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Influência da qualidade do sêmen criopreservado equino sobre a taxa de prenhez, hemodinâmica uterina e endometrite pós-cobertura / Influence of equine cryopreserved semen quality on pregnancy rate, uterine hemodynamic and post breeding endometritisRecalde, Elena Carolina Serrano 30 June 2014 (has links)
As condições do trato reprodutivo da fêmea, assim como a qualidade do sêmen são fatores que interferem na fertilidade. Os objetivos deste trabalho foram avaliar a resposta inflamatória uterina e a taxa de prenhez em éguas, considerando hemodinâmica uterina e citologia endometrial após a inseminação artificial (IA) com sêmen congelado de alta e baixa qualidade. Para o presente estudo foram realizados dois experimentos. No Experimento 1 foram utilizadas 15 éguas distribuídas de forma aleatória em quatro grupos: CT - controle: mimetização do procedimento de IA (n=7), DIL: infusão intrauterina de diluidor a base de leite desnatado (n=7), ALTA: IA com sêmen de alta qualidade (n=7), e BAIXA: IA com sêmen de baixa qualidade (n=7). A avaliação uterina foi realizada por ultrassonografia transretal com Doppler nos modos Espectral e Color-flow em sete momentos: prévio à indução da ovulação (TIO), imediatamente antes da IA (TIA), 2 (T2), 6 (T6), 12 (T12), 24 (T24) e 48 horas (T48) após a IA. Foram considerados os valores de índice de resistência (RI) da artéria uterina e de escore de vascularização (EV) uterino. A citologia uterina foi realizada 6 h após a IA. No Experimento 2 foram utilizadas 12 éguas, e seus ciclos foram distribuídos nos tratamentos: CT - controle: mimetização do procedimento de IA (n=8), DIL: infusão intrauterina de diluidor (n=8), ALTA: IA com sêmen de alta qualidade (n=8), e BAIXA: IA com sêmen de baixa qualidade (n=8). O delineamento experimental foi em Quadrado Latino 4X4. A avaliação uterina foi realizada por ultrassonografia transretal com Doppler modos Espectral e Color-flow em três momentos: prévio à indução da ovulação (TIO), imediatamente antes da IA (TIA) e 6 horas (T6) após a IA. A citologia uterina foi realizada 6 h após a IA. Neste experimento foi considerada a taxa de prenhez comparando-se os ciclos utilizados para os grupos ALTA e BAIXA. O diagnóstico de gestação foi realizado 14 dias após a ovulação. Foi utilizado o procedimento misto (PROC MIXED) do SAS (Versão 9.3) para a análise estatística e foi considerada diferença significativa quando p ≤0,05. No Experimento 1, não foi encontrada diferença estatística entre os grupos para os valores de hemodinâmica uterina RI e EV. Houve aumento significativo de células inflamatórias no endométrio de éguas inseminadas com sêmen de baixa qualidade, porém não diferiu do grupo de éguas inseminadas com sêmen de alta qualidade. No Experimento 2, não houve diferença estatística entre os grupos para os valores de RI, mas se observou maior EV uterino nos grupos inseminados com sêmen de alta e de baixa qualidade quando comparados com o grupo controle. Na citologia uterina não foi encontrada diferença estatística. A taxa de prenhez para os grupos ALTA (81,82%) e BAIXA (54,55%) não foi estatisticamente diferente. Conclui-se que a deposição de sêmen no útero, leva a um processo inflamatório do endométrio, e esta pode alterar a hemodinâmica uterina detectável por ultrassonografia Doppler em éguas. Não existe diferença significativa na resposta inflamatória entre as qualidades de sêmen. A inseminação com sêmen congelado de menor qualidade não altera a taxa de prenhez, mas são necessários mais estudos para verificar esta diferença. / The female reproductive tract soundness, as well as semen quality are factors that interfere in fertility. The aims of this study were to evaluate inflammatory uterine response and pregnancy rate in mares, considering uterine hemodynamics and endometrial cytology after artificial insemination (IA) with frozen semen of high and low quality. Two experiments were executed for the present study. For Experiment 1, fifteen mares were randomly distributed between four groups: CT - control: mimic of the procedure of IA (n=7), DIL: intrauterine infusion of skim milk semen extender (n=7), ALTA: IA with high quality semen (n=7), and BAIXA: IA with low quality semen (n=7). Uterine evaluation was done using transrectal Doppler ultrasonography by Spectral and Color-flow modes on seven moments: immediately before ovulation induction (TIO), immediately before AI (TIA), 2 (T2), 6 (T6), 12 (T12), 24 (T24) and 48 (T48) hours after IA. There were considered the numerical values of resistance index (RI) of uterine arteries and vascularity scores (EV) of uterine horns. Endometrial cytology was done at 6 h after IA. On Experiment 2, were used 12 mares which their cycles were distributed between the treatments: CT - control: only mimic of IA procedure (n=8), DIL: intrauterine infusion of semen extender (n=8), ALTA: IA with high quality semen (n=8), and BAIXA: IA with low quality semen (n=8). The experimental design was a Latin Square. Uterine evaluation was done using transrectal Doppler ultrasonography by Spectral and Color-flow modes on three moments: before ovulation induction (TIO), immediately before AI (TIA) and 6 hours (T6) after IA. Endometrial cytology was done at 6 h after IA. In this experiment it was considered the pregnancy rate comparing the cycles for groups ALTA e BAIXA. The pregnancy diagnosis was done 14 days after ovulation. It was used the Mixed Procedure (PROC MIXED) from SAS (Version 9.3) for statistical analysis and significant difference was considered when p≤0.05. On Experiment 1, no statistical difference was found between the groups for uterine hemodynamic values RI and EV. There was a significant increase of inflammatory cells on the mares inseminated with low quality semen, however it wasnt different from the group if mares inseminated with high quality semen. On Experiment 2, there was no statistical difference between the groups for RI values, but the uterine EV was higher for the groups inseminated with high and low quality when compared with the control group. There was no statistical difference on endometrial cytology. There was no statistical difference on pregnancy rate for the groups ALTA (81,82%) and BAIXA (54,55%). As a conclusion, the deposition of semen in the uterus causes an inflammatory process on the endometrium, and it could alter uterine dynamics detected by Doppler ultrasonography in mares. It does not exist significant difference on the inflammatory response between semen qualities. Low quality frozen-semen insemination does not alter pregnancy rate, but more studies are necessary in order to verify this difference.
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Investigação da hidrólise enzimática de derivados da quinizarina por espectroscopia e microscopia de fluorescência / Enzymatic hydrolysis of quinizarin diester investigated by spectroscopy and microscopy fluorescenceSabatini, Carolina Aparecida 13 September 2012 (has links)
A cinética enzimática dos derivados de quinizarina com cadeias homólogas por lipases imobilizadas foi investigada por espectroscopia de fluorescência. Este estudo foi realizado em nível macroscópico e microscópico. Para o estudo macroscópico, foi utilizada a lipase suportada CALB (Novozyme® 435) e para o estudo microscópico a lipase Rhizopus niveus imobilizada em nanopartículas de sílica. Os derivados de quinizarina são espécies que não apresentam fluorescência, porém, quando são hidrolisados, tornam-se fluorescentes (quinizarina). Com um modelo cinético considerando um mecanismo de dois processos sequenciais do tipo Michaelis-Menten, foi possível fazer uma descrição adequada da evolução temporal da formação da quinizarina. O tempo médio de reação da hidrólise enzimática, em nível macroscópico, foi determinado para os derivados diacetato, dibutirato, dihexanoato e dioctanoato de quinizarina nos solventes hexano, ciclo-hexano e decalina saturados com água. No estudo microscópico, a lipase de Rhizopus niveus foi incorporada em nanopartículas de sílica de 200nm. A hidrólise enzimática foi monitorada por imagens e pela flutuação da intensidade de fluorescência com o tempo, por meio da microscopia de fluorescência confocal. Os resultados mostraram que, após a adição do substrato (derivados da quinizarina), começam a aparecer regiões fluorescentes devido ao trabalho enzimático (formação da quinizarina). As imagens de microscopia de fluorescência confocal não mostraram uma nítida diferença entre os substratos avaliados. Entretanto, o estudo da flutuação da intensidade de fluorescência mostrou que há uma diferença entre os substratos e que é possível estimar constantes de tempo de relaxação da atividade enzimática. Além disso, a atividade da lipase depende da forma em que a mesma está distribuída nas nanopartículas (ligada ou adsorvida) e também do tamanho da cadeia de alquílica que compões os derivados. O decaimento de fluorescência da quinizarina produzida pela hidrólise dos derivados pela lipase foi adquirido por microscopia de fluorescência confocal usando excitação de 2-fótons. / The kinetics of enzymatic hydrolysis of quinizarin diester by supported lipase dispersed beads in organic solvents was investigated by fluorescence spectroscopy. This study was performed on macroscopic and microscopic levels. For the macroscopic study was used CALB immobilized lipase (Novozyme ® 435) on acrylate beads, and for microscopic study Rhizopus niveus lipase immobilized on silica nanoparticles. The quinizarin derivatives (substrates) are non-fluorescent species, and only the end product quinizarin has fluorescence. A kinetic model considering two sequential Michaelis-Menten mechanisms provides a suitable description of the time evolution of the quinizarin formation monitored by emission spectroscopy and photon counting measurements. The average reaction time of the enzymatic hydrolysis was determined for quinizarin diacetate, dibutirate, dihexanoate and dioctanoate in hexane, cyclohexane and decaline water saturated solvents. In the microscopic study, the Rhizopus niveus lipase was dispersed into and bound silica mesoporous 200nm particles. In both systems, dispersed silica nanoparticles and a small fraction of aggregates are found in thin film. The enzyme activity was monitored by images and fluctuations of fluorescence intensity over time using confocal fluorescence microscopy. The results showed that after addition of substrate fluorescent spots due to enzyme activity start to appear. Confocal fluorescence images showed no clear difference among substrates. However, the study of fluorescence intensity fluctuations showed that enzyme activity depends on the type of substrate and enzyme support. In addition, the lipase activity depends on the form in which it is distributed in the nanoparticles (bound or entrapped) and the size of the alkyl diester derivatives. The fluorescence decay of quinizarin produced by lipase hydrolysis of diester was measured by confocal fluorescence microscopy using 2-photon pulse excitation.
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Uso da melatonina e do ácido ferúlico como promotores da função do espermatozoide equino criopreservado / Use of melatonin and ferulic acid as promoters of cryopreserved equine spermRenata Lançoni 22 May 2015 (has links)
As espécies reativas de oxigênio (ROS) podem ser responsáveis por causar danos às membranas dos espermatozoides, fragmentação de DNA, entre outros fatores, influenciando assim na fertilidade principalmente no processo de criopreservação do sêmen. A melatonina (MEL) e o ácido ferúlico (AF) são potentes agentes antioxidantes que poderiam atuar no controle da produção de ROS no sêmen equino. Este estudo teve como objetivo avaliar o efeito dos antioxidantes AF e MEL na criopreservação do sêmen equino. Foram utilizados 5 ejaculados de 4 garanhões. Dentre os tratamentos aplicados, foram utilizadas duas concentrações de cada antioxidante (AF 0,5mM, AF 1,2mM, MEL 2mM e MEL 1µM) além do controle (diluidor de congelação convencional BotuCrio®), totalizando 5 tratamentos. As variáveis analisadas foram cinética espermática pelo sistema CASA (programa SCA - Sperm Class Analyser), morfologia, integridade de membranas plasmática, acrossomal e potencial de membrana mitocondrial, com o uso das sondas fluorescentes PI, Hoescht 33342, FITC-PSA e JC-1 além da produção de (ROS) pelo espermatozoide com a sonda fluorescente CellRox Deep Red®. Comparações entre os tratamentos foram realizadas pelo modelo linear generalizado (PROC GLM) do SAS (Versão 9.3) e as diferenças entre eles foram localizadas através do teste de Duncan. A probabilidade de P0,05 foi considerada como diferença significativa. Os resultados para características da motilidade tiveram diferença significativa em alguns aspectos, porém nenhum tratamento foi superior ao controle. Houve diminuição no percentual de defeitos maiores nas amostras tratadas com AF 1,2mM, MEL 2mM e MEL 1µM comparadas ao grupo controle. No que diz respeito à integridade de membranas, o tratamento MEL 1µM apresentou porcentagens significativamente melhores nas células com membrana plasmática intacta, acrossomo intacto e alto potencial de membrana mitocondrial, quando comparadas ao grupo controle. As células em estresse oxidativo não se diferenciaram entre os tratamentos. O uso da sonda fluorescente CellRox Deep Red® foi validado para espermatozoides de equinos. Foram utilizados 4 ejaculados de 4 garanhões aos quais eram submetidos aos tratamentos T0 (fração do ejaculado não submetida à indução do estresse oxidativo), T50 (50% da amostra não induzida e 50% induzida ao estresse oxidativo) e T100 (amostra induzida ao estresse oxidativo). Os dados de porcentagem de células positivas (com estresse oxidativo) foram submetidos à análise de regressão polinomial pelo modelo linear generalizado (PROC GLM) do SAS (Versão 9.3). O valor do coeficiente de determinação (R2) foi igual a 0,88 e a probabilidade de P0,05 foi considerada significativa. Pode-se concluir que o tratamento MEL 1µM contribui para a preservação da integridade de membranas espermáticas durante o processo de criopreservação do sêmen equino e que a sonda fluorescente CellRox Deep Red® é eficiente na detecção de espécies reativas de oxigênio no espermatozoide de garanhões. / Reactive oxygen species (ROS) can be responsible for causing damage to the membranes of sperm, DNA fragmentation, among other factors influencing fertility especially in cryopreservation. Melatonin (MEL) and ferulic acid (FA) are potent antioxidants that could act in the control of ROS production in equine semen. This study aimed to evaluate the effect of antioxidants AF and MEL in cryopreservation of equine semen. Five ejaculates from four stallions were used. Among the treatments, we used two concentrations of each antioxidant (AF 0.5mM, AF 1.2mM, MEL 2 mM and MEL 1µM) beyond the control (conventional freezing extender BotuCrio®), totaling five treatments. The parameters analyzed were sperm kinetics with the CASA system (SCA program - Sperm Class Analyzer), morphology, plasma and acrossomal membrane integrity mitochondrial membrane potential, using fluorescent probes PI, Hoechst 33342, FITC-PSA and JC- 1 over production ROS by the sperm with the fluorescent probe CellRox Deep Red®. Comparisons between treatments were performed by generalized linear model (PROC GLM) of SAS (version 9.3) and the differences between them were located with the Duncan test. The probability of P0.05 was considered significant. The results for the motility characteristics were significant differences in some aspects, but no treatment was superior to the control. There was a decrease in the percentage of major defects in the samples treated with AF 1.2mM, MEL 2 mM and MEL 1µM compared to the control group. Regarding to membrane integrity, treatment MEL 1µM showed significantly better in percentages of cells with intact plasma membrane, intact acrosome and high mitochondrial membrane potential compared to the control group. Cells with oxidative stress not differ between treatments. The fluorescent probe CellRox Deep Red® was validated for equine sperm previously. Was used ejaculates of 4 stallion which were subjected to the treatments T0 (fraction of the ejaculate not subjected to induction of oxidative stress), T50 (50% sample uninduced and 50% induced to oxidative stress) and T100 (sample induced to oxidative stress). The data of percentage of positive cells (with oxidative stress) were submitted to polynomial regression analysis based on generalized linear model (GLM PROC) of SAS (version 9.3). The value of the coefficient of determination (R2) was 0.88 and a probability of P0.05 was considered significant. It can be conclude that the treatment MEL 1µM contributes to the preservation of the integrity of sperm membranes during the equine sperm cryopreservation process and the fluorescent probe CellRox Deep Red® is efficient in the detection of reactive oxygen species in stallions sperm.
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Tratamento da degeneração testicular em carneiros com suplementação de vitamina A ou laserterapia de baixa intensidade / Treatment of testicular degeneration in rams supplemented with vitamin A or low level laser therapyMaíra Bianchi Rodrigues Alves 30 May 2014 (has links)
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.
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Relação da qualidade do sêmen com a fertilidade após IATF em vacas de corte / Relationship of semen quality to fertility after TAI in beef cowsFelipe Barbosa dos Santos 09 December 2016 (has links)
A criopreservação do sêmen resulta em danos à estrutura espermática, sendo nítida a importância da avaliação das partidas de sêmen antes de serem submetidas à inseminação artificial em tempo fixo (IATF). Todavia, nem sempre as avaliações convencionais do sêmen são suficientes para identificar partidas que possam resultar em baixa taxa de prenhez no campo, sendo necessária uma investigação mais profunda e acurada. Neste sentido, este experimento foi realizado com o objetivo de identificar partidas de sêmen que apresentam falhas na fertilidade, mesmo sendo aprovadas pelas avaliações convencionais. Foram realizadas análises convencionais (motilidade, vigor, concentração e morfologia espermática) de 72 partidas de sêmen de 22 touros antes da estação de monta. Destas, 55 partidas de 18 touros foram aprovadas para o uso na IATF, mas somente 28 partidas de 10 touros foram utilizadas. As partidas de sêmen utilizadas na IATF foram submetidas a outras avaliações: análise computadorizada da motilidade espermática (CASA), integridade das membranas plasmática e acrossomal e potencial de membrana mitocondrial (por sondas fluorescentes em microscopia de epifluorescência). Os dados foram analisados pelo Proc Mixed do SAS usando o Test T. As taxas de prenhez das diferentes partidas de sêmen variaram de 71 a 37%, sendo a média e desvio padrão das partidas de 55,57±7,57%. Os dados de fertilidade a campo permitiram a separação das partidas de sêmen como de Alta (>50% de prenhez) e Baixa (50% de prenhez) fertilidade, sendo comparadas quatro partidas de Alta e quatro de Baixa fertilidade. Os dados das características seminais de todas as partidas de sêmen foram submetidos à análise por boxplot e separados em quartis superior e inferior. Quando se comparou as partidas de Alta e Baixa fertilidade notou-se diferença na taxa de prenhez (p<0,01), mas não foi notada diferença para motilidade (p=0,91), vigor (p=0,63), concentração (p=0,27), número de espermatozoides por palheta (NEP, p=0,27), número de espermatozoides móveis e normais por palheta (p=0,18), defeitos maiores (p=0,17), defeitos totais (p=0,43), integridade de membrana plasmática (MPI, p=0,07), alto potencial de membrana mitocondrial (AP, p=0,94), motilidade total (MT, p=0,10), VCL (p=0,80), VSL (p=0,75), VAP (p=0,88), LIN (p=0,78), STR (p=0,71) e BCF (p=0,13). No entanto, foram encontradas diferenças entre os grupos (Alta e Baixa) para defeitos menores (p=0,05), espermatozoides com integridade das membranas plasmática e acrossomal e função mitocondrial (PIAIA)/Palheta (p=0,01), integridade de acrossomo (AI, p=0,03), motilidade progressiva (MP, p<0.01) e rápidos (p<0,01). Quando comparados os quartis superior e inferior das características seminais foram encontradas diferenças para concentração espermática (p<0,01), NEP (p<0,01), número de espermatozoides móveis por palheta (p<0,01), espermatozoides móveis e normais (p<0,01), defeitos maiores (p<0,01), defeitos menores (p=0,01), defeitos totais (p=0,05), MPI (p<0,01), AI (p<0,01), AP (p=0,03), PIAIA (p<0,01), PIAIA/palheta (p<0,01), mas esta divisão de grupos por quartis superior e inferior não apresentaram diferença sobre a fertilidade (p>0,05); sendo que somente. Entretanto, para MT (p<0,01) e MP (p<0,01) além da diferença entre os quartis foi notado efeito da fertilidade (MT, p=0,05 e MP, p=0,01), sendo maior para os de baixa fertilidade. Pode-se concluir que os padrões de qualidade de partidas de sêmen que apresentam fertilidade distinta podem ser semelhantes, além disso, que pode haver diferença na qualidade espermática entre as partidas que não influenciam a fertilidade. Desta forma, são necessárias outras análises mais acuradas para investigar as causas de falha na fertilidade de algumas partidas de sêmen. / Semen cryopreservation results in damage to sperm structure, and it is clear the importance of evaluating the semen batches before submitted it to timed artificial insemination (TAI). However, conventional semen evaluations are not always sufficient to identify batch that may result in reduced pregnancy rate in the field, requiring a more thorough and accurate investigation. Thus, this experiment was conducted in order to identify semen batches that have gaps in fertility, even being adopted by conventional assessments. Conventional analysis (motility, vigor, concentration and morphology) were performed of 72 semen batches from 22 bulls before the breeding season. Of these, 55 batches from 18 bulls were approved for use in TAI, but only 28 batches from 10 bulls were used. Semen batches used in TAI were subjected to further assessment: computer-assisted sperm analysis (CASA), integrity of plasma and acrosomal membranes and mitochondrial membrane potential (by fluorescent probes under epifluorescence microscopy). Data were analyzed by Proc Mixed of the SAS using Test \"T\". Pregnancy rates of different semen batches ranged 71-37%, and the mean and standard deviation of the batches was 55.57 ± 7.57%. Field fertility data allowed the separation of the semen batches as \"High\" (>50% pregnancy rate) and \"Low\" (50% pregnancy rate) fertility. It was compared four batches of \"High\" and four batches of \"Low\" fertility. Data of the seminal characteristics of all the semen batches were analyzed by boxplot and separated into upper and lower quartiles. When comparing the batches of \"High\" and \"Low\" fertility was noticed a difference in the pregnancy rate (p<0.01), but was not noticeable difference in motility (p=0.91), vigour (p=0.63), concentration (p=0.27), number of spermatozoa per straw (NEP, p=0.27), number of motile and normal sperm per straw (p=0.18), major defects (p=0.17), total defects (p=0.43) plasma membrane integrity (MPI, p=0.07), high mitochondrial membrane potential (AP, p=0.94), total motility (TM, p=0.10), VCL (p=0.80), VSL (p=0.75), VAP (p=0.88), LIN (p=0.78), STR (p=0.71) and BCF (p=0.13). However, differences were found between the groups (\"High\" and \"Low\") for minor defects (p=0.05), sperm with plasma and acrosomal membranes integrity and high mitochondrial membrane potential (PIAIA)/straw (p=0.01), acrosomal integrity (Al, p=0.03) progressive motility (PM, p <0.01) and rapid (p<0.01). When comparing the upper and lower quartiles of the seminal characteristics differences were found for sperm concentration (p <0.01), NEP (p <0.01), number of motile sperm per straw (p<0.01), motile and normal sperm (p<0.01), major defects (p<0.01), minor defects (p=0.01), total defects (p=0.05), MPI (p<0.01), AI (p<0.01), AP (p=0.03), PIAIA (p<0.01), PIAIA/straw (p<0.01), but this division groups by upper and lower quartiles showed no difference in fertility rate (p>0.05). However, to MT (p<0.01) and MP (p<0.01) the difference between quartiles of fertility effect was noted (MT, p=0.05 and MP, p=0.01), been higher for low fertility. It can be concluded that patterns of semen quality of batches that have distinct fertility may be similar, furthermore, there may be differences in sperm quality among batches that do not affect fertility. Thus, it takes other more accurate analysis to investigate the causes of failure on fertility of some semen batches.
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Highly Multiplexed Single Cell in situ Protein Analysis with Cleavable Fluorescent ProbesJanuary 2019 (has links)
abstract: Measurements of different molecular species from single cells have the potential to reveal cell-to-cell variations, which are precluded by population-based measurements. An increasing percentage of researches have been focused on proteins, for its central roles in biological processes. Immunofluorescence (IF) has been a well-established protein analysis platform. To gain comprehensive insights into cell biology and diagnostic pathology, a crucial direction would be to increase the multiplexity of current single cell protein analysis technologies.
An azide-based chemical cleavable linker has been introduced to design and synthesis novel fluorescent probes. These probes allow cyclic immunofluorescence staining which leads to the feasibility of highly multiplexed single cell in situ protein profiling. These highly multiplexed imaging-based platforms have the potential to quantify more than 100 protein targets in cultured cells and more than 50 protein targets in single cells in tissues.
This approach has been successfully applied in formalin-fixed paraffin-embedded (FFPE) brain tissues. Multiplexed protein expression level results reveal neuronal heterogeneity in the human hippocampus. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2019
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Développement de conjugués peptidiques fluorocarbonés pour augmenter la stabilité plasmatique de peptides visant des récepteurs couplés aux protéines G / Development of fluorocarbon peptide conjugates to increase the plasma stability of peptides targeting GPCRsEsteoulle, Lucie 05 October 2018 (has links)
Afin d’améliorer la stabilité plasmatique de peptides, nous avons développé une nouvelle stratégie basée sur l’introduction d’une chaîne fluorocarbonée dans la séquence d’un peptide natif. En appliquant le concept à l’apeline-17, un peptide présentant un intérêt potentiel pour le traitement de maladies cardiovasculaires, nous avons amélioré sa stabilité plasmatique de 4 min à plus de 24 h ainsi que son efficacité in vivo. L’étude du mécanisme de stabilisation a permis de mettre en évidence la liaison de la fluoroapeline à l’albumine, conduisant à la protection du peptide vis-à-vis de la protéolyse. Le concept a été appliqué à d’autres peptides tels que l’apeline-13, l’angiotensine II, l’ocytocine et la spexine, démontrant ainsi l’étendue et les limitations de la méthode. Enfin, nous avons également conçu des sondes fluorescentes « turn-on » originales capables de révéler leur fluorescence uniquement après liaison au récepteur ciblé. Ces sondes pourront nous servir, par la suite, pour l’étude in vivo de la biodistribution des fluoropeptides. / In order to improve the plasma stability of peptides, we have developed a new strategy based on the introduction of a fluorocarbon chain in the sequence of a native peptide. By applying this concept to apelin-17, a peptide showing a potential interest for the treatment of cardiovascular diseases, we have improved its plasma stability from 4.6 min to more than 24 h as well as its in vivo efficacy. The mechanism leading to the increase of plasma stability has been carefully investigated demonstrating the binding of the fluoroapeline to the albumin, leading to protection towards roteolysis. The concept has been applied to other peptides such as apelin-13, angiotensin II, oxytocin and spexine, showing the extension and the limitations of this method. Finally, we have designed original fluorescent fluorogenic probes which turn on their fluorescence only after binding to the targeted receptor. These probes could be used for in vivo biodistribution studies of fluoropeptides.
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Visible Light-Triggered Carbon Monoxide-Releasing MoleculesPopova, Marina 01 May 2019 (has links)
Carbon monoxide (CO) is now well established as one of the signaling molecules in higher organisms, including humans. Due to its physiological roles, CO is now accepted as a potential therapeutic agent. The use of CO gas has been studied in multiple clinical trials. Vasodilation, anti-inflammatory, anti-apoptotic, anti-proliferative and cytoprotective effects are just a few of the pharmacological actions attributed to CO gas in various models of diseases. Use of inhaled CO gas as a therapeutic has many limitations which necessitate the development of a new approach for CO delivery. In order to handle CO safely, compounds that release CO (CO-releasing molecules, CORMs) have been developed. CORMs that release CO only when triggered, and with the ability to target certain tissue sites, are of particular interest. Our lab is developing molecules that release CO only when illuminated with visible light (photoCORMs). These photoCORMs are based on a motif found in naturally-occurring flavonols, which are chemical compounds found in wide variety of foods including fruits, vegetables, tea and dark chocolate.
The research presented in this dissertation outlines the results of studies on extended flavonols as CO release agents. The specific studies described herein focus on understanding visible light-induced CO-releasing flavonols in terms of their: a) structure/reactivity relationships, especially in biological environments; b) interactions with metal ions and proteins; c) reaction pathway of CO release; and d) their properties when combined with a CO-sensing motif.
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The preparation and evaluation of N-acetylneuraminic acid derivatives as probes of sialic acid-recognizing proteinsCiccotosto, Silvana January 2004 (has links)
Abstract not available
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Development of Dynamic DNA Probes for High-Content in situ Proteomic AnalysesSchweller, Ryan 06 September 2012 (has links)
Dynamic DNA complexes are able to undergo multiple hybridization and dissociation events through a process called strand displacement. This unique property has facilitated the creation of programmable molecular detection systems and chemical logic gates encoded by nucleotide sequence. This work examines whether the ability to selective exchange oligonucleotides among different thermodynamically-stable DNA complexes can be harnessed to create a new class of imaging probes that permit fluorescent reporters to be sequentially activated (“turned on”) and erased (“turned off”). Here, dynamic DNA complexes detect a specific DNA-conjugated antibody and undergo strand displacement to liberate a quencher strand and activate a fluorescent reporter. Subsequently, incubation with an erasing complex allows the fluorophore to be stripped from the target strand, quenched, and washed away. This simple capability therefore allows the same fluorescent dyes to be used multiple times to detect different markers within the same sample via sequential rounds of fluorescence imaging.
We evaluated and optimized several DNA complex designs to function efficiently for in situ molecular analyses. We also applied our DNA probes to immunofluorescence imaging using DNA-conjugated antibodies and demonstrated the ability to at least double the number of detectable markers on a single sample. Finally, the probe complexes were reconfigured to act as AND-gates for the detection of co-localized proteins. Given the ability to visualize large numbers of cellular markers using dynamic DNA probe complexes, high-content proteomic analyses can be performed on a single sample, enhancing the power of fluorescence imaging techniques. Furthermore, dynamic DNA complexes offer new avenues to incorporate DNA-based computations and logic for in situ molecular imaging and analyses.
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