Thesis (Ph.D.)--University of Wollongong, 2006. / Typescript. Includes bibliographical references: leaf 208-224.

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.

Submitted by Lais Lucas Fernandes (lais-f@hotmail.com) on 2018-07-02T03:45:59Z No. of bitstreams: 1 BONECO DEFINITIVO CORRIGIDO com ficha.pdf: 3250792 bytes, checksum: c27c416f86a495d9eadf4074c1b1f411 (MD5) / Approved for entry into archive by Maria Lucia Martins Frederico null (mlucia@fca.unesp.br) on 2018-07-02T17:41:54Z (GMT) No. of bitstreams: 1 fernandes_ll_me-botfca.pdf: 3250846 bytes, checksum: 59b007beb7ecad527250296a9d0d2348 (MD5) / Made available in DSpace on 2018-07-02T17:41:54Z (GMT). No. of bitstreams: 1 fernandes_ll_me-botfca.pdf: 3250846 bytes, checksum: 59b007beb7ecad527250296a9d0d2348 (MD5) Previous issue date: 2018-05-17 / O estudo visou comparar e avaliar protocolos de produtos autógenos sanguíneos em aves, com base naqueles existentes para mamíferos. No Experimento 1 foram analisados dois protocolos para obtenção de plasma rico em trombócitos e leucócitos (L-PRT). Utilizaram-se 30 aves divididas em três Grupos equitativos: G1 - papagaios; G2 - tucanos-toco; G3 - galinhas domésticas. No protocolo 1, a primeira centrifugação foi a 220 gravidade (g) durante 10 minutos e a segunda a 660 g por 10 minutos. Após a segunda centrifugação, foi descartado 2/3 do sobrenadante, permanecendo apenas o L-PRT. No protocolo 2, a primeira centrifugação foi a 120 g durante 5 minutos e a segunda a 240 g por 5 minutos. Concluiu-se que houve diferenças na concentração de trombócitos entre as espécies, porém independente do protocolo a maior concentração foi nas galinhas, e entre os Protocolos o 2 foi o mais efetivo. No Experimento 2 foram produzidas e avaliadas histologicamente membranas de fibrina rica em trombócitos e leucócitos (L-TRF). Empregaram-se 40 aves divididas em quatro grupos equitativos: G1 – araras, G2 - galinhas domésticas, G3 – papagaios, G4 - tucanos-toco. Para cada ave foi coletado 0,5 ml de sangue, que foi depositado em tubo de vidro sem anticoagulante e centrifugado a 3000 rpm por 10 minutos. Membranas de L-TRF obtidas pela compressão dos cóagulos com gaze foram processadas para análise histológica. Foi possível concluir que é possível produzir membranas de L-TRF nas espécies de aves estudadas, porém histologicamente as proporções dos elementos avaliados foram similares apenas nas galinhas domésticas e papagaios. / This study aimed to compare and evaluate protocols of autogenous blood products in birds, based on protocols developed for mammals. In Experiment 1, two protocols were evaluated for obtaining Leukocyte- and Thrombocyte-Rich Plasma (L-TRP). Thirty birds were divided into three equally sized groups: G1 - parrots; G2 - toco toucans; G3 - domestic chickens. In Protocol 1 the first centrifugation was at 220 gravity (g) for 10 minutes and the second one at 660 g for 10 minutes. After the second centrifugation, 2/3 of the supernatant was discarded, leaving only the L-TRP. In protocol 2, the first centrifugation was at 120 g for 5 minutes and the second one at 240 g for 5 minutes. In conclusion, there were differences in thrombocyte concentration among the species, but independently of the protocol, the highest concentration was in chickens. Between the protocols, Protocol 2 was the most effective. In Experiment 2, Leukocyte- and Thrombocyte-Rich Fibrin (L-TRF) membranes were developed and assessed histologically. Forty birds were divided into four equally sized groups: G1 – macaws, G2 - domestic chickens, G3 – parrots, G4 - toco toucans. A total of 0.5 mL of blood was collected from each bird, which was put into glass tube without anticoagulant and centrifuged at 3000 rpm for 10 minutes. L-TRF membranes produced after compression of the clot were processed for histological analysis. In conclusion, L-TRF membranes can be produced in the evaluated avian species, but the ratio of the elements evaluated histologically were similar only in domesticated chickens and parrots.

Submitted by Suzane Peres Campanholi null (supc@hotmail.com) on 2016-06-02T21:33:16Z No. of bitstreams: 1 Dissertação Mestrado Suzane - Versão final pro repositório UNESP.pdf: 1223510 bytes, checksum: 26ebdc009120ee189335639e58233246 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-06-06T16:46:24Z (GMT) No. of bitstreams: 1 campanholi_sp_me_jabo.pdf: 1223510 bytes, checksum: 26ebdc009120ee189335639e58233246 (MD5) / Made available in DSpace on 2016-06-06T16:46:24Z (GMT). No. of bitstreams: 1 campanholi_sp_me_jabo.pdf: 1223510 bytes, checksum: 26ebdc009120ee189335639e58233246 (MD5) Previous issue date: 2016-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O plasma seminal já foi descrito como benéfico e ao mesmo tempo prejudicial aos espermatozoides e isto está relacionado com o tempo de contato entre eles. A criopreservação do sêmen em touros pode ser prejudicada por exposição contínua dos espermatozoides ao plasma seminal (PS) e a aplicação de métodos para sua remoção pode aumentar a qualidade dos espermatozoides recuperados pós-descongelação, melhorando os resultados de biotécnicas que utilizam sêmen criopreservado. Pelo fato da centrifugação causar muitos danos aos espermatozoides, a filtragem com Sperm Filter® apresenta-se como um novo método de remoção do PS que almeja melhores resultados, porém não foi encontrado relato da sua aplicação em bovinos até o momento. O objetivo deste estudo foi avaliar o efeito dos métodos de centrifugação e filtragem com Sperm Filter® sobre a criopreservação do sêmen bovino. Para isso, o sêmen de 38 touros Nelore foi colhido por eletroejaculação. Após a colheita, o sêmen foi fracionado em três alíquotas iguais para divisão em três grupos: controle (N), em que o sêmen foi diluído com PS; centrifugação (C), em que o PS foi removido por centrifugação a 600 X g por 10 minutos; e filtragem (F), em que o PS foi removido por filtragem com Sperm Filter®. As amostras foram criopreservadas, e pós-descongelação foram avaliados a cinética espermática, a integridade das membranas plasmática e acrossomal, o potencial mitocondrial, o estresse oxidativo e a capacidade fecundante através da produção in vitro de embriões (PIVE). Com relação à cinética, maiores valores da velocidade do trajeto (P = 0,0005) e velocidade progressiva (P = <0,0001) foram observados nos grupos C e F e os parâmetros frequência de batimento, retilinearidade e linearidade foram superiores no grupo F (P = 0,0008; P = 0,0133 e P = 0,0005, respectivamente). A integridade de membrana foi prejudicada pela centrifugação e filtragem do sêmen (P < 0,0001), porém o estresse oxidativo foi reduzido com esses métodos de remoção do PS (P < 0,0001). Na PIVE, as maiores taxas de desenvolvimento embrionário (blastocistos e blastocistos eclodidos) foram observadas no grupo N e F (P = 0,008 e P = 0,0042, respectivamente). Portanto, a remoção do plasma seminal pelo método da centrifugação reduz a qualidade do sêmen criopreservado bovino, interferindo negativamente na fertilidade dos espermatozoides e o método da filtragem com Sperm Filter® apresentou melhor resultado evidenciado por altas taxas de desenvolvimento embrionário. / Seminal plasma has been described as beneficial and harmful to the spermatozoa and this is related to the contact time between them. Cryopreservation of semen from bulls can be undermined by continuous exposure of sperm to the seminal plasma (SP) and the application of methods for removal can increase the quality of post-thaw sperm recovered, improving fertilization. Centrifugation cause a lot of damage to sperm and filtering with Sperm Filter® is a new SP removal method that aims to better results, but has not yet been applied in bull. The aim of this study was to evaluate the effect of the methods of centrifugation and filtering with Sperm Filter® on cryopreservation of bovine semen. For this, semen of 38 Nelore bulls was collected by electroejaculation. Semen was fractioned into three equal aliquots for division into three groups: control (N), wherein the semen was diluted with SP; centrifugation (C), in which the SP was removed by centrifugation at 600 X g for 10 minutes; and filtration (F), in which the SP was removed by filtration with Sperm Filter®. Samples were cryopreserved and was evaluated after thawing sperm kinetics, the integrity of plasma and acrosomal membranes, mitochondrial potential, oxidative stress and the fertilizing capacity by in vitro embryo production (IVP). The kinetics, higher values of the path velocity (P = 0.0005) and progressive speed (P = <0.0001) were observed in the groups C and F and the beat frequency parameters, straightness and linearity were higher in group F (P = 0.0008, P = 0.0133 and P = 0.0005, respectively). Membrane integrity was impaired by centrifugation and filtration of semen (P <0.0001), though oxidative stress was reduced with these SP removal methods (P <0.0001). In IVP, the highest rate of embryonic development (blastocyst and hatched blastocyst) were observed in the C and F group (P = 0.008 and P = 0.0042, respectively). Therefore, removal of seminal plasma by the method of centrifugation reduces the quality of semen cryopreserved cattle, a negative effect on fertility of sperm, and the method of filtering with Sperm Filter® showed better results evidenced by high rates of embryonic development.

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Agradecemos a compreensão. on 2016-09-22T17:06:43Z (GMT) / Submitted by RODRIGO DOS SANTOS ALBUQUERQUE null (rdsa20@gmail.com) on 2016-09-22T17:53:18Z No. of bitstreams: 1 Dissertação de mestrado Rodrigo Albuquerque.pdf: 1101110 bytes, checksum: 06cce8f64ce03aadf0ced70da5dcfcab (MD5) / Approved for entry into archive by Felipe Augusto Arakaki (arakaki@reitoria.unesp.br) on 2016-09-27T12:31:29Z (GMT) No. of bitstreams: 1 albuquerque_rs_me_jabo.pdf: 1101110 bytes, checksum: 06cce8f64ce03aadf0ced70da5dcfcab (MD5) / Made available in DSpace on 2016-09-27T12:31:29Z (GMT). No. of bitstreams: 1 albuquerque_rs_me_jabo.pdf: 1101110 bytes, checksum: 06cce8f64ce03aadf0ced70da5dcfcab (MD5) Previous issue date: 2016-03-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação para o Desenvolvimento da UNESP (FUNDUNESP) / A criopreservação provoca injúrias aos espermatozoides e necessita de métodos que minimizem esses danos. Embora seja bastante discutida, a remoção do plasma seminal tornou-se uma alternativa para melhorar a qualidade e viabilidade espermática após a congelação e vem sendo utilizada em outras espécies na tentativa de obter bons resultados. O objetivo deste estudo foi comparar a remoção do plasma seminal de bubalinos tanto por filtragem (SpermFilter®) quanto por centrifugação sobre a qualidade do sêmen descongelado de búfalos. Foram utilizados sete touros da raça Murrah, os quais foram submetidos à colheita de sêmen por vagina artificial uma vez por semana. Os ejaculados foram diluídos com Botu-Bov® e fracionados em alíquotas para confrontar três técnicas antes da congelação: grupo C (Criopreservação convencional com plasma seminal), grupo CE (Criopreservação removendo o plasma seminal por centrifugação) e grupo F (Criopreservação removendo o plasma seminal por filtragem). O sêmen fresco foi analisado quanto ao volume, aspecto, cor, concentração (câmara de Neubauer), turbilhonamento, motilidade progressiva, vigor e morfologia espermática. No sêmen descongelado, foram avaliados a cinética espermática pelo CASA, a integridade de membrana plasmática, acrossoma, potencial de membrana mitocondrial e quantificação de EROs por citometria de fluxo e, adicionalmente, foi estimado o grau de peroxidação lipídica pela técnica de TBARS. Os resultados revelam que a remoção de plasma seminal não proporcionou aumento da velocidade espermática, alteração no potencial de membrana mitocondrial e nem modificou o grau de peroxidação lipídica. Entretanto, a presença do plasma seminal (grupo controle) e a centrifugação provocou maiores índices de lesões na membrana plasmática, quando estas apresentam o acrossoma intacto (47,88±1,99% vs. 41,30±1,96%, respectivamente) em comparação ao grupo F (40,45±2,01%; p<0,07). A lesão em ambas as membranas (acrossomal e plasmática) foi mais evidente na filtragem (22,18±1,07) em relação ao grupo controle (15,23±1,06), sendo que a centrifugação não diferiu entre os grupos (17,43±1,04; p<0,01). A EROs foi mais evidente no grupo CE (56,68±4,53%) em relação ao grupo C e F (21,83±4,60% vs. 20,96±4,7%, respectivamente; p<0,01). Portanto, as hipóteses de que a remoção de plasma seminal melhoram a cinética espermática e proporcionam menores índices de lesões na membrana plasmática não foram confirmadas. Embora a filtragem tenha apresentado vantagem sobre a centrifugação gerando menor quantidade de EROs, não se justifica a remoção do plasma seminal em búfalos devido aos resultados serem muito semelhantes ao grupo controle. / Cryopreservation promotes injury to sperm and methods to minimize such damage are necessary. Although much discussed, removal of seminal plasma that has been successfully used in other species has become an alternative for improving sperm quality and viability after freezing. The aim of the present study was compare to removal seminal plasma in buffalo both by filtering (SpermFilter®) and by centrifugation on the quality frozen thawed buffalo semen. For this purpose, semen of seven Murrah buffalo-bulls, which were submitted to semen collection by artificial vagina once a week. The samples were diluted with Botu-Bov® and were fractionated in aliquots to confront three techniques before freezing: C group (conventional cryopreservation with seminal plasma), CE group (cryopreservation removing the seminal plasma centrifuge) F group (Cryopreservation removing the seminal plasma with Sperm Filter®). Fresh semen was analyzed for volume, appearance, color, concentration (Neubauer chamber), gross motility, progressive motility, vigor and sperm morphology. In the thawed sperm, sperm kinetics was evaluated by CASA, plasma membrane and acrosomal integrity, mitochondrial membrane potential and estimation of the levels of reactive oxygen species were assigned by flow cytometry and was estimated degree of lipid peroxidation by TBARS technique. The results show that the removal seminal plasma did not improve sperm kinetics, mitochondrial membrane potential and does not modify the degree of lipid peroxidation.However, the presence of seminal plasma (control group) and centrifugation resulted in higher rates of injury to the plasma membrane, where they have an intact acrosome (47,88±1,99% vs. 41,30±1,96%, respectively) compared to F group (40,45±2,01%; p<0.07). The damage in both membranes (acrosome and plasma) was more evident in filtering (22,18±1,07) compared to the control group (15,23±1,06), and centrifugation did not differ between groups (17,43±1,04; p<0.01). ROS production was more evident in the CE group (56,68±4,53%) compared to the C and F group (21.83±4.60% vs. 20.96±4.7%, respectively; p<0.01). Therefore, removal seminal plasmais not beneficial to sperm, as did not alter the sperm kinetics nor provided lower lesions rates in the plasma membrane, although the filter has presented advantage over centrifugation as generated lower amount of ROS, not justifies the use bothtechnique because the results are very similar to those with seminal plasma. / FUNDUNESP: 2523-002-14

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.

The result of cryopreservation of semen is crucial for patients in need of fertility preservation.The cryopreservation method is not optimized since only 10 % the sperms are expected tosurvive the treatment. The sperms are exposed to many risk factors such as oxidative stress,osmotic chock and ice crystallization. To minimize the risks, use of cryoprotectants is needed.The use of cryoprotectants helps the cell to dehydrate as penetrating cryoprotectants cancreate space between ice crystals and cell membrane.Two studies were performed. In study one, two different freezing medias (SpermFreezesolution and Cryoprotect II effect on sperm motility after freezing in were compared). Studytwo investigate whether the motility were best preserved if semen froze with all the contentsof the ejaculate or if the sample should be concentrated, with removal of seminal plasma,epithelium cells and dead sperm cells by gradient centrifugation.The project was performed according to recommended instructions for each freezing media.In total, 55 samples were collected for the first study and 23 samples were collected for thesecond study. The sperm motility was measured both before freezing and after thawing.The Cryoprotect II medium preserved the cells better than the SpermFreeze medium(p=0,006). Using SpermFreeze, higher rate of motility was obtained when centrifugation wereperformed before freezing (p=0,033), while this was not observed when using Cryoprotect II(p=0,055). In conclusion Nidacon preserved sperm more effectively than Vitrolife freezemedium. Vitrolife’s freezing medium preserved the samples better if centrifugation wereperformed before freezing. Nidacons freezing medium gave the same result for the samplesno matter centrifugation were performed before or after freezing.

Orientador: Ricardo de Lima Isaac / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Civil / Made available in DSpace on 2018-08-05T08:38:56Z (GMT). No. of bitstreams: 1 SilvaJunior_ArchimedesPereirada_M.pdf: 2766426 bytes, checksum: c4a329b55233959c0c769a4fd9c68911 (MD5) Previous issue date: 2003 / Resumo: Os principais resíduos de ETAs são os Iodos gerados nos decantadores, a água de lavagem dos filtros e o rejeito de limpeza dos tanques de produtos químicos. Os lodos mesmo classificados como resíduos sólidos, apresentam baixíssimo teor de sólidos. Sendo o custo do transporte e disposição final bastante elevado, a redução do volume constitui-se em alternativa, economicamente, viável. Os principais processos utilizados para redução de volume do Iodo são o adensamento, o condicionamento químico e a desaguamento. A pesquisa teve por objetivo comparar a tratabilidade do Iodo do decantador do tipo convencional e do decantador de alta taxa, da SANASA, na cidade de Campinas. Foi verificada a influência dos parâmetros que caracterizam a qualidade do Iodo, no seu desaguamento; o efeito do adensamento do Iodo por gravidade, com auxílio de polímeros e o efeito do condicionamento químico com polímeros no desaguamento, por centrifuga. O lodo bruto foi pré-condicionado com polímeros sintéticos, e também se investigou a eficiência destes no condicionamento do lodo já adensado, para sua centrifugação. O adensamento mostrou que o polímero aniônico foi o melhor para ambas as ETAs. A dosagem ótima foi a mesma para ambas as épocas, para as duas ETAs, sendo de 1mg/g para ETA-3 e 3g/Kg para ETA-4. O tempo de mistura, para ETA-3, foi maior na época seca (45s) do que no período de chuvas (30s). Para ETA-4 o tempo de mistura foi o mesmo (60s) para as duas épocas. O lodo adensado foi condicionado com polímero e submetido à centrifugação. Desta, concluiu-se que, para o período de chuvas e para os dois tipos de lodo a condição ótima foi: polímero catiônico, 1g/Kg, 5s de mistura e 200rpm de rotação. Na época de estiagem e lodo da ETA-3 a condição ótima foi dada por: polímero catiônico, 1g/Kg, 60s de mistura e 200rpm de rotação. No caso da ETA-4, a condição é dada por: polímero catiônico, 2,5g/Kg, 60 s de mistura e 100 rpm / Abstract: The main residues of WTPs are sludge from thickener, filter backwash waters and solids from basins of chemicals products. The sludge even labeled as solid residues present very low solids content. Like the costs of transportation and ultimate disposal are very high, to reduce the sludge volume is an economical feasible alternative. The principal methods used to reduce the sludge volume are thickening, chemical conditioning and dewatering. The aim of this research was to compare the handling of both sludge from the conventional and high-rate thickener. It was checked the influence of the parameters that give particular characteristics to the sludge in its dewatering; the gravity thickening with polymers aid and the influence of the polymers in the sludge dewatering with centrifuge. The raw sludge was conditioned with synthetics polymers. Also was checked whether that polymers were efficient in the thickened sludge conditioning aiming its centrifugation. The best polymer to the gravity thickening for both sludge (WTPs 3 and 4) was the anionic polymer. The same polymer dose was required for the both seasons studied (wet and dry) and for the two WTPs. It was 1mg/g to the WTP-3 and it was 3g/Kg to the WTP-4. The mixing time for the WTP-3 was greater in the dry season (45s) than the wet season (30s) and the mixing time was the same (60s) in both seasons for the WTP-4. The thickened sludge was conditioned with organic synthetic polymer and subjected to centrifugation. After this, was concluded that in the wet season and for the two kinds of sludge the optimum condition s given to: cationic polymer, 1g/Kg of polymer dose, mixing time of 5s and 200 rpm of centrifuge rotation. In the dry season and residues from WTP-3 the optimum condition is given to: cationic polymer, 1g/Kg of polymer dose, mixing time of 60s and 200 rpm of centrifuge rotation. For residues from WTP-4 the optimum condition is given to: cationic polymer, 2,5g/Kg of polymer dose, mixing time of 60s and 100 rpm of centrifuge rotation / Mestrado / Saneamento e Ambiente / Mestre em Engenharia Civil

The Refcoal process is being developed to produce graphite from coal. Coal is dissolved in dimethylformamide (DMF) and sodium hydroxide (NaOH) is used as additive. After separation, the extracted coal (Refcoal) is precipitated with water and dried. The extraction process and subsequent solid-liquid separation processes have to be as efficient and cost-effective as possible. The purpose of the study was therefore to complete research on various unresolved aspects of the processes as identified by the candidate and supervisor. Extraction at 95 °C (DMF:coal:NaOH = 100:10:1), has an induction period of approximately 60 minutes observed, after which the reaction rate increases considerably. The reaction reaches completion after 360 minutes. An increase in stirring rate decreases extraction time due to the elimination of external mass-transfer limitations. The progress curves obtained for extraction at 135 °C with lower solvent-to-coal ratios differ dramatically from those obtained in previous studies, which indicates that changes in the raw materials and the experimental set-up have a great influence on the extraction at higher temperatures and concentrations. These extractions at higher temperatures using DMF:coal:NaOH ratios between 100:30:3 and 100:30:2 take approximately 360 minutes to complete and do not have an induction period as is the case with the extractions at 95 °C. It was found that the optimum DMF:coal ratio for an operating temperature of 135 °C, is 10:3. The high-temperature extractions reach completion in different time periods, depending on the amount of NaOH added to the reaction mixture. When very low concentrations of NaOH are added, the extraction will take much longer to complete and vice versa. The amount of NaOH used influences various aspects of the process. The cost analysis of the process falls beyond the scope of this investigation, but it is recommended that a thorough financial study is done to determine the optimum balance between raw materials, heat load and plant availability. The relationships between the concentration of Refcoal in the Refcoal solution and the absorbance values measured are polynomial expressions ending in downward concaves. The kinetics for the low-concentration (DMF:coal:NaOH = 100:10:1) extraction are best described by an autocatalytic reaction rate equation which is a function of coal, coal complex and NaOH concentration. A good fit was also obtained for the high temperature extractions. The rate expression is a function of both the coal and NaOH concentrations, but not of the coal complex. The sedimentation test showed promising results. The use of a thickener instead of a centrifuge to separate the insoluble material from the Refcoal solution would be a feasible cost-saving method. Filtration of the Refcoal solution (after centrifugation) using suitable filter media decreases the amount of impurities in the Refcoal. Filtration constants were determined for the best filter medium. The use of a hydrocyclone to separate the insoluble material from the extract is not recommended as it did not give the required efficiency to make the process viable. It is recommended that more tests be done under different conditions. Useful expressions were obtained for the change in viscosity with temperature for three different concentrations of Refcoal solution. It was determined that the viscosity of the Refcoal solution increases with time and it is therefore recommended that this be taken into account when equipment is being designed and plant scheduling is being done. / Dissertation (MEng (Chemical Engineering))--University of Pretoria, 2007. / Chemical Engineering / MEng / unrestricted

A new process is being developed to produce graphite from prime coking coal. Coal is dissolved in dimethylformamide (DMF), on addition of sodium hydroxide. The minerals and undissolved coal are separated by centrifugation and filtration to give a solution (referred to as Refcoal solution or RCS). Over 90 wt % of the organic part of a flotation product, from the Tshikondeni mine, can be dissolved at temperatures ranging from room temperature to 135°C. The purified coal (referred to as Refcoal) and DMF need to be separated. the Refcoal to be coked and the DMF to be purified and recycled. This process should be as efficient as possible, whilst both products should be low in water content to minimise drying costs. The addition of water to the Refcoal solution causes precipitation to take place, forming a gel (referred to as Refcoal gel) liquid system. This mixture can be either centrifuged or filtered to give a denser gel, containing water, DMF and coal solids, and supernatant or filtrate, containing water and DMF. Different techniques and processes can be used to improve the separation of the DMF from the Refcoal by achieving a denser Refcoal gel: • Longer centrifugation times improve the density and therefore the separation, but this technique has its limits. • The use of low-temperature water improves the separation. • The use of syneresis could improve separation at a lower cost: heated standing tanks are used to expel the supernatant and therefore increase the density of the gel, thereby decreasing the required number of washing stages. • The addition of toluene at the beginning of a wash improved the removal of DMF by 20%, using centrifugation as separation method. • Pressure filtration gave a 20% improvement on centrifugation, with no additives. • The addition of toluene to the pressure filtration process gave another improvement of 15%, and after three stages the percentage of solids in the gel was 28%, the highest so far achieved. This method also resulted in the highest removal of DMF in the first stage (73% of the original DMF in the RCS was removed). Counter-current washing shows the greatest potential, using the least amount of water. The concentration of DMF in the wash solution, to gel the Refcoal solution, is a limitation of this process. If the concentration is too high, no gelling and therefore no separation can take place in the first stage. It is recommended that counter-current washing using pressure filtration should be investigated; however, this will be difficult on a laboratory scale due to the mass losses during transfers. / Dissertation (MEng (Chemical Engineering))--University of Pretoria, 2009. / Chemical Engineering / unrestricted