Chez la brebis, les BMPs inhibent la synthèse et la libération de FSH. La détection des ARNm de certaines BMPs dans l’hypophyse et de leurs récepteurs sur les cellules gonadotropes suggère une action paracrine/autocrine des BMPs sur la production de FSH. L’objectif de cette thèse a visé à déterminer l’importance des BMPs hypophysaires, en particulier BMP-4, dans la régulation de la sécrétion de FSH chez la brebis. Nous montrons que l’expression des ARNm du système BMP-4,analysée par RT-PCR en temps réel, ne varie pas au cours du cycle oestrien ou in vitro quelque soit le temps d’incubation (6h-48h) et le traitement (GnRH, oestradiol, activine) des cellules hypophysaires.Afin de déterminer si les cellules hypophysaires ovines sécrètent des BMPs, des milieux conditionnés(MC) hypophysaires ont été soumis à un test d’activité biologique reposant sur des cellules embryonnaires transfectées avec un élément de réponse BMPs couplé au gène rapporteur luciférase.Aucun effet des MC n’a été observé sur l’activité luciférase comparé au milieu non conditionnésuggérant l’absence ou la très faible activité BMP des MC, et cela quelque soit le temps d’incubation(6h-48h) et le traitement (GnRH, oestradiol, activine) des cellules hypophysaires. Toutefois, nous détectons une activité inhibitrice de BMPs qui est augmentée par la GnRH et l’oestradiol suggérant l’implication d’inhibiteurs de BMPs dans la régulation de la production de FSH. En conclusion,l’ensemble de nos résultats n’est pas en faveur d’un rôle de BMP-4 hypophysaire dans la synthèse de FSH chez l’adulte. Un rôle des BMPs au niveau hypophysaire via la voie endocrine peut s’envisager puisque nous avons mis en évidence une bioactivité de type BMP dans le sérum ovin. Les inhibiteurs BMPs produits par l’hypophyse, qui restent à identifier, pourraient moduler la biodisponibilité des BMPs atteignant l’hypophyse par la voie sanguine. / BMPs inhibit FSH synthesis and release in ewe. The detection of BMP mRNAs in the pituitary as well as the colocalisation of the two types of BMP receptors on gonadotrope cells suggest that these BMPs can exert paracrine/autocrine actions on FSH production. The aim of this thesis work was to determine the importance of pituitary BMPs in the regulation of FSH production in the ewe. We showed that the level of mRNAs for BMP-4 system, analyzed by real-time RT-PCR, did not vary across the oestrous cycle or in vitro whatever the incubation period (6h-48h) and the treatment (GnRH, oestradiol, activin) of pituitary cells. By using a bioactivity test based on embryonic mesenchymal cells transfected with an expression construct containing a BMP-responsive element fused to firefly luciferase reporter gene,we detected no BMP activity within conditioned media from pituitary cells whatever the incubation period (6h-48h) and the treatment (GnRH, oestradiol, activin) of the pituitary cells. However, we detected a BMP inhibitory activity which is increased by GnRH and oestradiol suggesting the implication of BMP inhibitor(s) in FSH regulation. In conclusion, the results are not in favor of a role for pituitary BMP-4 in the regulation of FSH synthesis in adult ewe. An endocrine action of BMPs at pituitary level can be evoked since BMP bioactivity was detected within ovine serum. BMP inhibitors that remain to be identified can modulate the bioavailability of BMPs reaching the pituitary by the blood way.

Ovine herpesvirus 2 (OvHV-2) is a gamma herpesvirus and is the causative agent of lymphoproliferative disease – sheep-associated malignant catarrhal fever in susceptible ruminants, including cattle. Sheep become persistently infected but do not show apparent clinical infection. MCF is characterized by marked T cell hyperplasia and proliferation of unrestricted cytotoxic large granular lymphocytes (LGLs) which leads to necrosis of infiltrated tissues and generally causes death of the host. Little is known about the underlying molecular basis of MCF pathogenesis or what controls the differences in clinical outcome of infection in two closely-related host species. MicroRNAs (miRNAs) constitute a large family of small, ~22nt, noncoding RNA molecules that regulate gene expression by targeting messenger RNAs post-transcriptianally in eukaryotes and viruses. Herpesvirus encoded miRNAs have been shown to play a role in regulating viral and cellular processes including cell cycle and may have a role in pathogenesis. OvHV-2 has also been found to encode for at least 46 OvHV-2 miRNAs in an immortalized bovine LGL cell line. 23 of these miRNAs have also been validated by northern blot analysis and RT qPCR. It was hypothesised that these OvHV-2 miRNAs may regulate viral and cellular genes expression and may play a role in MCF pathogenesis. The aim of this project was to determine if OvHV-2 miRNAs have functional targets within viral and host cell genes. Bio-informatic analysis has predicted several targets for these OvHV2 miRNAs in the 5’ and 3’ UTRs of several virus genes. Luciferase inhibition assay confirmed that out of 13 selected predicted targets, three (two targets ORF73 and one within ORF50) were positive and functional. A fourth predicted target was also found functional (ORF20), but its functionality could not be confirmed by knocking out the target site. A newly developed technique Crosslinking, Ligation And Sequencing of Hybrids (CLASH) was also used to identify miRNAs bound targets within cattle and sheep genome. High throughput sequencing and analysis of the hybrid data revealed many target genes. Four of those targeted genes, were validated by luciferase inhibition assays and three were found to be targeted by OvHV-2 miRNAs. This study gives the first evidence of viral miRNAs bound to their targets in cattle and sheep cells, by a highly sensitive technique-CLASH and provides a tool for studying differences in pathogenesis of two closely-related host species.

Teladorsagia circumcincta, an economically-important abomasal nematode of small ruminants in temperate regions worldwide, is currently controlled with a combination of anthelmintics and pasture management. Anthelmintic resistance has emerged and vaccination is a potential alternative control strategy, as protective immunity in sheep can be acquired after repeated exposure to the parasite. Abomasal mucosal IgA responses in immune sheep have been correlated with delayed worm development and reduced faecal egg counts. However, recombinant vaccine development against parasitic nematodes has had limited success, and one of the reasons may be unsuitable expression systems for antigen production leading to incomplete or inadequate post-translational modifications such as glycosylation and tertiary protein folding, resulting in incorrect epitope structures for antibody binding. In this thesis, to address this issue, “native” infective larval (L3) antigen targets of protective immune responses and synthetic peptide sequences which mimic structural epitopes on these antigens were identified. Abomasal mucosal IgA was used as a probe to identify native immunogenic antigens from T. circumcincta L3. IgA was purified from abomasal mucus of animals rendered immune by repeated experimental infection and a custom antibody-affinity column was created and used to purify antigens from an L3 somatic PBS-soluble extract. Affinity purified L3-antigen-specific IgA levels in sheep with varying levels of immunity to T. circumcincta were positively correlated (rs = 0.853, P < 0.001) with both the total IgA concentration in efferent gastric lymph after parasite challenge, and with the percentage of inhibited fourth-stage (L4) larvae present in the gastric glands of the immune hosts (rs = 0.534, P = 0.007). In contrast, a negative correlation between the levels of affinity-purified L3 antigen-specific IgA and total T. circumcincta burden was observed (rs = -0.565, P = 0.004). Proteomic analysis of the IgA-affinity purified L3 extract identified a number of proteins which represent potential vaccine candidate molecules in other helminth species, including paramyosin, superoxide dismutase, galectin, activation-associated secreted proteins and fatty-acid retinol-binding proteins. As a first step towards the development of a novel vaccine based on IgA-binding peptide mimics of native structural epitopes, phage display libraries were used to screen antibodies, from sheep rendered immune to T. circumcincta by experimental infection. These antibodies were affinity-purified before use and specifically bound T. circumcincta L3 glycans or, alternatively, surface antigens on exsheathed T. circumcincta L3. Five peptide sequences which mimic L3 antigenic epitopes were identified and positive correlations existed between peptide-specific IgA levels and both the total IgA concentration in efferent gastric lymph after parasite challenge and the percentage of inhibited L4 present (rs > 0.621, P < 0.001 to P < 0.05). In contrast, negative correlations between the levels of peptide-specific IgA and the total nematode burden were observed (rs > -0.528, P < 0.01 to P < 0.05). In conclusion, the selected phage clones may therefore represent vaccine candidates if they could be presented to the ovine immune system in an appropriate fashion.

Orientador: Sony Dimas Bicudo / Resumo: O objetivo do presente trabalho foi avaliar os corpos lúteos (CL) formados em decorrência da aspiração folicular guiada por laparoscopia (LOPU) na espécie ovina, quanto ao desenvolvimento morfológico, funcionalidade e potencial de desenvolvimento gestacional. Foram realizados dois experimentos, em ambos o estro das ovelhas foi sincronizado utilizando dispositivo intravaginal de acetato de medroxiprogesterona por 14 dias, aplicação de 140 μg cloprostenol no quarto dia e retirada do dispositivo no décimo quarto dia com concomitante administração de gonadotrofina coriônica equina (eCG). No Experimento I, 48 horas após a remoção do dispositivo realizou-se a LOPU dos folículos ovarinos em seis ovelhas (Grupo LOPU – LG), ou a LOPU associada com reposição de células em sete animais (Grupo reposição – RG) em cinco animais a ovulação foi espontânea (Grupo controle – CG). Foi realizada avaliação ultrassonográfica ovariana dos animais diariamente até o momento de uma nova ovulação e coleta de sangue para dosagem de progesterona (P4) plasmática a cada 48 horas até o decimo quinto dia. Os animais do LG (1,7±0,5) e RG (1,4±0,5) formaram mais CLs do que os animais do CG (1,0±0,0). A área lútea individual dos CLs foi menor no LG (0,69±0,30 cm2), em comparação ao RG (0,79±0,25 cm2) e CG (0,85±0,32 cm2). A área lútea total foi semelhante entre o LG (1,2±0,4 cm2) e RG (1,1±0,5 cm2), sendo ambas maiores que a do CG (0,9±0,3 cm2). A concentração de P4 plasmática não diferiu entre os grupos, fican... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of the present study was to evaluate the corpus luteum (CL) formed as a result of laparoscopic ovum pick-up (LOPU) in the ovine, regarding the morphological development, functionality and potential of gestational development. Two experiments were carried out, in both of them, the oestrous of the ewes was synchronized using a 14-day intravaginal device of medroxyprogesterone acetate, 140 μg cloprostenol on the fourth day and withdrawal of the device on the fourteenth day with concomitant administration of equine chorionic gonadotrophin (eCG). In Experiment I, 48 hours after the removal of the device, was realized a LOPU of the ovarian follicles in six sheep (LOPU Group - LG), or LOPU associated with cell replacement in seven animals (Replacement Group - RG) was performed in five animals a ovulation was spontaneous (Control Group - CG). Ultrasonographic evaluation of the animals was performed daily until the time of ovulation and blood collection for progesterone plasma concentration (P4) every 48 hours until the 15th day. LG animals (1.7 ± 0.5) and RG (1.4 ± 0.5) formed more CLs than CG animals (1.0 ± 0.0). The individual luteal area of CLs was lower in LG (0.69 ± 0.30 cm2), compared to RG (0.79 ± 0.25 cm2) and CG (0.85 ± 0.32 cm2). The total luteal area was similar between LG (1.2 ± 0.4 cm2) and RG (1.1 ± 0.5 cm2), both larger than CG (0.9 ± 0.3 cm2). Plasma P4 concentration did not differ between groups, being above 2 ng / mL in the static phase of CLs development. I... (Complete abstract click electronic access below) / Doutor

A degeneração testicular (DT) possui grande relevância dentre os distúrbios da reprodução e pode ser causada pelo aumento da temperatura testicular. Este provoca aumento do metabolismo celular, levando ao estresse oxidativo (EO) e apoptose. O tratamento usual consiste na retirada do agente causador e administração de antioxidantes; entretanto, pode não ser eficiente. Dessa forma, o presente estudo preconizou o tratamento da DT por meio de agentes com poder proliferativo: vitamina A e laserterapia de baixa intensidade (LTBI). Foram realizados três experimentos; o experimento 1 objetivou definir a dose de energia da LTBI necessária para a bioestimulação testicular. Foram utilizados seis carneiros distribuídos em três grupos: GC) insulação escrotal (IE) e sem tratamento (n=2); G28) IE e tratado com LTBI com 808 nm de comprimento de onda, 30 mW de potência e 28 J/cm² de densidade de energia por 15 dias a cada 48 horas (n=2); G56) IE e tratado com LTBI com 808 nm, 30 mW e 56 J/cm² por 15 dias a cada 48 horas (n=2). Foram feitas análises clínicas, reprodutivas e histopatológicas. Os dados foram submetidos à análise de variância (ANOVA) e teste de Tukey. Apesar da LTBI diminuir as taxas de espermatozoides com membrana acrossomal íntegra, esta foi eficiente em aumentar a população celular dos túbulos seminíferos no G28. Portanto, a LBTI provocou efeito bioestimulatório em testículos degenerados de carneiros. O experimento 2 objetivou validar a técnica de avaliação do EO espermático por meio da sonda fluorescente CellROX Deep Red®. Foram realizados dois experimentos; o primeiro utilizou ejaculados de três carneiros tratados em T0 (ejaculado não submetido à indução de EO), T50 (50% não induzido e 50% induzido ao EO) e T100 (submetido ao EO). Os dados foram submetidos à regressão linear. No segundo experimento foram utilizados 16 carneiros submetidos à IE. Foram feitas avaliações do EO antes e após a IE. Os dados foram submetidos à ANOVA e teste LSD de Fisher. O coeficiente de determinação foi de 0,728 e houve aumento do EO após a IE. Assim, a sonda CellROX® foi capaz de detectar o EO espermático. No experimento 3 foi proposto tratamento para a DT baseado na suplementação vitamínica ou LTBI. Foram utilizados 33 carneiros distribuídos em seis grupos: CC) sem IE e sem tratamento (n=5); CA) sem IE e tratado com vitamina A IM 120.000 UI/animal, duas vezes por semana durante três semanas (n=6); CL) sem IE e tratado com LTBI protocolo G28 (experimento 1) (n=5); IC) IE e sem tratamento (n = 5); IA) IE e tratado com vitamina A IM 120.000 UI/animal, duas vezes por semana durante três semanas (n=6); IL) IE e tratado com LTBI protocolo G28 (n=6). Foram realizadas análises clínicas, reprodutivas, hormonais e histopatológicas. Os dados foram analisados usando o procedimento de modelos mistos e os efeitos dos tratamentos foram avaliados utilizando contrastes ortogonais. Não houve efeito benéfico dos tratamentos para as características ultrassonográficas, qualidade espermática, concentração de testosterona e aspectos histopatológicos. Assim, os tratamentos não foram eficientes para melhorar a qualidade espermática nem promover a proliferação celular. / The testicular degeneration (TD) has great significance among the reproductive disorders and one of the main causes is the increase in testicular temperature. High testicular temperature results in increase cellular metabolism, leading to oxidative stress and apoptosis. The treatment consists in removing the causative agent and administration of antioxidants; however, it could be not efficient. The objective of this study is to recommend the treatment of TD by administering agents with proliferative action: vitamin A and low level laser therapy (LLLT). For this, three experiments were conducted. In experiment 1 the objective was to define the dose of energy for LLLT testicular biostimulation; it was used six rams distributed in three groups: GC) scrotal insulation (SI) and untreated (n=2); G28) SI and treated with LLLT with 808 nm, 30 mW and 28 J/cm ² of power density for 15 days every 48 hours (n=2); G56) SI and treated with LLLT with 808 nm, 30 mW and 56 J/cm² for 15 days every 48 hours (n=2). Clinics, reproductive and histopathological analyzes were done. Data were subjected to analysis of variance and Tukey test. The rates of sperm with intact acrosome membrane were decreased by LLLT, but the LLLT was effective in increasing the cell population of the seminiferous tubules in the G28. Thus, LLLT was able of causing stimulatory effect in degenerate testis of rams. The objective of experiment 2 was to assess the technique of evaluation of sperm oxidative stress (OS). This study was divided in two experiments; in experiment 1 was used ejaculates of three rams treated in T0 (ejaculate that was not submitted to OS induction), T50 (50% without OS and 50% inducted to OS) and T100 (entire submitted to OS induction). Data obtained were evaluated by linear regression analysis. In experiment 2, sixteen rams were submitted to SI. Analyses of OS were done before and after the SI. Data obtained were evaluated by analysis of variance and Fisher\'s LSD test. The determination coefficient was of 0.728 and there were increase in sperm showing OS after SI period. Thus, CellROX® fluorescent probe was able to detect sperm OS. The objective of experiment 3 was establish a treatment for TD based on vitamin A supplementation or LLLT; 33 rams were distributed in six groups: CC) no SI and non-treated (n=5); CA) no SI and treated with 120,000 IU/animal of IM vitamin A, twice a week for three weeks (n=6); CL) no SI and treated with LLLT G28 protocol (experiment 1) (n=5); IC) SI and untreated (n=5); IA) SI and treated with 120,000 IU/animal of IM vitamin A, twice a week for three weeks (n=6); IL) SI and treated with LLLT G28 protocol (n=6). Clinics, reproductive, hormonal and histopathological analyzes were performed. Data were analyzed using the mixed models procedure and treatments effects were evaluated using orthogonal contrasts. There was no beneficial effect of treatments for ultrasonographic characteristics, sperm quality, testosterone concentration and histopathological aspects. Thus, the treatments were not effective for improving sperm quality or promoting cell proliferation.

A ovinocultura é um sistema de criação muito antigo em nosso país e ainda continua apresentando uma série de problemas que dificultam a produção econômica desses animais e necessitam de urgentes soluções. Visando minorar estes prejuízos esta pesquisa foi idealizada com o intuito de realizar um estudo minuncioso das principais enfermidades reprodutivas parasitárias e bacterianas. Todos os animais foram submetidos ao exame clínico geral e específico do sistema reprodutor e colheram-se amostras de soro, sêmen, abortamentos, lóquios, secreções, fezes e zaragatoa prepucial e vaginal dependendo da enfermidade a ser estudada. Não foi possível o isolamento nos fetos de nenhum agente etiológico. Conclui-se que a toxoplasmose é a enfermidade de maior importância nos rebanhos de estado de São Paulo e a de maior fator de risco em casos de abortamentos, a neosporose estaria relacionada principalmente com animais que apresentam repetição de cio, leptospirose foi observado em baixa titulação e a mais prevalente foi a Leptospira australis sorovar australis proveniente de animais silvestres, a campilobacteriose e a brucelose são as duas enfermidades da reprodução com menor ocorrência no estado. Foi isolado o primeiro caso de endometrite no Brasil por Histophilus somni. Foi isolado o primeiro caso de orquite por Actinobacillus seminis no estado de São Paulo. / Sheep industry is a traditional system in Brazil and presents a series of issues that complicate economical exploitation and urgently need solution. In order to reduce losses this research was idealized to carefully study the major bacterial and parasitary reproductive diseases. All animals were submitted to a general and specific reproductive system clinical examination. Blood, semen, abortions, lochia, vaginal discharges, feces, and preputial/vaginal swabs were collected according to the studied disease. It was not possible to isolate any etiological agent from aborted fetuses. The conclusion was that toxoplasmosis was the major disease on São Paulo herds and has the biggest risk factor in abortions, neosporosis would be related to animals that present heat repetition, leptospirosis was observed with low antibodies titles and the most prevalent was Leptospira australis sorovar australis arising from wild animals, campilobacteriosis and brucellosis are both the reproductive diseases with the lowest occurrence in the state. The first endometritis case caused by Histophilus somni. In Brazil was isolated. The first orchitis case caused by Actinobacillus seminis in São Paulo state was isolated.

O objetivo deste trabalho foi avaliar de maneira detalhada e sistemática os perfis hormonais sanguíneos, fecais, urinários e salivares das progestinas e estrógenos durante o ciclo estral induzido de ovinos. Foram colhidas amostras diárias durante um período de 60 dias de sete fêmeas adultas (n=8) saudáveis e sexualmente maduras. Antes do início da fase de colheita das amostras, todos os animais foram submetidos ao protocolo de tratamento hormonal para indução e sincronização do cio durante dozes dias. O primeiro ciclo ovariano de cada animal desse experimento, detectado logo após a indução do cio foi descartado e seus valores não foram utilizados nas análises hormonais, pois poderiam estar sob o efeito dos hormônios exógenos. A concentração dos progestágenos foi determinada pelas técnicas analíticas de Radioimunoensaio (RIE) e Enzimaimunoensaio (EIE) e os estrógenos por RIE. Houve correlações entre as concentrações de progesterona medidas nas matrizes sérica e fecal, sérica e salivar, fecal e salivar (r=0,90, p<0,0001; r=0,90, p<0,0001; r=0,92, p<0,0001, respectivamente) durante os ciclos estrais observados (n=15). Obtivemos correlação (r=0,74, p<0,0001) entre as concentrações dos estrógenos quantificados nas matrizes sérica e fecal, mas não entre estas concentrações e aquelas medidas na matriz salivar. Não obtivemos nenhuma correlação entre as concentrações medidas na matriz urinária com as quantificadas nas outras matrizes para nenhum dos hormônios estudados. Obtivemos correlação entre as concentrações de progesterona medidas na matriz fecal pelos métodos de RIE e EIE (r=0,78, p<0,0001) e também na matriz salivar pelos dois métodos empregados (r=0,81, p<0,0001). Os resultados do presente experimento indicam que os imunoensaios utilizados podem ser utilizados para a avaliação das concentrações de progestágenos nas matrizes fecal e salivar durante o ciclo estral em ovinos. / The aim of the present work was evaluate the hormonal profiles of progestins and estrogens in blood, feces, urine and saliva during the induced estral cycle in ovine. Samples were collected daily a 60-day period from eight adult (n=8) cycling ewes. The animals were previously submitted to a protocol of estrus induction and synchronization for twelve days. In order to avoid the effect of exogenous hormones, the first cycle immediately after the synchronization was not considered for hormonal analysis. Progestagen concentrations were quantified by two analytical techniques, radioimmunoassay (RIA) and enzyme immunoassay (EIA). Estrogen concentrations were assessed by radioimmunoassay. Correlations in progesterone concentrations were found to be significant for serum and feces, serum and saliva and feces and saliva (r=0.90, p<0.0001; r=0.90, p<0.0001; r=0.92, p<0.0001, respectively) during the estrous cycles (n=15). Estrogen concentrations in the serum and feces were also positively correlated (r=0.74, p<0.0001). Salivary concentrations of estrogens were not correlated with fecal or serum concentrations of the same hormone. No correlation was found between urinary concentrations and concentrations found in other matrixes for both progestagens and estrogens. Concentrations of progestagens obtained using RIA and EIA were correlated on feces (r=0.78, p<0.0001) and saliva (r=0.81, p<0.0001). Results indicate that both immunoassays used in the present experiment can be used to evaluate progestagen concentrations on fecal and salivary matrixes during the estrous cycle of sheep.

A artroscopia tem função diagnóstica e terapêutica na doença articular. Mesmo com as modalidades de imagem mais modernas, ainda é considerada como padrão ouro no diagnóstico de problemas articulares em equinos. Sabe-se que as características físico-químicas das diferentes soluções de infusão podem determinar alterações estruturais na superfície da cartilagem. Apesar da existência de estudos baseados na busca de um fluido ideal para irrigação durante a artroscopia, com principal foco nas características bioquímicas do líquido, poucos trabalhos testaram características físicas destes fluidos, como a temperatura. Na primeira etapa deste trabalho foram avaliadas 24 articulações de ovinos submetidas à irrigação articular com solução de Ringer com lactato condicionado a quatro diferentes temperaturas (5, 12, 25 e 38 ºC). Para avaliação de cada grupo foram coletadas biopsias comparativas de membrana sinovial e de cartilagem com osso subcondral da articulação femoropatelar visando análise histológica e por microscopia eletrônica de varredura, respectivamente, imediatamente antes do início da infusão do líquido e após uma hora de infusão. Apesar de alterações na ultraestrutura da superfície articular terem sido observadas após lavagem das articulações, nenhuma diferença estatística entre os grupos foi identificada. Na segunda etapa do experimento foram testadas as temperaturas 5, 25 e 38 °C do Ringer com lactato utilizado para irrigação articular durante procedimentos artroscópicos em 12 articulações radiocárpicas de seis equinos. As articulações foram divididas em três grupos de quatro, sendo avaliados, além da membrana sinovial e cartilagem, o líquido sinovial previamente ao início da infusão com fluido condicionado e 12, 24 e 48 horas após o término da irrigação, visando análise comparativa das concentrações de IL-1&beta;, PGE2, TNF-&alpha;, condroitim sulfato (CS) e ácido hialurônico (AH). As alterações da superfície articular dos equinos decorrentes da irrigação também foram independentes da temperatura utilizada. Houve um aumento significativo das concentrações de PGE2 e IL-1&beta; no líquido sinovial às 12 horas em todos os grupos, exceto para IL-1&beta; no grupo de articulações irrigadas com fluido a 38 °C, que não apresentou alteração nas concentrações. A análise de TNF-&alpha; não revelou diferença significativa entre os grupos. As concentrações de CS aumentaram às 24 horas nos três grupos e foi estatisticamente maior no grupo irrigado a 38 °C. Não houve diferença significativa das concentrações de AH no líquido sinovial do grupo de articulações irrigadas a 25 °C, o que não ocorreu nos grupos 5 e 38 °C, havendo diminuição às 24 e 12 horas, respectivamente, retornando aos valores basais na coleta subsequente. As biopsias de membrana sinovial coletadas em ambas as etapas do experimento não demonstraram alteração estrutural ou migração celular que inferissem em resposta inflamatória ao insulto. As alterações da ultraestrutura da superfície da cartilagem estão mais relacionada ao fator mecânico da passagem do líquido que à temperatura de acondicionamento. Apesar de mais análises serem necessárias para definição da melhor temperatura a ser utilizada durante as artroscopias, a detecção de menores concentrações de IL-1&beta; nas articulações de equinos irrigadas com Ringer com lactato a 38 °C sugerem de que essa seja a temperatura menos deletéria para o ambiente articular. / Arthroscopy has a diagnostic and a therapeutic function in joint disease. Even with modern imaging modalities, it is still considered the gold standard for diagnosing equine joint problems. It is known that the physicochemical characteristics of different infusion solutions could influence structural modifications on cartilage surface. A number of studies have described the search for the ideal fluid to be used for irrigation during arthroscopy, focusing on the biochemical characteristics of the various liquids. However, few works have taken temperature into consideration when studying the physical characteristics of these fluids. Therefore, in the first phase of this study four different temperatures of Ringer lactate used for articular irrigation were assessed into four groups of six ovine joints each (5, 12, 25 and 38 ºC). To evaluate each group, comparative synovial membrane and osteochondral samples were collected from the patellofemoral joints for histological analysis and Scanning Electron Microscopy, respectively, immediately before and 1 hour after starting fluid infusion. Ultrastructural changes of the joint surfaces after irrigation were observed, but no statistical differences between groups were identified. In the second phase of this study, lactated Ringers solution was assessed at three different temperatures: 5, 25 and 38°C, for irrigation of 12 radiocarpal joints of six horses during arthroscopy. Three groups of four joints were studied to evaluate synovial membrane, cartilage and synovial liquid immediately before and 12, 24 and 48 hours after joint irrigation. These samples were subjected to comparative analysis of IL-1&beta, PGE2, TNF-&alpha, condroitin sulfate (CS) and hyaluronic acid (HA) concentrations. Changes of equine joint surfaces were also independent of temperature. There was a significant increase in the PGE2 and IL-1&beta; concentrations in the synovial liquid at 12 hours in all groups except for IL-1&beta; on the joints irrigated with fluid at 38°C, which did not show concentrations changes. There was no statistical difference of TNF-&alpha between groups. CS concentrations had a significant increase at 24 hours in all groups, meanwhile in the 38 °C group this increased was statistically greater. There was no statistical difference for HA concentrations on synovial liquid of the irrigated joints at 25 °C. In the 5 and 38 °C groups there were synovial fluid HA decrease at 24 and 12 hours, respectively, returning to basal values at the subsequent collect. The synovial membrane biopsies collected in the both steps of this study did not show any structural alterations or cellular migration that might interfere in response to inflammatory insult. Ultrastructural changes in articular most superficial zone are more related to mechanic action of fluid lavage than irrigation solutions temperature. Although more tests needs to be done, the lowest concentrations of IL-1&beta; in equine joints irrigated with Ringer lactate at 38 °C give some evidence that this is the less deleterious temperature to articular environment.

Os ácidos graxos poliinsaturados garantem a fluidez à membrana plasmática do espermatozóide. No entanto, as duplas ligações presentes, os tomam mais susceptíveis aos efeitos nocivos da peroxidação lipídica que produz grande quantidade de radicais livres. A adição do antioxidante Glutationa reduzida (GSH) poderia conferir proteção aos espermatozóides de ovinos submetidos a criopreservação contra danos, em membranas plasmáticas, acrossomais, DNA e atividade mitocondrial causados pelo estresse oxidativo. O objetivo do presente experimento foi: avaliar se a GSH protege os espermatozóides ovinos criopreservados contra danos causados pelo estresse oxidativo. Foram colhidos ejaculados de quatro carneiros adultos. As amostras foram avaliadas após a criopreservação (TI). As análises convencionais foram: concentração, motilidade, vigor e morfologia. As análises funcionais foram: integridade de membrana, integridade acrossomal, integridade de cromatina e atividade mitocondrial. O sêmen foi criopreservado utilizando o diluidor Tris-gema-critrato suplementado com GSH (controle, 1, 5 e 10mM). Uma alíquota de cada amostra foi submetida ao protocolo de peroxidação lipídica induzida e subseqüente quantificação das substâncias reativas ao ácido tiobarbitúrico(TBARS). As análises estatísticas foram realizadas utilizando o Sistema SAS para Windows. Os resultados indicam que não houve efeito do tratamento com GSH sobre as variáveis avaliadas pelos testes convencionais. A GSH diminuiu a proporção de acrossomas íntegros. Amostras tratadas com 5mM de GSH apresentaram menor percentual de células com membranas íntegras quando comparadas com as amostras controle e aquelas tratadas com 10 mM; , o percentual de células sem atividade mitocondrial foi influenciado pela GSH também não houve de efeito nas TBARS. As amostras do grupo controle foram mais susceptíveis à desnaturação da cromatina. Em conclusão, a adição do antioxidante Glutationa reduzida (GSH) confere proteção ao DNA e à atividade mitocondrial de espermatozóides de ovinos. / It is well known that sperm is extremely susceptible to the oxidative stress. This occurs especially due to the high content of poly-unsaturated fatty acids (PUF As) in its plasma membrane. The PUF As provide the necessary fluidity to the plasma membrane, which allows the sperm to move. However the double bonds present in those fatty acids are more susceptible to the oxidative stress. Therefore, sperm is highly dependent of the extra-celluIar environment antioxidant protection. Studies in human indicate that cryopreservation may lead to damages to the sperm due to the oxidative stress. The objective of this experiment was to study if the antioxidant glutathione (GSH) may protect ovine cryopreserved sperm against the damages caused by the oxidative stress. Semen samples of four rams were collected using an artificial vagina. Samples were then cryopreserved using Tris-egg yolk extender supplemented with different concentrations of reduced glutathione (control, 1, 5 and 10 mM). After thawing, samples were evaluated using conventional (motility and vigor) and functional tests (membrane integrity and mitochondrial activity). An aIiquot of each thawed sample was submitted to the protocol of induced lipid peroxidation using ascorbate (20 mM) and ferrous sulphate (4 mM), with the further measurement of the tiobarbituric acid reactive substances (TBARS), as an index of oxidative stress. This technique aims to evaIuate sperm oxidative status by its susceptibility to the lipid peroxidation. Statistical analysis was performed using SAS system for Windows. The results indicate that there was no effect of GSH on the variables assessed by conventional tests. GSH decreased the proportion of intact acrosomes. Samples treated with 5 mM GSH showed a lower percentage of cells with intact membranes when compared with control sampIes and those treated with 10 mM, the percentage of cells with mitochondriaI activity was affected by GSH and there was also no effect on TBARS. Samples from the control group were more susceptible to denaturation of chromatin. In conclusion, the addition of the antioxidant Glutathione (GSH) offers protection to DNA and mitochondrial activity of ovine sperm.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-02-10T12:25:05Z No. of bitstreams: 1 Pedro Augusto Marinho Patriota Lima.pdf: 1417191 bytes, checksum: 7e1d18fc8ee49410d1d129b817022e3f (MD5) / Made available in DSpace on 2017-02-10T12:25:05Z (GMT). No. of bitstreams: 1 Pedro Augusto Marinho Patriota Lima.pdf: 1417191 bytes, checksum: 7e1d18fc8ee49410d1d129b817022e3f (MD5) Previous issue date: 2013-02-08 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The gossypol is a Polyphenolic yellow pigment (C30H30O8), toxic that is present in all parts of cotton plant and its derivatives. Experiments show the reproductive side effects caused by feed containing high percentage of bran or cottonseed and claim that gossypol might affect in different ways the reproductive system, causing sperm abnormalities and subsequent infertility. In this work, it was examined the effects caused by diet rich in cottonseed containing gossypol on hair sheep ram semen Santa Inês Breed. The work was performed with 22 rams, sexually mature, which were divided into 2 groups, being the animals confined in individual stocks. The gossypol group (Gg = 15), was supplemented with 500g of gossypol cottonseed and the control group (Gc = 7), supplemented with 500g of corn. The two groups were supplemented during 104 days and underwent semen collections, by the method of artificial vagina and measurements of scrotal circumference (C.E.), before (7 days), during (at intervals of 15 days) and after (10 days) the supplementation period. Non o statistical difference was seen in the mean C.E. group being treatment and control group respectively 27.07 cm and 27.08 cm. The variable observed in the semen were: color, aspect, volume, whirling, motility, concentration, vigor and sperm morphology. For color and aspect analyses were qualitative and no differences were seen in these parameters. The mean for seminal volume for animals who consumed cottonseed was 1.04 ml and for control group was 0.94 ml with no significant difference. In the variable whirling, motility, vigor, concentration and morphology there was no statistical difference and the results was respectively 4.12-81.6%-4.26-3.57 x 109/ml sptz – 6.7% and 3.91 control group – 74.77%-4.07-3.48 x 109 sptz/ml-5.97%. After the last semen collection 5 animals from gossypol group and 2 animals from control group was submitted to unilateral orchiectomy. The material was used to perform the comparative Histopathological examination in historesin, No difference in seminiferous tubule diameter was seen between control and gossypol group. There was a significant difference in seminiferous epithelium height, having a larger measurement at gossypol epithelium compared with control group. The height of seminiferous epithelium is an effective feature for sperm production assessment, however in this study there was no significant difference in sperm concentration between control and treatment group demonstrating that this difference did not affect sperm production. Two out of the three castrated animals from gossypol group were used as a fertility test exposing them to 10 fertile ewes during 90 days, resulting in 6pregnancies. The results showed that supplementation with cottonseed gossypol rich-diet did not influence sperm quality, testicular morphology, fertilization capacity and seminiferous tubules morphology. / O gossipol é um pigmento polifenólico amarelo (C30H30O8), de natureza tóxica que está presente em todas as partes do algodoeiro e em seus derivados primários. Experimentos realizados mostram os efeitos colaterais na reprodução causadas por rações contendo alta porcentagem de farelo ou caroço de algodão e afirmam que o gossipol pode afetar de maneira multiforme o sistema reprodutivo, causando anormalidade nos espermatozoides e consequente infertilidade. Nesse trabalho foram analisados os efeitos causados pela dieta rica em caroço de algodão contendo gossipol no sêmen de ovinos deslanados da Raça Santa Inês. Para isso, o trabalho foi desenvolvido com 22 ovinos machos, em idade reprodutiva, os quais foram divididos em dois grupos, sendo os animais confinados em baias individuais. O grupo gossipol (Gg = 15), suplementado com 500g de caroço de algodão e o grupo controle (Gc = 7), suplementado com 500g de milho. Os dois grupos foram suplementados durante 104 dias e foram submetidos a coletas de sêmen, pelo método da vagina artificial e aferições de circunferência escrotal (C.E.), antes (7dias), durante (em intervalos de 15 dias) e depois (14 dias) do período de suplementação. Não houve diferença estatística na média de circunferência escrotal (C.E.) sendo a média do grupo tratamento e do grupo controle respectivamente 27,07 cm e 27,08 cm. As variáveis observadas no sêmen foram: coloração, aspecto, volume, turbilhonamento, motilidade, concentração, vigor e morfologia espermática. Para coloração e aspecto as análises foram qualitativas e não foram observadas diferenças nessas variáveis. A média do volume seminal para os animais que consumiram caroço de algodão foi de 1,04 ml e para os que não consumiram foi de 0,94 ml. Nas variáveis de turbilhonamento, motilidade, vigor, concentração e morfologia não houve diferença significativa sendo os valores do grupo tratamento respectivamente 4,12 – 81,6% - 4,26 – 3,57 x 109sptz /ml – 6,7% e do grupo controle 3,91 – 74,77% – 4,07 – 3,48 x 109sptz/ml – 5,97%. Após o término da última coleta de sêmen cinco animais sofreram orquiectomia unilateral, sendo dois do grupo controle e três do grupo gossipol. O material foi utilizado para realização de exame histopatológico comparativo em historesina, onde não apresentou diferença significativa entre tratamento e controle quanto ao diâmetro de túbulo seminífero, e apresentou diferença na altura do epitélio seminífero. Tendo o grupo gossipol maiores medidas de epitélio do que o grupo controle. A altura do epitélio seminífero é uma característica efetiva para a avaliação da produção espermática, entretanto nesse estudo não houve diferença significativa na concentração espermática entre grupo controle e tratamento demonstrando que essa diferença não afetou a produção espermática. Dois dos três animais castrados do grupo gossipol foram utilizados para um teste de fertilidade, onde cobriram 10 ovelhas férteis durante 90 dias, resultando em 6 prenheses positivas. Os resultados da pesquisa mostram que a suplementação com caroço de algodão não influenciou a qualidade espermática, a morfometria testicular, a capacidade fertilizante dos espermatozoides e a morfologia dos túbulos seminíferos.