O presente trabalho foi composto de dois estudos distintos. O Estudo I, com um foco conservacionista, teve como objetivo a avaliação e comparação de diferentes métodos de criopreservação de sêmen de bugio-preto (Alouatta caraya). Para tanto, o estudo foi dividido em dois experimentos: Experimento I composto de dois ensaios no primeiro ensaio foram comparados dois diluidores comerciais BotuBOV e Test-yolk buffer (TYB) e no segundo ensaio foram comparados dois métodos de criopreservação de sêmen congelação lenta e vitrificação; Experimento II avaliação dos efeitos da adição de DHA e de Trolox (análogo da vitamina E) ao diluidor para criopreservação de sêmen. O diluidor TYB apresentou melhores resultados quando comparados ao BotuBOV. A congelação lenta apresentou melhores resultados quando comparada à vitrificação. Não houve diferença entre o diluidor controle e os diluidores com Trolox, DHA ou combinação dos dois (DHAT), com exceção da integridade de acrossoma, que foi significativamente menor para o diluidor DHAT. Conclui-se que são necessários mais estudos, com utilização de outras doses de DHA e Trolox, além de outros antioxidantes. O Estudo II, com foco em pesquisa biomédica, teve como objetivo a criopreservação de sêmen de macaco-rhesus (Macaca mulata) e avaliação da qualidade pós-descongelação por meio de fecundação in vitro (FIV). Para tanto, o estudo foi dividido em três experimentos: Experimento I avaliação e comparação de dois métodos de criopreservação de sêmen congelação lenta e vitrificação; Experimento II avaliação e comparação de quatro métodos de preparação do sêmen pós-descongelação lavagem simples (LS), swim-up (SU), separação por gradiente de densidade (SGD) e filtragem em lã de vidro (FLV); Experimento III avaliação da qualidade seminal pós-descongelação por meio de FIV. A congelação lenta apresentou melhores resultados que a vitrificação (p<0,05). LS apresentou os melhores resultados, seguido por SGD e SU, enquanto FLV apresentou os piores resultados. LS e SGD foram utilizados para avaliação da qualidade seminal por meio de FIV, utilizando sêmen fresco como controle. As taxas de fecundação (média±EPM%) para oócitos MI inseminados com sêmen fresco (43.5±16.4) foram significativamente maiores (p<0,05) que LS (2.0±2.0), mas não diferiram de SGD (25.1±14.2). Não houve diferença na taxa de blastocistos (média±EPM%) entre os tratamentos (variação de 0 a 11.9±7.9). As taxas de fecundação para oócitos MII inseminados com sêmen fresco (41.4±3.6) também foram significativamente maiores que SGD e LS (18.4±6.9 e 12.7±7.7, respectivamente), assim como a taxa de blastocistos (64.7±13.6; 4.7±4.7; 30.9±13.8, respectivamente). Conclui-se que, espermatozoides criopreservados foram capazes de fertilizar oócitos e os embriões atingiram o estágio de blastocisto. A SGD selecionou espermatozoides pós-descongelação de melhor qualidade para FIV em macacos-rhesus, quando comparada à LS / This work was divided in two studies. The objective of Study I was to test and compare different cryopreservation methods for sperm from black-and-gold howler monkeys (Alouatta caraya), with a focus on species conservation. The study was divided in two experiments. Experiment I composed of two trials the first trial compared two commercial extenders BotuBOV and Test-yolk buffer (TYB), and the second trial compared two cryopreservation methods slow freezing and vitrification; Experiment II evaluation of the effects of DHA and Trolox (vitamin E analog) as additives to the freezing extender. TYB had better results when compared to BotuBOV. Slow freezing had better results when compared to vitrification. There was no difference between control extender (TYB) and extender containing Trolox, DHA or a combination of both (DHAT), except for acrosome integrity, which was significantly lower for DHAT. In conclusion, more studies are necessary, using other doses of DHA and Trolox, as well as other antioxidants. The objective of Study II was to assess the quality of frozen-thawed sperm from rhesus macaques (Macaca mulatta) by in vitro fertilization (IVF). The study was divided in three experiments. Experiment I evaluation and comparison of two cryopreservation methods slow freezing and vitrification; Experiment II evaluation and comparison of four preparation methods for frozen-thawed sperm simple wash (SW), swim-up (SU), density gradient centrifugation (DGC), and glass wool filtration (GWF); and Experiment III evaluation of frozen-thawed sperm quality by IVF. Slow freezing had better results when compared to vitrification (p<0,05). SW had better results, followed by DGC and SU, while GWF had the worse results. SW and DGC were further evaluated by IVF. Fertilization rates (mean±SEM%) with MI oocytes using fresh sperm were significantly higher (43.5±16.4) than with SW (2.0±2.0) and did not differ from DGC (25.1±14.2). There was no difference in blastocyst rates between treatments (range 0 to 11.9±7.9). Fertilization rates with MII ova were also significantly higher with fresh sperm (41.4±3.6) than DGC and SW (18.4±6.9 and 12.7±7.7, respectively), and more blastocysts developed from MIIs fertilized with fresh sperm (64.7±13.6) than SW and DGC (4.7±4.7 and 30.9±13.8, respectively). In conclusion, frozen-thawed sperm were able to fertilize oocytes and embryos reached the blastocyst stage. DGC yielded better frozen-thawed sperm for IVF in rhesus macaques, when compared with SW

Research on in vitro embryo production (IVP) in the equine is impeded by the limited availability of mature oocytes as the mare is mono ovulating and superovulation is still difficult (Dippert and Squires, 1994; Bezard et al., 1995; Alvarenga et al., 2001b). Despite recent improvement in IVM of equine oocytes, success rates of IVM in that species remain low in all culture media tested compared to other species (Goudet et al., 2000b). However, most studies have focused on the percentage of oocytes reaching the metaphase II stage (nuclear maturation) but few concentrated on the final oocyte competence as measured by its ability to develop into a blastocyst and further establish a pregnancy. Blastocyst production rate is influenced not only by culture environment but also by oocyte maturation conditions. Under in vitro culture conditions, oxidative modifications of cell components via increased ROS represent a major culture induced stress (Johnson and Nasr-Esfahani, 1994). Anti-oxidant systems can attenuate the deleterious effects of oxidative stress by scavenging ROS (Del Corso et al., 1994). Glutathione, a tripeptide thiol, is the major non-protein sulfydryl compound in mammalian cells that plays an important role in protecting the cell from oxidative damage (Meister and Tate, 1976; Meister and Anderson, 1983). It has been suggested that GSH content in oocytes may serve as a reservoir protecting the zygote and the early embryos from oxidative damage before genomic activation and de novo GSH synthesis occur (Furnus et al., 1998; de Matos and Furnus, 2000). The addition of GSH synthesis precursors, such as cysteamine, a thiol compound, to IVM media has been shown to improve IVP in various species (Takahashi et al., 1993; de Matos et al., 1995; Grupen et al., 1995; de Matos et al., 2002a; de Matos et al., 2002b; de Matos et al., 2003; Gasparrini et al., 2003; Oyamada and Fukui, 2004; Balasubramanian and Rho, 2007; Anand et al., 2008; Singhal et al., 2008; Zhou et al., 2008). Very little information on the use of thiol compounds in the equine is available. Conventional in vitro fertilization (IVF) has not been successful in the mare, and a repeatable IVF technique has not yet been developed (Alm et al., 2001). To overcome the limitation of conventional IVF procedures, other methods to produce embryos from oocytes, either in vivo or in vitro, have been investigated. Among these, intra cytoplasmic sperm injection (ICSI) has permitted efficient equine in vitro blastocyst production (Galli et al., 2002; Lazzari et al., 2002; Choi et al., 2006a; Choi et al., 2006c). However, ICSI requires specific equipment and skills. Transfer of an immature oocyte into the preovulatory follicle of an inseminated recipient mare (Intra-Follicular Oocyte Transfer, IFOT) has produced embryos but the success rate was low (Hinrichs and Digiorgio, 1991). Similarly, oocyte transfer (OT) into the oviduct of an inseminated recipient mare was investigated (McKinnon et al., 1988; Carnevale, 1996; Hinrichs et al., 1997; Carnevale et al., 2001; Carnevale et al., 2003; Carnevale, 2004), and commercial programs using OT for mares with reproductive abnormalities are now available (Carnevale et al., 2001). Unfortunately, IFOT is poorly documented in the literature and reports of OT have been published by various laboratories and under various conditions, making comparisons between results and choosing among these as substitutive techniques to ICSI or embryo transfer difficult. The first aim of the present work was to investigate if there is an influence of supplementation with 100 µM of cysteamine on conventional IVF success rate. Cumulus oocytes complexes (COCs) retrieved by transvaginal ultrasound guided aspiration were matured in vitro with or without cysteamine supplementation and were then submitted to conventional IVF using either calcium ionophore or heparin as capacitation treatment for spermatozoa. A total of 131 oocytes were evaluated for evidence of sperm penetration. Both techniques (ionophore or heparin) yielded 6% of IVF and results were similar both for the cysteamine and the control group. This success rate of IVF is low compared to some published data (Palmer et al., 1991; Dell'Aquila et al., 1996; McPartlin et al., 2009) but similar to what others reported in the literature (Choi et al., 1994; Dell'Aquila et al., 1997a). Although, it seems likely that cysteamine did not significantly improve IVF rates under our conditions, our general success rates for IVF procedures may be too low for us to conclude definitely about the effect of cysteamine. As ICSI was not available to us, the second aim of this work was to determine what in vivo technique could best bypass the lack of an efficient conventional IVF procedure. We compared embryo production following transfer of in vivo recovered oocytes (1) into a recipients oviduct or (2) into her preovulatory follicle either immediately after ovum pick up or (3) after in vitro maturation. Recipients were inseminated with fresh semen of a stallion with a known normal fertility. Ten days after transfer, rates of embryos collected in excess to the number of ovulations were calculated and compared for each group. Embryo collection rates were 32.5% (13/40), 5.5% (3/55) and 12.8% (6/47) for OT, post-IVM and immediate IFOT respectively. OT significantly yielded more embryos than immediate and post-IVM IFOT did. These results show that, when ICSI is not an option, intra-oviductal oocyte transfer is to be preferred to IFOT, as an in vivo alternative, to bypass the inadequacy of conventional in vitro fertilization and to assess oocyte developmental competence. After it was established that in comparison to IFOT, OT is the most reliable in vivo alternative to in vitro fertilization where ICSI technology is not available, this technique was used to assess the effect of cysteamine supplementation on nuclear maturation and oocyte competence. The third aim of this work was to investigate the influence of supplementation with 100 µM of cysteamine on in vitro nuclear and cytoplasmic maturation by specific DNA staining and the ability of oocytes to undergo in vivo fertilization after OT. Oocytes were collected by transvaginal ultrasound guided aspiration and matured in vitro with (cysteamine group) or without (control group) cysteamine. The nuclear stage after DNA Hoechst staining and the embryo yield following OT were used as a criterion for assessing nuclear and cytoplasmic maturation, respectively. Overall maturation rate was 52%, which is rates reported in the literature ranging from 40 to 70% in the equine (Goudet et al., 1997a; Bogh et al., 2002; Hinrichs et al., 2005; Galli et al., 2007). Nuclear maturation was not statistically different (p>0.05) between oocytes cultured with or without cysteamine (55% and 47% respectively). From 57 oocytes transferred to the oviduct in each group, the number of embryos collected was 10 (17%) in the control group and 5 in the cysteamine group (9%). Those two percentages were not statistically different (p>0.05). Contrary to the data described in other domestic species, there was no effect of cysteamine on in vitro nuclear maturation, or in vivo embryonic development under our conditions. Under our conditions, the addition of 100 µM of cysteamine to a classic culture medium does not improve equine oocyte maturation or embryonic development after OT. The same dose failed to increase GSH content in the equine (Luciano et al., 2006). However, the effect of cysteamine supplementation is highly species and concentration dependant. The inadequacy of the chosen concentration may explain that equine embryo production has not been increased by the cysteamine under our conditions as opposed to what has been observed in many other species. Alternatively, we can hypothesize that some substances present in the IVM medium can interfere with GSH synthesis. This has been suggested for FSH and estradiol (Bing et al., 2001) and, although our maturation medium is not supplemented with gonadotropins or estradiol, factors contained in fetal calf serum or EGF might also have an effect on GSH synthesis. Considering its beneficial effects in many other species, supplementation with cysteamine to different IVM media should be further investigated in the equine. Ideally combining different concentrations and ICSI or OT in order to determine an optimal concentration and its effects on oocyte developmental competence.

Perdas embrionárias e alterações gestacionais são frequentemente observadas em prenhezes de embriões bovinos manipulados in vitro. Sabe-se que tais anormalidades são resultantes de alterações epigenéticas ocasionadas pela manipulação dos gametas e/ou do embrião durante as técnicas de reprodução assistida (ARTs), com destaque para as técnicas de fecundação in vitro (FIV) e da clonagem por transferência nuclear de células somáticas (SCNT). Tais alterações resultam em distúrbios no desenvolvimento do concepto em algum momento crítico entre a fertilização e o parto, fornecendo bons modelos de estudos sobre a fisiopatologia de perdas embrionárias e dos distúrbios de desenvolvimento. Caracterizam-se como momentos críticos após a transferência do embrião (TE) o desenvolvimento embrionário no útero, o reconhecimento materno da gestação, a placentação e o desenvolvimento da placenta e do feto, os quais tem de ser transpassados sem nenhuma falha, permitindo um desenvolvimento normal do concepto até o termo. Sendo assim, o presente trabalho abordou três fases distintas do amplo período gestacional em prenhezes por ARTs. O Estudo 1 foi realizado durante o período peri-reconhecimento materno da gestação e objetivou descrever a abundância de expressão de genes estimulados pelo interferon tau (ISGs) de células mononucleares do sangue periférico (PBMCs) maternas em gestações oriundas de ARTs no primeiro mês de gestação; o Estudo 2 compreendeu os primeiros 35 dias de gestação e objetivou descrever as mudanças morfológicas e vasculares do complexo útero-concepto-ovário e o estímulo à expressão de ISGs em PBMCs em gestações de conceptos clonados por SCNT com diferentes fenótipos de desenvolvimento, sendo esses denominados gestação anembrionada e CL persistente; e o Estudo 3 foi conduzido durante o período pré-parto e objetivou descrever as alterações na produção de esteroides sexuais e corticosteroides em gestações produzidas por ARTs. Três hipóteses foram testadas: (1) Gestações de conceptos clonados por SCNT apresentam uma baixa e mais tardia estimulação de ISGs em PBMCs maternas quando comparadas com gestações de conceptos produzidos por FIV e IA; (2) O concepto clonado por SCNT apresenta um menor estímulo sobre mudanças morfológicas e vasculares do complexo útero-ovário e ISGs em PBMCs maternas durante os primeiros 35 dias de gestação, quando comparado com conceptos oriundos de IA; e (3) Gestações de embriões oriundos de ARTs apresentam alterações na dinâmica esteroidogênica no pré-parto quando comparados com gestações de IA. No estudo 1 foram coletadas amostras de sangue de gestações produzidas por inseminação artificial (IA), FIV e clonagem por SCNT, nos dias 15, 18, 20, 22, 24, 26, 28 e 31 pós-ovulação e foi realizada mensuração da abundância de transcritos de ISGs (OAS1 e ISG15) em PBMCs maternas. No estudo 2, gestações produzidas por IA e clonagem por SCNT, foram submetidas a escaneamentos ultrassonográficos dos ovários, útero e concepto a cada 3 dias do dia 14 ao 35 (dia 0 = ovulação) e amostras de sangue foram coletadas nos dias 15, 18, 20, 22, 24, 26, 28 e 31 para mensuração da abundância de transcritos de ISGs (OAS1 e ISG15) em PBMCs maternas. No estudo 3, foram coletadas amostras de sangue no último mês em gestações naturais, oriundas de FIV e de clonagem por SCNT para análise hormonal de 10 esteroides utilizando o método de espectrometria de massas multi-hormonal de alta resolução LC-MS/MS. O primeiro estudo mostrou semelhanças na expressão de genes estimulados pelo IFNT em gestações oriundas de ARTs e produzidas por IA. Entretanto, a estimulação nas gestações oriundas de ARTs aparentou ser quatro dias mais prolongada, sugerindo uma maior funcionalidade do trofectoderma em conceptos oriundos de ARTs. O segundo estudo demonstrou um aumento na expressão de ISGs em PBMCs maternas tanto em gestações de conceptos normais quanto em anormais, justificando a manutenção da função luteal mesmo na ausência de detecção do concepto por ultrasonografia. No terceiro estudo, demonstrou-se alterações na esteroidogênese nas gestações de embriões FIV e clonados no último mês de gestação, sendo essas compatíveis com a hiperativação da enzima aromatase durante todo o último mês de gestações oriundas de FIV e hiperativação das enzimas P450C11 e P450C21 trinta dias antes do parto em gestações oriundas de clonagem por SCNT. O presente estudo concluiu que conceptos oriundos de FIV e clonagem por SCNT apresentam um prolongamento no estímulo de ISGs pelo IFNT, conceptos clonados anormalos apresentam estímulo de ISGs, o que justifica a manutenção da função luteal, e, por fim, a cascata esteroidonênica que culmina com o parto apresenta-se alterada em gestações oriundas de FIV e clonagem por SCNT. / Pregnancy losses and gestational abnormalities are frequently observed in pregnancies from in vitro produced embryos in bovines. It is known that these abnormalities are due to epigenetic changes from the manipulation of gametes and/or embryo during the use of assisted reproduction techniques (ARTs), especially for the in vitro fertilization (IFV) and cloning by somatic cells nuclear transfer (SCNT). These changes results in disturbances of conceptus development in any critical stage between the fertilization and parturition, which provides good models for the study of physiopathology of embryo losses and disturbances of development. Critical stages after the embryo transfer (ET) to the uterus are characterized as the maternal recognition of pregnancy, placentation, and fetal-placental development, which needs to be surpassed without failures, in order to develop a normal conceptus until term. Therefore, the present work approached three distinct phases of the wide gestational period in pregnancies from ARTs. The Study 1 was conducted during the maternal peri-recongnition of pregnancy period and aimed to describe the expression of interferon stimulated genes (ISGs) in maternal peripheral blood mononuclear cells (PBMCs) in pregnancies derived of ARTs; the Study 2 comprise the first 35 days of pregnancy and aimed to describe morphological and vascular changes of the complex uterus-ovaries-conceptus, as well as the expression of ISGs in maternal PBMCs in pregnancies of conceptus cloned by SCNT with different phenotypes of development, denominated as anembryonic gestation and persistent CL; the Study 3 was conducted during the pre-partum period and aimed to describe changes in the production of sexual steroids and corticosteroids during the last month of pregnancies derived of ARTs. Three hypothesis were tested: (1) Pregnancies of conceptus cloned by SCNT presented a decrease and delay in the stimulation of ISGs in maternal PBMCs when compared with conceptuses produced by IFV and AI; (2) Stimulus from the conceptus for changes in the morphology and vasculature of the the uterus-ovarian complex, detected by ultrasonography in B and Doppler modes, and the stimulation of ISGs in maternal PBMCs during the first 35 days of pregnancy of conceptus cloned by SCNT are less intense when compared with conceptus derived from AI; and (3) Pregnancies derived of ARTs present changes in the steroidogenic dynamics in the pre-partum, when compared with pregnancies derived from AI. In Study 1 blood samples were collected from pregnancies produced by AI, IVF, and cloning by SCNT, at days 15, 18, 20, 22, 24, 26, 28, and 31 post-ovulation for the measurement of abundance of transcripts of ISGs (OAS1 and ISG15) in maternal PBMCs. In Study 2, pregnancies derived of AI and cloning by SCNT, were submitted to ultrasonographic scans for the evaluation and description of morphological and vascular changes in ovaries, uterus, and conceptus every 3 days from day 14 to 35 (day 0 = ovulation) and blood samples were collected on days 15, 18, 20, 22, 24, 26, 28 e 31 for the measurement of the abundance of transcripts of ISGs (OAS1 and ISG15) in maternal PBMCs. In Study 3, blood samples were collected during the last month of pregnancies naturally conceived, derived of IVF, and cloned by SCNT for the analysis of 10 steroids using the method of mass spectrometry high resolution LC-MS/MS. The first study showed similarities in the ISGs expression stimulation in pregnancies derived of ARTs and AI. However, the stimulation in the ART derived pregnancies was apparently 4 days longer, suggesting a greater placental function in conceptus derived of ARTs. The second study showed an increase in ISG expression in both normal and abnormal conceptus development, which justifies the maintenance of CL in the absence of a conceptus structure detected by ultrasonography. In the third study, was detected changes in the steroidogenesis of pregnancies derived of IFV and cloning by SCNT during the last month of pregnancy, which are compatible with the hyperactivation of the aromatase enzyme during the last month of IFV derived pregnancies, and hyperactivation of the enzymes P450C11 and P450C21 thirty days before parturition in pregnancies derived of cloning by SCNT. The present study concludes that conceptus derived of IFV and cloning by SCNT present a prolonged stimulus of ISGs, cloned conceptus with anomalous development presents a stimulus of ISGs, which justifies the CL function maintenance, and, ultimately, the steroidogenic cascade that culminates with the term is altered in pregnancies derived from IFV and cloning by SCNT.

No description available.

No description available.

Depuis sa découverte il y a plus de 30 ans, le Virus de l’Immunodéficience Humaine est à l’origine d’une importante mortalité dans le monde. De par la difficulté de tester l’efficacité de formulations thérapeutiques et/ou vaccinales directement chez l’homme, des études d’infections modèles du VIH, comme celle du Virus de l’Immunodéficience Féline (FIV), ont été entreprises ces dernières années. Au-delà de son intérêt vétérinaire, l’étude du FIV représente un avantage important pour trouver un moyen de contrôler les infections par les lentivirus tel que le VIH. Elle peut permettre de développer et surtout de tester l’efficacité des vaccins et/ou thérapies spécifiques chez le chat, dont le SIDA mime les symptômes et les modifications hématologiques rencontrés chez l’homme. Ce manuscrit s’est intéressé à l’étude structurale de deux familles de protéines virales de ces virus, les protéines lentivirales précoces (protéine Tat du VIH) et tardives (domaines Capside CA et Matrice MA de Gag du FIV). L’étude structurale de ces protéines et leur compréhension fonctionnelle au sein de l’hôte pourront à l’avenir ouvrir de nouvelles voies thérapeutiques et/ou vaccinales contre les lentivirus, palliant ainsi les problèmes existants de résistances virales / Since its discovery 30 years ago, the Human Immunodeficiency Virus is the cause of an important mortality worldwide. Because of the difficulty to test the efficiency of therapeutical and/or vaccinal formulations directly in humans, studies of models of HIV infections, such as the Feline Immunodeficiency Virus (FIV), have been performed in recent years. In addition to its veterinary interest, the study of FIV is an important issue to find a way to control infections by lentiviruses such as HIV. It can help to develop and test the efficiency of specific therapies and/or vaccines for cats, where AIDS mimics the symptoms and hematologic changes observed in humans. This manuscript describes the structural study of two types of viral proteins of these viruses, early lentiviral proteins (HIV Tat protein) and late lentiviral proteins (CA capsid and MA Matrix domains of FIV Gag). The structural study of these proteins and their functional understanding into the host will open new therapeutic and/or vaccine strategies against these lentiviruses in the future, in order to overcome the existing problems of viral resistance

O vírus da imunodeficiência felina (FIV) é um lentivirus que infecta gatos domésticos (Felis catus), causando uma imunodeficiência progressiva análoga a AIDS (Síndrome da Imunodeficiência Adquirida Humana). A ampla heterogeneidade molecular do FIV e a alta capacidade de promover mutações sob pressões imunológicas, farmacológicas ou ambientais são características inerentes aos lentivirus. A identificação do subtipo de vírus e o conhecimento da diversidade genética das cepas circulantes são fundamentais para o desenvolvimento estratégico de vacinas capazes de resultar na imunização do hospedeiro e no estabelecimento de testes diagnósticos. Objetivando isolar o material genético e realizar a caracterização molecular do vírus da imunodeficiência felina foram coletadas e analisadas amostras de sangue periférico de felinos portadores do FIV, co-habitantes de um abrigo aberto de felinos, em São Paulo, SP, em quatro momentos distintos, T0 (zero), no momento inicial da avaliação e seis, dez e quinze meses após a coleta inicial, correspondendo aos momentos T1, T2 e T3, respectivamente. Foram realizados testes hematológicos e bioquímicos nas quatro coletas com a finalidade de avaliar a evolução clínica da infecção. Adicionalmente foi realizado um estudo de variabilidade genética do FIV, com base no sequenciamento dos produtos amplificados dos gene env obtidos no estudo. Os envelopes clonados foram utilizados para transfectar células resultando na expressão das proteínas do envelope que possibilitaram estudos com os receptores celulares utilizados pelos isolados brasileiros. As análises das seqüências virais mostraram que todas as amostras, do abrigo, pertencem ao subtipo B. Foi observado um baixo percentual de mudança, da região estudada do vírus entre as quatro coletas. O fenômeno de &quot;quasispecies&quot; virais, bastante estudado no HIV, pode ser documentado em nossas amostras. Nos exames hematológicos e bioquímicos; hematócrito, hemoglobina, contagem total de leucócitos, proteína total e gamaglobulinas; dos animais infectados pelo FIV observou-se mudanças entre a primeira e quarta coleta demonstrando assim a importância dos testes utilizados no acompanhamento da infecção pelo FIV. Com relação aos dados com os receptores do FIV, os resultados apontam uma menor complexidade na interação entre os envelopes dos isolados do estudo com o receptor CD134 para proceder a infecção quando comparados com cepas virulentas do FIV. / FIV is an important viral pathogen that infects the domestic cat and causes a slow progressive degeneration of the immune system which eventually leads to a disease comparable to acquired immune deficiency syndrome (AIDS) in humans. Similar to all retroviruses, FIV has a relatively high evolutionary rate and genomic heterogeneity. The determination of subtype and the knowledge of genetic diversity of the current strains are very important to developing a protective vaccine and for the routine diagnosis of infection. The aim of this study was to isolate and characterize samples of feline immunodeficiency virus from cats from an open shelter in Sao Paulo, Brazil. All cats infected with FIV from this shelter were sampled on August 26th, 2007 (T0) and also six (T1), ten (T2) and fifteen (T3) months after the basal sampling (T0). In each sample, blood was analyzed for the following: complete hematology, clinical chemistry and serum protein electrophoresis. Hematological and clinical chemistry parameters were analyzed to determine laboratory parameters characteristic of disease progression which allow a better description of the chronic phase of the infection. Furthermore, analyses of the variants from each sample were performed in order to estimate the degree of divergence following infection with Brazilian strains. The FIV envelope glycoprotein gene from Brazilian FIV isolates cloned were transfected to investigate the receptor usage. The sequences of all virus of the study belong to subtype B. Little sequence variation was observed in circulating viruses between the samples from each infected cat. Quasispecies of FIV have been detected in this study. The following hematological and clinical chemistry parameters were changed in the FIV-infected cats between the first blood sampling and last blood sampling: packed cell volume (PCV), hemoglobin, total white blood cells (WBC), total protein and gamma globulin fractions. Monitoring of hematological and clinical chemistry parameters may prove useful for the evaluation of disease progress. Regarding receptors, our data are consistent with isolates of the study requiring a less complex interaction with CD134 for infection to proceed compared to the virulent FIV isolates.

La capacitación espermática puede ser mimetizada in vitro eliminando el plasma seminal por distintos sistemas de lavado. Entre los tratamientos espermáticos empleados habitualmente en los laboratorios para capacitar a los espermatozoides se encuentran los lavados que se realizan con medios enriquecidos con albúmina o a través de gradientes de Percoll. El objetivo de este trabajo ha sido determinar los cambios que acontecen en los espermatozoides (procedentes de epidídimo y eyaculados) sometidos a tres sistemas de capacitación in vitro evaluados mediante una batería de técnicas que determinan distintos estadios de la capacitación espermática.De los resultados obtenidos se desprende que tanto la procedencia de los espermatozoides (epidídimo o eyaculado) como el tratamiento de capacitación al que se les somete afecta en gran medida a los resultados de la penetración in vitro y por lo tanto, la capacitación se produce de manera diferente entre estos grupos. / Sperm capacitation may be defined as a set of molecular modifications that occurs in the spermatozoa, after maturation in the epididymis, which enables them to fertilize the oocyte. In vitro, this process can be imitated in vitro by separation of the seminal plasma by different systems of washing. Between the sperm treatments routinely used in the laboratories, semen samples are washed of albumin or centrifugations through a Percoll. The aim of this work was examine and characterize the changes that happen in the sperms (from epididymis and ejaculated) submitted to three systems of in vitro capacitation, evaluated by means of a battery of tests to determine different levels of the sperm capacitation. Sperm capacitation was dependent on sperm treatment, whetherepididymal or ejaculated and to whichever parameter measured.Nevertheless, all these parameters, in spite of the fact that they have beendescribed as tools to evaluate the sperm capacitation, really are not capable of discriminating or indicating the level of capacitation.

El objetivo de este trabajo consistió en describir el papel del sistema plasminógeno/plasmina (PLG/PLA) en la fecundación bovina y porcina. Mediante fecundación in vitro, demostramos que la presencia de PLG ó PLA en el medio de coincubación de los gametos disminuía la penetración de los espermatozoides en los ovocitos y su unión a la zona pelúcida (ZP). Esta disminución no se debía a alteraciones de la funcionalidad espermática ni a cambios en la resistencia de la ZP a la proteolisis, sino a que la PLA provocaba la liberación de los espermatozoides adheridos a la ZP. Mediante inmunofluorescencia indirecta detectamos la presencia de PLG y sus activadores en la ZP y en el oolema de los ovocitos antes de la fecundación. Tras la fecundación, dicha presencia disminuyó o desapareció por completo, por lo que proponemos que el sistema PLG/PLA se activa durante la interacción espermatozoide-ovocito y contribuye a regular la polispermia. / The aim of this study was to describe the role of the plasminogen/plasmin system (PLG/PLA) in bovine and porcine fertilization. Through in vitro fertilization, we demonstrated that the presence of PLG or PLA in the incubation medium of gametes decreased penetration of oocytes and sperm binding to the zona pellucida (ZP). This decrease was not due to alterations in sperm function or changes in the ZP resistance to proteolysis, but the PLA caused the release of sperm previously bound to the ZP. By indirect immunofluorescence we detected the presence of PLG and its activators in the ZP and oolema of the oocytes before fertilization. After fertilization, this presence diminished or disappeared completely, so we propose that the PLG/PLA system is activated during sperm-oocyte interaction and contributes to the regulation of polyspermy.

O vírus da imunodeficiência felina (FIV) é um lentivirus que infecta gatos domésticos (Felis catus), causando uma imunodeficiência progressiva análoga a AIDS (Síndrome da Imunodeficiência Adquirida Humana). A ampla heterogeneidade molecular do FIV e a alta capacidade de promover mutações sob pressões imunológicas, farmacológicas ou ambientais são características inerentes aos lentivirus. A identificação do subtipo de vírus e o conhecimento da diversidade genética das cepas circulantes são fundamentais para o desenvolvimento estratégico de vacinas capazes de resultar na imunização do hospedeiro e no estabelecimento de testes diagnósticos. Objetivando isolar o material genético e realizar a caracterização molecular do vírus da imunodeficiência felina foram coletadas e analisadas amostras de sangue periférico de felinos portadores do FIV, co-habitantes de um abrigo aberto de felinos, em São Paulo, SP, em quatro momentos distintos, T0 (zero), no momento inicial da avaliação e seis, dez e quinze meses após a coleta inicial, correspondendo aos momentos T1, T2 e T3, respectivamente. Foram realizados testes hematológicos e bioquímicos nas quatro coletas com a finalidade de avaliar a evolução clínica da infecção. Adicionalmente foi realizado um estudo de variabilidade genética do FIV, com base no sequenciamento dos produtos amplificados dos gene env obtidos no estudo. Os envelopes clonados foram utilizados para transfectar células resultando na expressão das proteínas do envelope que possibilitaram estudos com os receptores celulares utilizados pelos isolados brasileiros. As análises das seqüências virais mostraram que todas as amostras, do abrigo, pertencem ao subtipo B. Foi observado um baixo percentual de mudança, da região estudada do vírus entre as quatro coletas. O fenômeno de &quot;quasispecies&quot; virais, bastante estudado no HIV, pode ser documentado em nossas amostras. Nos exames hematológicos e bioquímicos; hematócrito, hemoglobina, contagem total de leucócitos, proteína total e gamaglobulinas; dos animais infectados pelo FIV observou-se mudanças entre a primeira e quarta coleta demonstrando assim a importância dos testes utilizados no acompanhamento da infecção pelo FIV. Com relação aos dados com os receptores do FIV, os resultados apontam uma menor complexidade na interação entre os envelopes dos isolados do estudo com o receptor CD134 para proceder a infecção quando comparados com cepas virulentas do FIV. / FIV is an important viral pathogen that infects the domestic cat and causes a slow progressive degeneration of the immune system which eventually leads to a disease comparable to acquired immune deficiency syndrome (AIDS) in humans. Similar to all retroviruses, FIV has a relatively high evolutionary rate and genomic heterogeneity. The determination of subtype and the knowledge of genetic diversity of the current strains are very important to developing a protective vaccine and for the routine diagnosis of infection. The aim of this study was to isolate and characterize samples of feline immunodeficiency virus from cats from an open shelter in Sao Paulo, Brazil. All cats infected with FIV from this shelter were sampled on August 26th, 2007 (T0) and also six (T1), ten (T2) and fifteen (T3) months after the basal sampling (T0). In each sample, blood was analyzed for the following: complete hematology, clinical chemistry and serum protein electrophoresis. Hematological and clinical chemistry parameters were analyzed to determine laboratory parameters characteristic of disease progression which allow a better description of the chronic phase of the infection. Furthermore, analyses of the variants from each sample were performed in order to estimate the degree of divergence following infection with Brazilian strains. The FIV envelope glycoprotein gene from Brazilian FIV isolates cloned were transfected to investigate the receptor usage. The sequences of all virus of the study belong to subtype B. Little sequence variation was observed in circulating viruses between the samples from each infected cat. Quasispecies of FIV have been detected in this study. The following hematological and clinical chemistry parameters were changed in the FIV-infected cats between the first blood sampling and last blood sampling: packed cell volume (PCV), hemoglobin, total white blood cells (WBC), total protein and gamma globulin fractions. Monitoring of hematological and clinical chemistry parameters may prove useful for the evaluation of disease progress. Regarding receptors, our data are consistent with isolates of the study requiring a less complex interaction with CD134 for infection to proceed compared to the virulent FIV isolates.