A pirapitinga (Piaractus brachypomus) Ã uma espÃcie de peixe exÃtico introduzido no nordeste brasileiro o qual tem grande importÃncia comercial, devido a seu hÃbito alimentar, excelente crescimento, resistÃncia ao manejo em cativeiro e Ãs enfermidades. A criopreservaÃÃo de seu sÃmen pode contribuir nos aspectos econÃmico, genÃtico e meio ambiental da regiÃo e da espÃcie. O objetivo deste trabalho foi desenvolver um protocolo adequado de congelaÃÃo seminal para poder obter motilidades prÃximas ao sÃmen a fresco. Trabalhou-se com o sÃmen d e19 machos maduros de dezembro de 2007 a marÃo de 2008, determinando as caracterÃsticas seminais; a toxicidade dos crioprotetores DMSO 10% e metilglicol 10%, diluÃdos em glicose 5%, ACP 104 e soluÃÃo Ringer das diluiÃÃes de 1:3, 1:5 e 1:7 durante 15 minutos a 4Â C; a criopreservaÃÃo a -196Â C com as mesmas condiluiÃÃes usando palhetas de 0,25 e 0,5 mL, congeladas em vapores de nitrogÃnio lÃquido em caixas de isopor ou em âdry shipperâ, sendo depois transferidas para botijÃes criogÃnicos durante 15 dias. Foram avaliadas as motilidades e velocidades atravÃs da anÃlise seminal auxiliado por computador (CASA) pÃs-descongelaÃÃo; e a morfologia seminal fresco e pÃs-descongelaÃÃo. Os parÃmetros seminais a fresco observados foram: motilidade objetiva mÃdia de 94%, concentraÃÃo de 94 x 10 spzt/mL, volume d e7 mL, Osmolaridade de 317 mOsm/Kg e ph de 8,4. Nos testes de toxicidade nÃo foram encontradas diferenÃas significativas entre o T3 na diluiÃÃo 1:3 comparado com o grupo controle (P=0.06), apresentando motilidade de 80% e VCL de 95 um/s. O melhor mÃtodo de congelamento foi o âdry shipperâ utlizando palhetas de 0,5 mL registrando motilidade de 625 para o T6 na diluiÃÃo de 1:7, entretando, apresentaram baixas velocidades para todos os tratamentos, com melhores resultados de VCL, VSL e VAP para o T6. No sÃmen fresco a morfoanomalia mais encontrada foi a cauda dobrada (20%) e no sÃmen pÃs-descongelaÃÃo, a cauda enrolada (23%). Comparando-se os resultados de morfoanomalia antes e deposi do pÃs-descongelamento foram observadas diferenÃas significativas entre as amostras (P<0,05). Concluiu-se que o sÃmen da Piratininga pode ser preservado em Ringer+Retilglicol 105 em 1:3, sem apresentar altas toxicidades, e criopreservado em Glicose+DMSO 10% utilizando o âdry shipperâ. / The pirapitinga (Piaractus brachypomus) is a species of exotic fish introduced in northeastern Brazil which has great commercial importance due to their feeding habits, excellent growth, resistance management and diseases in captivity. The cryopreservation of their semen may contribute to the economic, environmental and genetic and species of the region. The objective of this study was to develop a protocol suitable for freezing sperm motility to get close to the fresh semen. Worked with the mature male sperm d e19 December 2007 to March 2008, determining the seminal characteristics, the toxicity of the cryoprotectants DMSO and 10% methylglycol 10%, diluted in glucose 5%, ACP 104 and the dilution of Ringer solution 1:3, 1:5 and 1:7 for 15 minutes at 4 Â C, the cryopreservation to -196 Â C with the same condiluiÃÃes using straws of 0.25 and 0.5 mL, frozen in liquid nitrogen vapors in styrofoam boxes or "dry shipper" and then transferred to cryogenic botijÃes for 15 days. We evaluated the motility and velocity through the computer-aided sperm analysis (CASA) after thawing, and sperm morphology fresh and post-thawing. The seminal parameters were observed in fresh: motility average objective of 94%, concentration of 94 x 10 spzt / mL, d e7 mL volume, osmolarity of 317 mOsm / kg and pH of 8.4. In toxicity tests were not significant differences between the T3 in 1:3 dilution compared to the control group (P = 0.06), showing 80% motility and 95 of a VCL / s. The best method of freezing was the "dry shipper" user straws of 0.5 mL recording motility of 625 for T6 in dilution of 1:7, entertain, showed low rates for all treatments, with best results of VCL, VSL and VAP for the T6. In the fresh semen was found more morfoanomalia the tail folded (20%) and the sperm after thawing, the coiled tail (23%). Comparing the results of morfoanomalia deposited before and after the thawing were significant differences between samples (P <0.05). It was concluded that the semen can be preserved in Piratininga Ringer + Retilglicol 105 in 1:3, without high toxicity, and cryopreserved in Glucose + 10% DMSO using the "dry shipper".

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Os objetivos deste estudo foram: 1) avaliar os aspectos da interaÃÃo das Binder of SPerm proteins (BSP) presentes no plasma seminal de caprinos e as principais proteÃnas do leite; 2) determinar se as BSP homÃlogas do plasma seminal de equinos, suÃnos e ovinos possuem propriedades de ligaÃÃo as proteÃnas do leite semelhante Ãs BSP bovinas; 3) investigar a interaÃÃo das proteÃnas do plasma seminal de caprinos e os componentes dos diluidores à base de lecitina de soja; 4) identificar as proteÃnas do plasma seminal de touros Curraleiro (PÃ-duro) associadas à circunferÃncia escrotal e os parÃmetros seminais. Para tanto, o leite desnatado tratado termicamente foi incubado com as proteÃnas do plasma seminal de caprinos e, aplicados em coluna cromatogrÃfica de gel filtraÃÃo na presenÃa e na ausÃncia de cÃlcio. AlÃm disso, o leite desnatado foi fracionado em duas fraÃÃes, MF-1 (proteÃnas do soro de leite) e MF-2 (caseÃnas), e posteriormente incubadas com as proteÃnas do plasma seminal de caprinos, seguida pela aplicaÃÃo em coluna de gel filtraÃÃo. As fraÃÃes foram eluÃdas e analisadas por Western blot. Adicionalmente, outras anÃlises foram realizadas para avaliar as propriedades desta ligaÃÃo. No primeiro procedimento, uma placa de ELISA foi sensibilizada com leite desnatado tratado termicamente e, em seguida, incubadas com diluiÃÃes em sÃrie de BSP purificadas e plasma seminal caprino, seguido por incubaÃÃo com anticorpos primÃrio e secundÃrios. Na segunda anÃlise, o leite desnatado, caseÃnas, &#945;-lactalbumina, &#946;-lactoglobulina diluÃdas em sÃrie foram gotejadas sobre uma membrana de nitrocelulose e, posteriormente, as membranas foram incubadas com plasma seminal de caprinos. A interaÃÃo foi detectada pela incubaÃÃo com anticorpos primÃrios e secundÃrios. No terceiro experimento, os espermatozoides epididimÃrios foram incubados com: (1) citrato/glicose + plasma seminal caprino, (2) plasma seminal caprino + diluidor leite, e (3) diluidor leite + plasma seminal de caprino. ApÃs o perÃodo de incubaÃÃo, as proteÃnas da membrana de espermatozoides foram extraÃdas e analisadas por Western blot. No segundo estudo, o leite desnatado foi incubado com as proteÃnas do plasma seminal de bovino, equino, suÃno e ovino, separadamente, e em seguida, foram aplicados em coluna cromatografia de filtraÃÃo em gel. AlÃm disso, a interaÃÃo de componentes de diluidores à base de lecitina de soja (Andromed e BioxcellÂ) foi investigada por cromatografia de filtraÃÃo em gel e ultracentrifugaÃÃo. Uma alÃquota de diluidor à base de lecitina de soja foi incubada com as proteÃnas do plasma seminal de caprinos e aplicadas em coluna de gel filtraÃÃo. As fraÃÃes foram eluÃdas e, apÃs este procedimento, as amostras foram analisadas por Western blot. A anÃlise por ultracentrifugaÃÃo foi realizada na ausÃncia e na presenÃa de vesÃculas de fosfolipÃdios oriundas de diluidores a base de lecitina de soja, Andromed e BioxcellÂ. As vesÃculas isoladas foram incubadas com as proteÃnas do plasma seminal de caprino. ApÃs a ultracentrifugaÃÃo, a presenÃa de BSP no sobrenadante e no sedimento foi analisada por Western Blot. Adicionalmente, o plasma seminal de dez touros Curraleiro (PÃ-duro) foi analisado por 2-D eletroforese. Os gÃis corados por Coomassie foram analisados no aplicativo PDQuestÂ. Nossos resultados demonstraram que as proteÃnas de BSP do plasma seminal caprino se ligam Ãs proteÃna do leite, especialmente, as caseÃnas e &#946;-lactoglobulina. AlÃm disso, estas proteÃnas interagem com as vesÃculas de fosfolipÃdios presentes nos diluidores Andromed e BioxcellÂ. As BSP de bovinos, equinos e suÃnos tambÃm se ligam aos componentes do leite. Jà as BSP de ovinos nÃo interagem com os componentes do leite desnatado. Nossas analises demonstraram que vinte e sete spots proteÃcos se correlacionam com pelo menos um parÃmetro reprodutivo. Em resumo, nossos resultados indicam que as BSP de caprinos se ligam Ãs proteÃnas do leite e aos fosfolipÃdios do diluidor Andromed e BioxcellÂ. Isto sugere que estes componentes tÃm um papel significativo na proteÃÃo de espermatozoide de caprinos, pois sequestram as BSP presentes no sÃmen. Nossos resultados sugerem que as BSP homÃlogas presentes no plasma seminal de equinos e suÃnos apresentam propriedades de ligaÃÃo Ãs proteÃnas do leite semelhantes Ãs BSP bovinas. AlÃm disso, o plasma seminal de touros Curraleiro (PÃ-duro) tem potenciais proteÃnas relacionadas com parÃmetros de qualidade seminal. Estes resultados sÃo de grande interesse, visto que elucida aspectos relacionados aos mecanismos de proteÃÃo dos espermatozoides pelos diluidores, para o desenvolvimento de novos diluidores livre de produtos de origem animal e na identificaÃÃo de proteÃnas com potencial à marcadores de fertilidade no plasma seminal de bovino. / The objectives of this study were: 1) to evaluate aspects of interaction between goat seminal plasma proteins and milk proteins; 2) to determine if homolog Binder of Sperm Proteins (BSP) from stallion, boar and ram seminal share the binding properties of the bovine BSP for the milk proteins; the mechanism of bovine sperm protection by milk is similar in different species of mammals; 3) to investigate the interactions of BSP proteins present in goat seminal plasma and phospholipids from soybean lecithin-based extender; 4) to identify seminal plasma proteins associated with scrotal circumference and sperm parameters. Heated skimmed milk was incubated with goat seminal plasma proteins and loaded onto Sepharose CL-4B column both in the presence and absence of calcium. Fractions were eluted and evaluated by Western blots using anti-goat BSP antibodies. Also, we fractionated milk into two fractions, MF-1 (mostly whey proteins) and MF-2 (mostly caseins), which were incubated with goat seminal plasma proteins and passed through gel filtration columns. Eluted fractions were then analysed by immunoblotting with anti-BSP antibodies. In addition, a series of analysis was conducted to evaluate binding properties involving milk and seminal plasma proteins. In the first procedure, an ELISA plate was saturated with heated skimmed milk and then incubated with serial dilutions of purified BSP proteins, goat seminal plasma proteins and anti-goat BSP. In a second analysis, heated skimmed milk, casein, &#945;-lactalbumin and &#946;-lactoglobulin were spotted onto a nitrocellulose membrane with serial dilutions. Membranes were then overlaid with goat seminal plasma proteins. Binding between milk and seminal plasma components were detected by successive incubations with primary and secondary antibodies. A third set of analysis was conducted to evaluate interactions among ejaculated and caudal epididymal sperm, milk and goat seminal plasma proteins. Epididymal sperm from each animal was incubated with: (1) citrate-glucose medium followed by seminal plasma; (2) with seminal plasma followed by milk extender; (3) with milk extender followed by seminal plasma (Fig. 1). Both ejaculated and epididymal goat sperm were also incubated with citrate-glucose medium without seminal plasma, as positive and negative controls, respectively. Following incubations, sperm membrane proteins were extracted and the presence of goat BSP proteins was assessed by Western blot Furthermore, heated skim milk was incubated with seminal plasma proteins from bulls, stallions, boars and rams and then loaded onto gel filtration column. The proteins were eluted and fractions were analysed by immunoblotting. Moreover, the interaction of hospholipids vesicles from soybean lecithin-based extender (Andromed and BioxcellÂ) and goat BSP proteins was investigated by gel filtration chromatography and ultracentrifugation analysis. An appropriate volume of soybean-based extender was incubated with goat seminal plasma proteins and passed through a gel filtration column. Proteins were eluted and fractions analysed by Western blot. Ultracentrifugation analysis was performed in the absence or in the presence of phospholipid vesicles. These vesicles were isolated from soybean lecithin extender and incubated with goat seminal proteins. The presence of goat BSP proteins in the supernatant and in the pellet was assessed by Western blot. In addition, seminal samples from ten Curraleiro Bulls were subjected to 2-D electrophoresis and Coomassie blue-stained gels analyzed with PDQuest software. Our results demonstrated that goat BSP proteins bound to milk protein, specially caseins and &#946;-lactoglobulin. In addition, these proteins interact with phospholipids vesicles of Andromed and BioxcellÂ. Bull, stallion and boar BSP proteins also bind to milk protein, whereas ram BSP proteins did not bind. Also, we identified that twenty and seven protein spots presented significant correlation with at least one reproductive parameter. In summary, our results indicate that goat BSP proteins bind to milk proteins and phospholipids from Andromed and Bioxcell semen extender. This suggests that these components play a significant role in protecting goat sperm by sequestrating all BSP proteins in semen. We also demonstrated that BSP homologs in boar, stallion seminal plasma share the binding properties of the bovine BSP for the milk proteins. Additionally, Curraleiro seminal plasma has potential proteins related to quality sperm parameters and they should be tested as putative fertility markers. These findings are of considerable interest in view of the mechanisms of sperm protection by extender constituents, for the development of novel extender free of animal components and the identification of proteins as potential biomarkers of fertility in bovine seminal plasma.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O presente trabalho tem como objetivo determinar o melhor mÃtodo de congelaÃÃo e envase de sÃmen humano, quando comparado o mÃtodo rÃpido com o lento e o envase em palhetas de 0,25ml com criotubos de 2ml, utilizando como variÃveis a morfologia e motilidade dos espermatozÃides antes e apÃs a criopreservaÃÃo. Trata-se de um estudo experimental, de validaÃÃo de tÃcnica laboratorial. Foi desenvolvido no Centro de ReproduÃÃo Assistida do CearÃ. Foram analisadas dezoito amostras de sÃmen de dezoito voluntÃrios. As amostras foram submetidas à anÃlise da morfologia e motilidade espermÃtica prÃvia à criopreservaÃÃo. Foi determinada ainda uma curva de motilidade espermÃtica nas primeiras trÃs horas. O meio TEST-YOLK foi utilizado como crioprotetor. Depois de adicionado o crioprotetor, o sÃmen foi dividido em partes iguais e envasado em criotubos e palhetas. Metade dos criotubos e das palhetas foi submetida a criopreservaÃÃo pelo mÃtodo rÃpido (RT e RP, respectivamente) e a outra metade pelo mÃtodo lento (LT e LP, respectivamente). Em seguida, as amostras foram descongeladas e determinadas as morfologias, bem como as motilidades logo apÃs a descongelaÃÃo e a cada hora durante trÃs horas. AnÃlise estatÃstica foi realizada pelo programa de SPSS for Windows versÃo 11.0.0. Houve uma queda, estatisticamente significante, da motilidade apÃs a criopreservaÃÃo, motilidade inicial de 58,1% e motilidades nos diferentes tratamentos de 30,3% (LT), 21,1% (LP), 27% (RT) e 19,2% (RP). TambÃm houve uma diminuiÃÃo significante da morfologia normal apÃs a criopreservaÃÃo, em relaÃÃo à morfologia inicial (14,2%), mas nÃo foi observada diferenÃa entre os mÃtodos (12,8%, 12,6%, 12,6% e 12,4%; RP, RT, LP e LT, respectivamente). O mÃtodo LT foi o que obteve uma curva de motilidade espermÃtica mais prÃxima da observada no sÃmen in natura. Conclui-se que o mÃtodo LT foi o que menos afetou a motilidade espermÃtica e que, apesar de ter sido observada uma piora significativa da morfologia espermÃtica apÃs a criopreservaÃÃo, nÃo houve diferenÃa entre os mÃtodos. Palavras-chaves: CriopreservaÃÃo; PreservaÃÃo do SÃmen; EspermatozÃide; ReproduÃÃo / This research has as objective determines the best freezing method and storage of human semen, when compared the fast method with the slow method and the storage in 0,25ml straws with cryotubes of 2ml, using as variables the morphology and motility of the spermatozoa before and after the cryopreservation. It is an experimental study, laboratorial technique validation. It was developed in the Assisted Reproduction Center of CearÃ. Eighteen semen samples from eighteen men were analyzed. The samples were submitted to the analysis of the morphology and motility previous the cryopreservation. It was still determined a spermatozoa motility curve in the first three hours. TEST-YOLK was used as cryoprotective media. After having added the TEST-YOLK media, the semen was divided in same parts and storage in cryotubes and straws. Half of the cryotubes and of the straws was cryopreserved by the fast method (RT and RP, respectively) and the other half by the slow method (LT and LP, respectively). The samples were thawed and analyzed the morphologies, as well as the motilities soon after the thawed and every hour for three hours. Statistical analysis was done by the SPSS program - Windows version 11.0.0. Motility of spermatozoa decrease after the cryopreservation, initial motility was 58,1% and motilities in the different treatments were 30,3% (LT), 21,1% (LP), 27% (RT) and 19,2% (RP). There was a significant decrease of the normal morphology after the cryopreservation, in relation to the initial morphology, but difference was not observed among the methods (RP=12,8%, RT=12,6%, LP=12,6% e LT=12,4%). The motility of LT method was closer than that obtained in the fresh semen. The method LT showed the best recovery of motile cells after freezing and thawing. There was not difference among the methods when appraised the morphology. Key-Word: Cryopreservation; Semen preservation; Spermatozoa; Reproduction.