 (2200 X g). No Experimento 2, as amostras de sêmen de cada touro foram processadas individualmente e centrifugadas em: F1 (9000 X g) e F4 (2200 X g). Todas as amostras espermáticas foram avaliadas quanto a concentração, motilidade, vigor, morfologia, espécies reativas de oxigênio (EROs) e integridade da membrana plasmática, sendo no experimento 2 avaliados ainda a peroxidação lipídica, defesas antioxidantes e desenvolvimento embrionário. Não foi observada diferença na concentração espermática nos sêmens submetidos a diferentes forças centrifugação. No Experimento 1, a percentagem de espermatozoides móveis foi superior (p <0,05) após centrifugação em F3 e F4 e a produção de EROs de F1 foi superior (p <0,05) em comparação com outras forças. No Experimento 2, quando o sêmen de cada touro foi processado individualmente, não foram observadas diferenças significativas nos parâmetros de qualidade espermática, peroxidação lipídica, defesas antioxidantes, taxa de clivagem e tempo médio da primeira clivagem entre F1 e F4. No entanto, o aumento da força de centrifugação reduziu a taxa de penetração e a fertilização normal (P<0,05). Este trabalho demonstrou pela primeira vez que a força de centrifugação de 2200 X g aumentou a penetração e a taxa de fecundação em relação à força de centrifugação habitual (9000 X g) utilizadas na separação de espermatozoides por gradientes descontínuos Percoll ® em bovinos. Estes resultados sugerem que esta força de centrifugação pode ser utilizada com sucesso na PIV de embriões bovinos, uma vez que não reduz a recuperação de espermatozoides e aumenta a taxa de fecundação. / Centrifugation by Percoll® gradient is the most widely used method in the preparation of bull sperm for the purpose of in vitro fertilization, however at the moment, no scientific study determined the best centrifugation speed to remove seminal plasma and other constituents, i.e. the speed at which the loss of sperm cells is minimized and where the spermatozoa in the pellet still remain functional. The present study was designed to examine the efficiency of different centrifugation forces in sperm separation Percoll by methods evaluating sperm quality parameters and subsequent development and quality of bovine in vitro production (IVP) embryos. Additionally, we evaluated oxidative stress parameters to verify a possible relationship with the findings in the sperm evaluation. In Experiment 1, the semen samples from each bull were pooled and submitted to centrifugation in discontinuous gradients Percoll (30,60 and 90%) at different forces: F1 (9000 X g), F2 (6500 X g), F3 (4500 X g) and F4 (2200 X g). In Experiment 2, the semen samples from each bull were done separately and submitted to: F1 (9000 X g) and F4 (2200 X g). All sperm samples were evaluated to the concentration, motility, vigor, morphology, reactive oxygen species (ROS) and integrity of the plasma membrane, and in experiment 2 also were evaluated lipid peroxidation, antioxidants assays and embryo development. No difference was observed in the concentration of sperm submitted to different centrifugation forces. In Experiment 1, the total percentage of motile sperm was increased (p < 0.05) after centrifugation in F3 and F4 and the ROS production to F1 was superior (p < 0.05) compared to other forces. In Experiment 2, when the bull semen was processed individually, no significant difference was observed in the sperm quality parameters assessment, lipid peroxidation, antioxidants assays, cleavage rate and average time of the first cleavage between F1 and F4, sires or interaction between them after Percoll. However, the increased force centrifugation reduced the rate of penetration and normal fertilization (P < 0.05). This work demonstrated for the first time that 2200 X g centrifugation force enhanced the penetration and fertilization rates in relation to usual centrifugation force (9000 X g) using sperm separation by discontinuous Percoll gradients in bovine. These findings suggest that this centrifugation force could be used with successful in the bovine IVP embryo since it does not reduce sperm recuperation and increases the fecundation rate.

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-03-08T20:08:58Z No. of bitstreams: 1 127110020.pdf: 531553 bytes, checksum: 0170d654c8a7662821baf128927374e8 (MD5) / Made available in DSpace on 2015-03-08T20:08:58Z (GMT). No. of bitstreams: 1 127110020.pdf: 531553 bytes, checksum: 0170d654c8a7662821baf128927374e8 (MD5) Previous issue date: 2014-08-12 / A técnica de seleção espermática para fecundação in vitro (FIV) deve proporcionar além, da recuperação eficiente de espermatozoides, a manutenção da integridade celular. Diversos métodos vêm sendo propostos para a seleção de espermatozoides bovinos destinados a FIV, sendo o método de centrifugação em gradientes de densidade descontínuos de Percoll® o mais utilizado. Em bovinos esta técnica de seleção vem sendo amplamente modificada no que se refere ao volume, número de gradientes, tempo e força de centrifugação, visando aumentar a recuperação espermática, proporcionando um melhor rendimento da dose. Contudo, a etapa de centrifugação, presente nestes protocolos, pode causar danos irreversíveis aos espermatozoides e assim influenciar nas taxas de fecundação. O presente estudo teve como objetivo determinar a influência da força e do tempo de centrifugação, assim como a associação do meio Cushion Fluid® na segunda centrifugação na seleção espermática por gradientes de Mini Percoll® modificado, através da avaliação de características morfofuncionais, formação de espécies reativas ao oxigênio (EROs), defesas antioxidantes, taxa de fecundação in vitro e desenvolvimento embrionário em três experimentos. No experimento I, um pool contendo sêmen de dois touros Bos taurus taurus com fertilidade conhecida foi submetido ao método de separação por Mini Percoll®, sendo as amostras divididas em quatro grupos e submetidos a duas forças (2200 ou 9000 X g) e dois tempos de centrifugação (1 e 3 minutos). No experimento II e III, um pool de sêmen de dois touros Bos taurus taurus com fertilidade conhecida foram processados pelo método de Mini Percoll® modificado, sendo adicionado uma quantidade de 150μL de Cushion Fluid® (Minitübe, Tiefenbach, Germany) durante a segunda centrifugação. Foram realizadas avaliações morfofuncionais e bioquímicas das amostras do sêmen destinado a FIV, assim como a capacidade fecundante e o desenvolvimento embrionário dos oócitos fecundados com os espermatozoides selecionados. No primeiro experimento foi observada uma melhora no vigor dos espermatozoides após o gradiente de Mini Percoll®, para todos os tratamentos. No segundo experimento, a taxa de recuperação espermática não diferiu entre os tratamentos, sendo 42,0% ± 4,9 para T1 e 38,6% ± 3,6 para T2 (grupo com adição do Cushion Fluid® na segunda centrifugação):. Na morfologia espermática o T2 (5,1 ± 1,1) apresentou menor taxa de espermatozoides com defeitos maiores do que os tratamentos T0 (Pré-Percoll) e T1 (8,8 ± 0,8 e 5,8 ± 1,0). Neste mesmo experimento, os níveis de EROs apresentaram-se aumentados (P < 0,05) no meio com Cushion Fluid® (T2), no entanto, a avaliação das defesas antioxidantes não diferiram entre os tratamentos. No experimento III, não foram observadas diferenças na taxa de fecundação. Este trabalho demonstrou que o uso de uma menor força e tempo (2200 X g/1 min.) de centrifugação pode ser utilizado sem interferir nas taxas de recuperação espermática. O uso do meio Cushion Fluid® durante a centrifugação de espermatozoides bovinos não influenciou a taxa de fecundação e desenvolvimento embrionário até 48 horas. / The technique of sperm selection for in vitro fertilization (IVF) should provide addition, the sperm efficient recovery, the maintenance of cellular integrity. Several methods have been proposed for bovine sperm selection for IVF, and the discontinuous density gradients Percoll® ( Sigma Aldrich – St. Louis MO, USA) centrifugation method on the most used. In cattle this selection technique has been extensively modified with respect to the volume, number of gradients, time, and centrifugal force, to increase the sperm recovery, providing a better yield of the dose. However, the centrifugation step present in these protocols, may cause irreversible damage to sperm and thus influence fertilization rate. The present study aimed to determine the influence of centrifugation force and time, as well as the association of the Cushion Fluid® centrifugation on sperm selection by gradients Mini Percoll® modified by evaluating morphological and functional characteristics, ROS formation, antioxidant defenses, rate of in vitro fertilization and embryo development. Three experiments carry out. In experiment I, a pool containing two semen Bos taurus taurus bulls was subjected Mini Percoll® separation method, samples being divided into four groups, with two centrifugal forces (2200 or 9000 X g) and two times (1 and 3 min). In experiment II and III, the pool has been processed by the Mini Percoll® modified method, being added to an amount of 150μLix of Cushion Fluid® (Minitübe, Tiefenbach, Germany) in the second centrifugation in the treated group. Morphofunctional and biochemical evaluation of semen samples for IVF, as well as the fertilizing capacity and embryo development of oocytes fertilized with sperms were selected performed. In the first experiment an improvement was observed in the vigor of sperm after gradient Mini Percoll® for all treatments. In the second experiment, the recovery rate sperm did not differ between treatments (T1: 42.0 ± 4.9 and T2 (with adding Cushion Fluid® the second centrifugation): 38.6 ± 3.6). In sperm morphology T2 (5.1 ± 1.1) showed a lower sperm rate with major defects than T0 (Pre-Percoll) and T1 (8.8 ± 0.8 and 5.8 ± 1.0 treatments ). In this same experiment, ROS levels were presented increased (P <0.05) in the medium with Cushion Fluid® (T2), however, the evaluation of antioxidant defenses did not differ between treatments. In experiment III, no differences in fertilization rate were observed. This study demonstrated that the use of a smaller force and time (2200 X g / 1 min.) to centrifugation can be used without interfering with the sperm recovery rate. The use of the medium Cushion Fluid® during centrifugation of bovine sperm did not influence the fertilization rate and embryo development to 48 hours.

O sêmen eqüino é muito utilizado na forma refrigerada, porém, para alguns garanhões, a longevidade espermática é baixa. Durante a refrigeração o espermatozóide pode ser afetado por vários fatores, que podem causar a perda da integridade da membrana plasmática, danos no acrossomo e disfunções mitocondriais, resultando em perda irreversível de motilidade e capacidade fecundante. A composição e concentração do plasma seminal podem causar efeitos deletérios ao espermatozóide, sendo a centrifugação do sêmen uma alternativa para minimizar estes efeitos. Este experimento foi realizado para verificar o efeito da centrifugação nas características de movimento, integridade e peroxidação lipídica das membranas do espermatozóide eqüino refrigerado a 5°C por até 72h. Foram realizadas cinco colheitas de sêmen de quatro garanhões. O sêmen foi avaliado quanto ao volume e concentração, e em seguida dividido em dois tratamentos, sem centrifugação (S) e centrifugado (C) (300g/10min). Ambos foram diluídos na concentração final de 30 x106 eptz/mL em diluidor a base de leite desnatado e então refrigerados. Foram analisadas as características do movimento espermático (CASA), morfologia espermática (DIC), integridade das membranas plasmática e acrossomal (PI/FITC-PSA), potencial de membrana mitocondrial (JC-1) e peroxidação lipídica das membranas (C11-BODIPY581/591), sendo estas três últimas análises por citometria de fluxo. As análises foram realizadas no tempo 0 e 24, 48 e 72 horas após a refrigeração. O delineamento experimental foi em blocos generalizados adicionado do fator medidas repetidas no tempo. Os dados foram analisados pelo programa SAS, submetidos à análise de variância e às interações determinadas pelo teste de Greenhouse-Geisser ao nível de 5% de significância. O espermatozóide do sêmen centrifugado apresentou valores médios maiores de motilidade total e progressiva, ALH, STR e LIN (P<0,05), porém os valores das velocidades e BCF foram menores (P<0,05). Houve efeito do tempo nas características de movimento espermático (P<0,05), exceto para STR. Não houve efeito da centrifugação e nem do tempo quanto às características morfológicas dos espermatozóides refrigerados, esta só diferiu entre os garanhões (P<0,05). Por sua vez, a integridade das membranas plasmática e acrossomal e o potencial de membrana mitocondrial espermáticas foram maiores nos sêmen centrifugado (P<0,05) e suas porcentagens decresceram com o tempo (P<0,05). A peroxidação lipídica da membrana do espermatozóide refrigerado aumentou somente nas primeiras 24h (P<0,05) e não foi influenciada pela centrifugação. Conclui-se que a centrifugação e a retirada do plasma seminal melhoram a qualidade do sêmen refrigerado eqüino, principalmente em garanhões com baixa longevidade, sendo esta influenciada pelas características iniciais da morfologia espermática. Ainda, tempo de refrigeração induz a um declínio da qualidade seminal. E, por fim, a peroxidação lipídica da membrana do espermatozóide parece não ser a principal causa de alterações da integridade das membranas e do potencial de membrana mitocondrial durante a refrigeração do sêmen eqüino. / Equine cooled semen is frequently used, although the longevity for some stallions is low. During the cooling process the spermatozoon can be affected by several factors that can cause loss of plasma membrane integrity, acrosomal damage and mitochondrial dysfunctions, resulting in irreversible loss of motility and fertilizing capacity. The composition and concentration of seminal plasma could cause harmful effects to the sperm, being semen centrifugation an alternative to minimize these effects. This experiment was designed to verify the effects of the centrifugation in motion characteristics, membranes integrity and lipid peroxidation of cooled equine sperm to 5°C until 72 hours. Five ejaculates from four stallions were collected. The semen was evaluated for volume and concentration, then split in two treatments, no centrifugation (N) and with centrifugation (W) (300g/10min). Both were diluted in the final concentration 30 x106 spermatozoa/mL in a skin milk extender and then cooled. The following analyses were performed: the sperm motion characteristics (CASA), morphology (DIC), plasma and acrosomal membranes integrity (PI/FITC-PSA), mitochondrial membrane potential (JC-1) and membrane lipid peroxidation (C11-BODIPY581/591), the last three analyses were assessed in flow citometry. The analyses were performed in time 0 and 24, 48 and 72 hours after cooling. The experimental design was random blocks with repeated-measures. The data were analyzed by the SAS program, submitted to analysis of variance and the interactions by the test of Greenhouse-Geisser at the level of 5% of significance. The values were larger in W semen for total and progressive motility, ALH, STR and LIN (P<0,05), however the values of the three types of speed and BCF were smaller (P<0,05). There was effect of time in the motion characteristics of sperm (P<0,05), except for STR. There was not any effect of centrifugation and time for the morphologic characteristics of cooled spermatozoa, that only differed among stallions (P<0,05). On the other hand, the sperm integrity of plasma and acrosomal membranes and the potential of mitochondrial membrane were larger in W semen (P<0,05) and their percentages decreased within time (P<0,05). The membrane lipidic peroxidation of the cooled sperm only increased in the first 24 hours (P<0,05) and it was not influenced by centrifugation. In conclusion, the centrifugation and withdrawal of the seminal plasma improve semen quality of the cooled equine sperm, mainly in stallions with low longevity, which is influenced by the initials characteristics of sperm morphology. Moreover, time of cooling induces a decline of seminal quality. Finally, the membrane lipidic peroxidation of the cooled sperm seems not to be the main cause of alterations in the sperm integrity of plasma and acrosomal membranes and the potential of mitochondrial membrane during the cooling of equine semen.

The vestibular system senses changes in head position and is responsible for the brain’s perception of verticality. Vestibular dysfunction is caused by deficits in the semicircular canals and/or otolith end-organs with resulting symptoms including dizziness, vertigo, and unsteadiness. Current vestibular rehabilitation focuses on compensation of the semicircular canal-mediated vestibulo-ocular reflex through gaze and balance exercises. Little is known about rehabilitation of otolith organ function, yet research findings suggest that fall risk may be related to otolith dysfunction. A recent case study demonstrated improvement of vertical perception and balance following off-axis rotation in a rotary chair, showing that such stimulation may be useful for compensation of otolith organ dysfunction. The purpose of our research was to further investigate off-axis rotation as a possible treatment method by evaluating subjective visual vertical (SVV) in healthy controls. Two distance parameters (3.5 cm off-axis and 7.0 cm off-axis) were applied to the rotary chair, with results measured through the SVV test, visual analog scales (VAS), and the balance tilt test (BTT). The magnitude of SVV shift following off-axis rotation was measured in both the 3.5 cm and 7.0 cm off-axis experiments. The greater distance parameter (7.0 cm) did not increase SVV shift magnitude more than the 3.5 cm parameter; yet, resulted in greater symptom intensity as measured through the VAS. These findings led to the conclusion that a distance parameter of 3.5 cm off-axis is optimal for stimulating the otolith organs. This discovery may be helpful in future research utilizing off-axis rotation as a possible treatment method for vestibular patients suffering from otolith dysfunction.

[Truncated abstract] The feasibility of using interlocked light gauge sheet piles to form a deep cross-sectional wall embedded in a residual slope or with a berm support is explored. This thesis compares the performance of a large section modulus sheet pile wall as an alternative to a concrete diaphragm wall, acting as an embedded cantilever wall on a slope (ECWS) by means of experimental centrifuge tests, numerical models and analytical methods. Abaqus (Hibbitt, Karlsson and Sorensen Inc, 1997) was used to conduct extensive numerical trials on the structural performance of the sheet pile wall model prior to the actual physical testing. The Abaqus results showed that the integrity of the interlock and reduced modulus action (RMA) due to slippage along the interlocked joint did not cause premature buckling of the thin wall even at the ultimate load. Further, a comparative study using centrifuge tests on 1:30 scaled models and Plaxis analysis demonstrated that under the worst condition with high water table, the rigid sheet pile wall of 1.32 m cross-sectional width carried a higher ultimate surcharge load with a much lower top of wall deflection, compared to a more flexible 0.6 m thick cracked concrete diaphragm wall. The analysis of the wall/soil/slope interactions for an ECWS involves many inter-dependent variables in addition to the complications of considering an adjacent slope or a berm support. It is difficult for existing analytical approaches to take all these factors into account, and some form of numerical analysis, calibrated through field data and results from centrifuge model tests is necessary. From the observations of the centrifuge tests and finite element analysis, major assumptions about the failure of a stiff ECWS in a rotational mode were deduced and adopted in the proposed limiting equilibrium method (Leq). The plane strain Leq ECWS Abstract ii analysis is based on the framework of minimum upper bound limiting equilibrium with planar failure planes and a Mohr-Coulomb soil model. As compared to the traditional limit equilibrium analysis, the Leq method is a fully coupled analysis using the shear strength reduction technique (SSR). New formulations are proposed for the development of horizontal active and passive pressure distributions based on the experimental and FE models. The proposed active pressure profile used is derived by combining the Coulomb and Krey method, and empirically back-figured to curve-fit the centrifuge tests by Morris (2005). The proposed passive pressure profile of a rigid rotational wall in failure is adjusted to allow for an adjacent slope or berm support through a presumed elasto-plastic deformation instead of a linear rigid translation of the passive wedge. ... A parametric study was later undertaken using the Leq method to develop a series of non-dimensionalised graphs to study and draw summarised conclusions on the behaviour of the ECWS. The final conclusions on the comparative study of the centrifuge tests, Plaxis and Leq analyses demonstrated that the alternative light gauge steel sheet pile performed very well as an ECWS. A key factor in the performance of the sheet pile wall was attributed to the large 1.32 m cross-sectional width of the interlocked sections. This provided high bending stiffness and high moment stability from shear stresses acting on the back and front faces of the wall.

Hydrogels, which are polymeric cross-linked networks that swell in aqueous environments, are versatile materials that can contain a variety of chemical functionalities, mechanical properties, and topographical features. Microgels are the stable colloidal form of hydrogel materials that range in size from approximately 100 nm to a few microns in diameter. While they also can exhibit similar properties to those of macrogels, microgels can be used as building blocks in a bottom-up approach to assemble films of higher complexity. In this dissertation, work is focused on understanding the assembly and behavior of microgel thin films as non-fouling surfaces, centrifugally deposited materials, self-healing coatings, and degradable constructs. Non-fouling films were assembled using PEG cross-linked microgels to reduce non-specific protein adsorption and mitigate cellular adhesion. These constructs were assembled in a polyelectrolyte multi-layered fashion, of alternating anionic microgels and cationic linear polymer, to effectively block the substrate from the biological environment and consequently exhibited control over cellular adhesion with the surface. The utility and application of these non-fouling microgel coatings on functional implants was also explored. Centrifugal deposition was used to rapidly generate non-fouling microgel multi-layered interfaces on planar surfaces, and upon closer inspection of the microgel monolayers, it was found that the centrifugally deposited films contained closer-packed microgel assemblies with microgels of smaller footprint size, compared to microgels that are passively adsorbed to the surface. Microgels that are centrifugally deposited may adopt a higher energy chain conformation than passively adsorbed microgels, and this higher energy chain conformation may translate into the multi-layered materials. Nonetheless, the centrifugally deposited non-fouling microgel multi-layered films were found to effectively block macrophage adhesion. Films were also assembled in a polyelectrolyte fashion on soft substrates, and were observed to become significantly damaged under mechanical manipulation (poking, bending, or stretching), but then self-heal upon addition of water. By altering the building blocks of the polyelectrolyte multi-layered films, such as the molecular weight of the polycation between microgel layers or by using anionic rigid spheres as the particle in the assembly, changes in the observed film damage suggest that particle-linear polymer interpenetration and polyvalency likely play an important role in the strength and integrity of the microgel thin films. Fluorescently-labeled microgels were also used to interrogate how the films reorganize in the lateral direction, and these early studies suggest that the microgel multi-layered films reorganize when damaged and also possibly when they are undamaged and simply incubated in an aqueous environment. Additional studies were also conducted on microgels synthesized with a hydrolyzable cross-linker, and by supporting these degradable constructs on substrates, detailed single-particle morphological changes during erosion could be interrogated in complex media such as serum. This work, as a collection, demonstrates the ability to obtain information about microgel thin film assemblies and their behavior using microscopy techniques such as ambient and in liquid atomic force microscopy, brightfield optical microscopy, and fluorescence microscopy. The observations made here illustrate how microgels can be used to fabrication thin films that can be utilized in biological applications (non-fouling, self-healing, and erodable constructs), and how different deposition methods (centrifugal deposition and polyelectrolyte multi-layers) can dictate their behavior.

Das Wasser der Elbe auf der Höhe von Dresden enthält anorganische Kolloidpartikel, deren Partikelgrößenverteilung nahezu den gesamten kolloidalen Definitionsbereich (1 nm bis 1 µm) überstreicht und die vor allem aus sekundär ausgeschiedener amorpher Kieselsäure sowie aus Oxyhydroxiden des Fe, Al und Mn bestehen. Als wichtigstes Schwermetall führen sie Zn. Ihre Konzentration liegt unter 5×10-1 mg/l, um etwa Faktor 30 unter der Konzentration der Schwebstoffe des Elbwassers (Partikel >1 µm). Wegen ihrer hohen spezifischen Oberfläche sind die Kolloidpartikel als potentielle Träger für Schadstoffe trotz dieser geringeren Massekonzentration nicht gegenüber den Schwebstoffen zu vernachlässigen. Die in der Elbe gemessene Partikelgrößenverteilung ähnelt derjenigen, die zu einem früheren Zeitpunkt im Rhein gefunden worden war. Auch die chemische Zusammensetzung der Kolloidpartikel in der Elbe ist der der Partikel des Rheins ähnlich. Die Konzentration der Kolloidpartikel in der Elbe war aber um Faktor 5 bis 10 höher als im Rhein. Grund für letzteres ist wahrscheinlich der höhere Gehalt des Elbwassers an gelöstem Kohlenstoff (DOC). Im "Bulk" eines Flusses sind kolloidgetragene Schadstoffe - anders als schwebstoffgetragene - vermutlich fast genauso mobil wie echt gelöste. Unterschiede zwischen den Transportgeschwindigkeiten der kolloidgetragenen und der echt gelösten Spurenstoffe treten in bestimmten Situationen auf, in denen das Wasser den "Bulk" eines Flusses verlässt (Sickerbereich unter dem Fluss, Ästuar). Es werden Schlussfolgerungen über die Rolle von Kolloidpartikeln im Ökosystem eines Flusses gezogen und noch bestehende Forschungsdesiderate benannt.

Actuellement, le secteur des nanotechnologies est en développement intense. Les nanoparticules de dioxyde de titane et d¿or occupent une part importante de ce secteur, avec des applications utilisant principalement leurs propriétés optiques et catalytiques. Les synergies pouvant exister entre les nanoparticules d¿or et leur support, en particulier via l¿effet plasmon, permettent de modifier la réponse optique de dispositifs catalytiques ou photovoltaïques. Une nouvelle technique polyvalente de mise en forme de film mésoporeux à base de nanoparticules de dioxyde de titane et d¿or a été développée. L¿utilisation de différentes méthodes de synthèse des nanoparticules a permis le contrôle de la morphologie et des propriétés physiques des films déposés par centrifugation. Finalement, l¿adéquation de ces films à leur utilisation en tant qu¿anode de cellules solaires à colorant a été évaluée.