Dissertations / Theses on the topic 'Ram Semen'

A theoretical study showed that Al can greatly affect the efficiency of sheep breeding schemes provided fertility is maintained at the highest levels. Factors that affect the survival of ram spermatozoa during preservation were studied. A pH range between 6 and 7 was well tolerated. The addition of 4% (v/v) glycerol to the diluted ram semen in Tris buffer lowered the motility and survival of spermatozoa during 5 hours of storage at 30'C. Following insemination of chilled ram semen, with and without glycerol in the diluent, lambing percentages of 59% and 73% respectively were obtained. Ram semen was frozen in 0.25 ml straws using various cooling combinations. The optimal procedure was found to be to cool rapidly from 5'C to -120'C at -20'C/min. When semen so treated was compared in a fertility trial with semen frozen by the pellet method of Evans and Maxwell (1987), lambing percentages of 14% and 18% respectively were obtained. Attempts were made to formulate a vitrifying diluent for ram semen. A method was developed for the assessment of semen in highly concentrated cryoprotective solutions. Semen tolerated 10% concentrations of each of glycerol, acetamide and propylene glycol applied together, but when concentrations were raised above this level sperm mortality was very high. A simple spectrophotometric procedure for the objective assessment of vigour of ram semen was developed and tested. Raffinose 66 mM in the freezing diluent improved the post-thawing revival rate of spermatozoa from 46% to 71%, and increased the post-thawing recovery of the swimmingup vigour (P< 0.01). Raffinose treatment reduced the ATP content of semen but did not reduce the rate of glucose oxidation by diluted spermatozoa at either the pre-freezing or post-thawing stages. Frozen storage of ram spermatozoa as pellets was best achieved using two volumes of Tris buffer diluent containing 18% (v/v) egg-yolk, 6% (v/v) glycerol and 66 mM raffinose to one volume of semen. The diluted semen was chilled to 5'C and frozen as 0.10 ml pellets on dry ice. For frozen storage of ram semen in 0.25 ml straws, best results were obtained when the Tris buffer diluent contained 18% (v/v) egg-yolk, 9% (v/v) glycerol and 66 mM raffmose, and cooling was at a rate of -30'C/min from 5'C to -120'C. Non-return rates were 21%, 20% and 31% for ewes inseminated with semen samples frozen as standard 0.20 ml pellets, as raffinose containing 0.10 ml pellets, and as raffinose containing 0.25 ml straws respectively. Of the in vitro tests, only the swim-up test was correlated with non-return rates (r=0.904, P< 0.1). Post-thawing survival of the spermatozoa was improved by the addition of raffinose which had no deleterious effect on fertility.

Bibliography: leaves 267-292. This study aimed at finding better ways of storing ram semen at refrigerator or room temperature with particular reference to ingredients readily available in Indonesia, namely coconut extract and quail egg yolk. Coconut extract showed consistent advantages with regard to sperm motility and quail egg yolk was as effective as hen egg yolk. Investigations were extended to examine storage for subnormal semen such as would be produced during periods of heat stress. Motility was assessed visually and using a Hamilton Thorn semen evaluation apparatus.

Includes bibliographical references (leaves 274-316) Examines several aspects of male reproduction in the sheep, and how these are related to fertility in the female when semen is introduced by natural mating or artificial insemination.

Com os processos de preservação do sêmen, os espermatozoides sofrem danos devido à indução de alterações estruturais e funcionais na membrana e, aliada a estrutura anatômica do cérvix da ovelha, repercute na redução de fertilidade após a inseminação artificial. Muitos protocolos de congelação e de refrigeração aliados a inúmeros diluentes, vêm sendo estudados, a fim de diminuir os danos causados a essas células e aumentar sua capacidade fertilizante. Para otimizar os processos de congelação e de refrigeração, os objetivos deste estudo foram determinar (1) a influência do método de congelação na motilidade, viabilidade, potencial de membrana mitocondrial e nos defeitos acrossomais do semen ovino criopreservado e (2) a influência de três diluentes comerciais na motilidade, viabilidade, potencial de membrana mitocondrial e nos defeitos acrossomais do sêmen ovino refrigerado. Doze ejaculados foram coletados, diluídos e congelados utilizando (A) uma curva manual de congelação (em vapor de nitrogênio liquido) e (B) um congelador programável (CL-8800, Cryologic Freeze Control®). Na curva A, as amostras foram refrigeradas e congeladas à 30°C/min. Na curva B, houve uma redução de temperatura de 20°C até -50°C durante 37min. Imediatamente após as curvas de criopreservação, as amostras foram submersas em nitrogênio líquido (-196ºC). Para o descongelamento, as palhetas foram submetidas à 37°C por 20seg e analisadas. Para os protocolos de refrigeração, o sêmen diluído em Botubov®, Bovimix® e TYB® foi armazenado à 5°C e à 15°C, e avaliado em 0, 24, 48 e 72h pós coleta. As análises de viabilidade e potencial de membrana mitocondrial (MMP) foram realizadas por citometria de fluxo, e a motilidade e os defeitos acrossomais foram avaliados microscopicamente. Motilidade (29%), viabilidade (18%) e MMP (26%) dos espermatozoides criopreservados com o diluente Bovimix® e a curva B foram significativamente (P<0,05) superiores do que todos os outros tratamentos. As amostras refrigeradas e diluídas com o diluente Bovimix® apresentaram resultados superiores, tanto à 5°C quanto à 15°C, para MMP (44% e 51%, respectivamente) e viabilidade (60% e 53%, respectivamente) 72h pós coleta, quando comparado com as amostras diluídas com Botubov® e TYB®. Em conclusão, os protocolos de congelação/descongelação e de refrigeração utilizados nestes experimentos, reduziram a motilidade, viabilidade, MMP e aumentaram os defeitos acrossomais quando comparados com o sêmen fresco. Os protocolos de congelação/descongelação prejudicaram mais a função espermática quando comparado com os protocolos de refrigeração. Por fim, os resultados sugerem que as amostras espermáticas diluídas com o diluente Bovimix® causou menos danos às células espermáticas do que as amostras diluídas com os diluentes Botubov® e TYB®, tanto nos protocolos de congelação, quanto nos protocolos de refrigeração.
The process of sperm preservation damages spermatozoa due to induction of structural and functional changes in the membrane, and allied to the ewe cervix anatomy, reflects the reduction in fertility after artificial insemination. Many semen preservation protocols including freezing and cooling rates coupled with different extenders have been studied in order to reduce sperm cryo-damage, increase the fertilizing capacity and increase the time of storage. To improve freezing and cooling processes, the objectives of this study were to determine (1) the influence of freezing method on sperm motility, viability, mitochondrial membrane potential and defected acrosome of cryopreserved sperm and (2) the influence of three commercial extenders on the motility, viability, mitochondrial membrane potential and defected acrosome of liquid storaged and frozen-thawed sperm. Twelve semen samples were collected from two mature rams, pooled, and diluted in Botubov®, Bovimix® and TYB®, and frozen using (A) a manual freezing method (liquid nitrogen vapor) and (B) an automated programmable freezer (CL-8800, Cryologic Freeze Control®). For curve A, semen samples were maintained at 37°C and then cooled/frozen at 30°C/min. For curve B, the temperature reduced from 20°C to -50°C during 37min. Immediately, after cryopreservation curves, samples were plunged and stored into liquid nitrogen (-196ºC). Then, the straws were thawed at 37°C for 20sec, and analyzed. For cooling studies, pooled ram semen was diluted in Botubov®, Bovimix® and TYB®, stored at 5°C and at 15°C, and evaluated at 0, 24, 48 and 72 h post collection. For viability and mitochondrial membrane potential (MMP) assessments, semen samples were stained and analyzed by flow cytometry. Motility and defected acrosome assessments were analyzed microscopically. Motility (29%), viability (18%) and MMP (26%) of spermatozoa cryopreserved in Bovimix® coupled to curve B were significantly (P<0.05) higher than all other treated groups. Semen samples diluted with Bovimix® and stored at 5°C and at 15°C yielded better MMP (44% and 51%, respectively) than the samples diluted in Botubov® and in TYB®, and also showed higher viability characteristics at 5°C and at 15°C (60% and 53%, respectively) 72h post collection. In conclusion, freezing/thawing and cooling protocols used in these experiments reduced sperm motility, viability, MMP and increased the defects of acrosomes when compared to fresh semen. Freezing/thawing protocols harmed more spermatozoa function than cooling protocols. Furthermore, our results suggest that the semen samples diluted in the extender Bovimix® caused the least cellular injury than the samples diluted in Botubov® and in TYB® on freezing and on cooling protocols.

O melhoramento genético ovino carece de uma maior conexão genética entre os rebanhos, o que pode solucionado pelo uso mais intensivo do sêmen congelado. Entretanto, com os níveis tecnológicos atualmente disponíveis o sêmen congelado apenas pode ser empregado com eficácia diretamente no útero, dependendo de sincronização de estros e mão de obra especializada para as inseminações via laparoscopia. Uma outra alternativa é o uso de sêmen congelado com 200 milhões de espermatozóides por dose com inseminações efetuadas cerca de doze horas após a identificação do estro, preconizado para a inseminação artificial dos ovinos na Noruega pelos próprios produtores. O presente ensaio consta de uma adaptação local deste modelo com o objetivo geral de verificar a viabilidade de uso da inseminação cervical superficial com sêmen ovino congelado numa dose de 0,25 ml contendo 200 x 106 de espermatozóides em palhetas de 0,50 ml após cio natural, empregando baixo uso de insumos e mão de obra semi-qualificada. As observações foram procedidas em duas propriedades no Rio Grande do Sul, incluindo 1419 ovelhas das raças Merino, Ideal e Texel. Os resultados obtidos indicaram que as diferenças nas taxas de não retorno entre carneiros poderá ser minimizada através da utilização de outros métodos de análise e seleção de carneiros mais férteis após os procedimentos de congelamento/descongelamento, e, adicionalmente que a constatação de uma taxa de não retorno entre 25-35% nas distintas condições investigadas, permitem inferir que os sistemas investigados podem ser utilizados para interligar geneticamente rebanhos via uso de sêmen congelado.
A sheep improvement program depends on genetic connexion among flocks, which could be done by extensive use of frozen semen. However, nowadays-available technology just recommends the employment of frozen semen directly inside the uterine ambient through laparoscopy. Alternatively, there is a model recommended for Norway producers including frozen semen with 200 millions of sperms used in cervical superficial inseminations up to twelve hours from oestrus detection. The present essay is an local adaption of this system aiming to verify the viability of the employment of frozen semen for sheep reproduction in a volume of 0,25 ml, with 200 x 106 sperms in 0,50 ml straws, after natural oestrus, using low input resource and semi-qualified artificial insemination technicians. The observations were done in the two properties located at Rio Grande do Sul, including 1419 ewes from Merino, Ideal and Texel breeds. The results obtained indicate that the differences in non return rates among rams could be minimized by the employment of other methods of ram selection before the freezing procedures, and additionally the observed 25-35% non return rates in the distinct conditions investigated, permits to infer that the tested system could be useful to connect genetically the flocks using frozen semen.

En esta tesis doctoral, el objetivo principal fue proporcionar más información sobre los procesos de crioconservación de espermatozoides de Aranesa y Xisqueta así como explorara estrategias para loa mejora de su calidad tras la descongelación previo a su aplicación. Para ello, se evaluó el efecto estacional y la implantación de melatonina en sus características reproductivas. Además, la calidad del semen conservado a 50C durante 24h en un medio de congelación obtenido de diferentes machos fuera y dentro de la época reproductiva fue analizado. El objetivo fue proporcionar información útil sobre el efecto de la refrigeración en la calidad de los espermatozoides mantenidos durante cierto tiempo antes de su crioconservación o como alternativa al semen congelado para su uso en IA. Este estudio demostró que los implantes de melatonina en los machos fuera de la época reproductiva mejoraron casi todos los parámetros espermáticos estudiados durante su conservación mediante refrigeración. Asimismo, en esta tesis, la eficacia de 2 métodos de recogida de semen, electroeyaculación (EE) vs. Vagina Artificial (AV)) sobre la calidad del semen fresco y descongelado de Aranesa ha sido analizada, con el objetivo de conocer la posibilidad de crear de forma rápida un banco de semen, especialmente a partir de un elevado número de machos no entrenados, aportando información esencial acerca de estos métodos de recogida de semen sobre la calidad de los espermatozoides pre- y post-descongelación. Nuestros resultados demuestran la inclusión del método de EE como alternativa viable y más rápida para la recogida de semen dentro del desarrollo del programa de conservación y establecimiento de criobanco de semen de Aranesa. Otro estudio de la presente tesis evaluó la calidad seminal de los espermatozoides ovinos criopreservados con o sin plasma seminal bajo condiciones controladas y sujetos a un test de evaluación termal durante 4h tras la descongelación, siendo el objetivo de este test resistencia el proporcionar un mayor conocimiento sobre su capacidad de supervivencia en el tracto genital femenino y de fecundar al óvulo. Este estudio demostró que la presencia de plasma seminal plasma previa a la congelación fue beneficiosa para el mantenimiento de la motilidad espermática tras la descongelación y muy útil para la creación del banco de semen. Por último, se evaluó también el efecto del lavado por centrifugación de los espermatozoides descongelados y la subsiguiente suplementación con plasma seminal (20%) recogido por EE fuera y dentro de la época reproductiva y por VA durante la estación reproductiva, seguido de un periodo de incubación de 90 min. El objetivo era conocer si la suplementación de las dosis seminales con plasma seminal de distinto origen tras un test de Resistencia térmica a 37oC podría mejorar la calidad y supervivencia de los espermatozoides descongelados durante su paso a lo largo del tracto genital femenino. Este estudio demostró que la adición de plasma seminal, independientemente de su origen, mantiene las funciones espermáticas y características de motilidad durante el periodo de incubación tras la descongelación. De la misma manera, se observó que el lavado por centrifugación o la eliminación del diluyente tras la descongelación no es necesario. Podemos concluir que la aplicación de implantes de melatonina a los sementales durante la época no reproductiva proporcionó un efecto beneficioso en los espermatozoides de morueco durante la refrigeración, la inclusión de la electoeyaculación como método de recogida de semen de la raza Aranesa para el establecimiento de un banco de semen es recomendable, y finalmente, la presencia de plasma seminal previo a la criopreservación o su adición tras la descongelación de los espermatozoides es importante para el mantenimiento de la motilidad y demás funciones espermáticas.
The new interest towards the characterization and conservation of Aranesa and Xisqueta ram breeds was constituted by ex situ programs using techniques such as semen cryopreservation following its ultimate use for artificial insemination. However, there are still concerns due to issues regarding frozen-thawed sperm quality. Therefore, with the present technological advances made on sperm evaluating techniques, a wealth of information on their sperm characteristics may be achieved, improving the application of refrigerated and frozen-thawed semen technology. In this doctoral thesis, the general aim was to provide some more information on Aranesa and Xisqueta sperm processes for cryopreservation as well as exploring some strategies towards improving their post-thawed sperm quality prior to testing for further application. To this regard, we evaluated the seasonal effect and melatonin implantation on their reproductive characteristics. In addition, the quality of sperm stored at 50C for 24h in a cryopreservation media from the various ram donors during a non-breeding and breeding season was determined. The goal was to provide some useful information on the effect of chilled liquid storage on sperm quality held for a period of time prior to cryopreservation or as an alternative to frozen-thawed semen for AI. This study demonstrated that melatonin implantation to rams during the non-breeding season improved almost all sperm parameters studied during chilled liquid semen storage. Furthermore, in this thesis, the efficacy of two collection methods electroejaculation (EE) vs. artificial vagina (AV)) on fresh and post-thawed sperm quality of Aranesa ram sperm. The objective being that for rapid constitution of the sperm bank especially from a large number of untrained males within a short period of time, information on semen recovery methods on pre- and post-thawed sperm quality is essential. Our finding supports the inclusion of EE method as a viable alternative and quicker method for semen collection towards the ongoing conservation program and establishment of Aranesa sperm cryobank. Another study in this thesis evaluated the sperm motility and qualitative characteristics of ram spermatozoa cryopreserved with or without seminal plasma under a controlled condition and subjected to a 4 h post-thawing thermal evaluation test. The goal being that exposing these frozen-thawed sperm to thermal resistance test will provide some knowledge on their survivability in the female genital tract towards fertilizing the ovum. This study demonstrated that the presence of seminal plasma prior to cryopreservation was beneficial in maintaining post thawed sperm motility, and as such, may be useful for ex situ ram sperm cryopreservation towards its use for artificial insemination. The last study in this thesis evaluated the effect of washing frozen-thawed sperm by centrifugation and subsequently supplementing with whole seminal plasma (20%) collected by EE from a non-breeding and breeding season or by AV from a breeding season, following a 90 min incubation period. The aim being that supplementing these sperm samples with different seminal plasma source following a thermal resistance test at 37oC will provide some insight as regards their quality and survivability in the female genital tract. This study demonstrates that supplementing frozen-thawed sperm with seminal plasma irrespective of its source maintained sperm functions and motility characteristics during a post-thawing incubation period. Furthermore, washing by centrifugation or removal of freezing component after thawing was not necessary. We can therefore conclude that melatonin implants on male during a non-breeding season provided a beneficial effect on ram sperm during chilled liquid storage, the inclusion of EE as method for semen collection towards the establishment of a sperm bank is recommendable, and finally, the presence of seminal plasma prior to cryopreservation or its addition to post-thawed sperm was important in maintaining sperm motility and functions.

La finalidad del presente estudio fue evaluar en 2 tiempos la calidad espermática de muestras de semen de ovino tratadas por la técnica de gradiente de densidad con respecto a muestras no tratadas. Se utilizó un total de 102 eyaculados de carnero que presentaron en promedio un volumen de 1.12 ± 0.34 ml, color blanquecino, aspecto cremoso, pH de 6.73 ± 0.25, concentración inicial de 3.54 x 109 ± 5.30 x 108 esp/ml y motilidad masal alrededor del grado 4. Posteriormente, cada eyaculado se dividió en un grupo control y experimental de volúmenes iguales. El semen del grupo control fue diluido con Triladyl® y el experimental, tratado con la técnica de gradiente de densidad. La tasa de recuperación espermática después del tratamiento fue del 31.50 ± 10.89%. Se encontró un aumento significativo (p<0.05) en la motilidad individual progresiva, el porcentaje de espermatozoides vivos y de espermatozoides con membrana intacta en las muestras tratadas con respecto al grupo control (92.07 ± 1.81% vs. 85.74 ± 1.75%, 78.42 ± 5.13% vs. 75.19 ± 4.59%, 78.55 ± 4.34% vs. 73.37 ± 4.48%, respectivamente); así como una disminución significativa (p<0.05) en el porcentaje de espermatozoides anormales en las muestras del grupo experimental con respecto al grupo control (8.41 ± 1.33% vs. 9.94 ± 1.73%). El resto de muestras de ambos grupos fue distribuido en pajillas de 0.25 ml y enfriadas hasta alcanzar los 5°C para su refrigeración por 24 horas. Las muestras tratadas con gradiente de densidad presentaron un aumento significativos (p<0.05), con respecto al grupo control, en motilidad individual progresiva, el porcentaje de espermatozoides vivos y el porcentaje de espermatozoides con membrana intacta (86.94 ± 3.14% vs. 82.44 ± 2.26%, 71.24 ± 4.11% vs. 68.82 ± 4.15%, 70.87 ± 3.11% vs. 67.98 ± 4.42%, respectivamente). En conclusión, la técnica de gradiente favoreció la obtención de un mayor número de espermatozoides vivos, de mejor motilidad y con membrana intacta, así como la disminución del número de espermatozoides anormales tanto en muestras frescas como refrigeradas. The purpose of the present study was to assess at 2 evaluation times the sperm quality from ram semen treated with the density gradient technique and to compare it against non-treated ram semen. A total of 102 ejaculates were evaluated. The mean macroscopic parameters were: volume of 1.12 ± 0.34 ml, creamy white color, creamy-dense appearance, pH of 6.73 ± 0.25, initial concentration of 3.54 x 109 ± 5.30 x 108 spz/ml and mass motility around grade 4. Then, each ejaculate was divided into two equal volume groups: the control and experimental group. For the control group, the sample was diluted in Triladyl®. The experimental group was treated with the density gradient technique. The recovery rate after treatment was 31.50 ± 10.89%.The results showed that, at zero hour, samples treated undergo a significant increase (p <0.05), compared with the control group, in individual progressive motility, percentage of living spermatozoa and the percentage of spermatozoa with intact membrane (92.07 ± 1.81% vs. 85.74 ± 1.75%, 78.42 ± 5.13% vs. 75.19 ± 4.59%, 78.55 ± 4.34% vs. 73.37 ± 4.48%, respectively). There was also a significant decrease (p <0.05) in the percentage of abnormal in samples from the experimental group compared to control group spermatozoa (8.41 ± 1.33% vs. 9.94 ± 1.73%). The remaining samples, from both the control and experimental group, were distributed in 0.25 ml straws and cooled to 5°C for 24 hours refrigeration. Then, the samples were evaluated. After 24 hours at 5°C, the samples treated with the density gradient technique showed significantly greater values (p <0.05), compared with the control group, in individual progressive motility, percentage of living spermatozoa and the percentage of spermatozoa with intact membrane (86.94 ± 3.14% vs. 82.44 ± 2.26%, 71.24 ± 4.11% vs. 68.82 ± 4.15%, 70.87 ± 3.11% vs. 67.98 ± 4.42%, respectively). In conclusion, in the present study, the density gradient technique allowed to obtain better a greater number of living spermatozoa with better motility, intact membrane and with lower abnormalities in fresh and refrigerated samples.

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The low pregnancy rates obtained with artificial insemination using frozen ram semen make its routine use unfeasible. As the cryopreservation process causes injuries in the sperm cells, the development of extenders that include state-of-the art cryoprotectant solutions is justified. This study tested the inclusion of low density lipoprotein (LDL) and trehalose in extenders for freezing ram semen, by evaluating parameters of post-thawing semen quality. In the first experiment, 3 treatments were compared: Tris including 20% egg yolk and 5% glycerol (T1); Tris including 10 mM trehalose (T2); and T1 including 50 mM trehalose (T3). Sperm motility did not differ across treatments (P > 0.05), but T2 presented higher proportion of sperm cells having membrane integrity (P < 0.05). In the second experiment, 4 treatments were compared: Tris including 20% egg yolk (T1); T1 including 5% glycerol (T2); T1 including 100 mM trehalose (T3); and t1 including 100 mM trehalose and 5% glycerol (T4). Sperm motility and membrane integrity were higher for T2, T3 and T4 than for T1 (P < 0.05), but acrossome integrity did not differ among treatments (P > 0.05). In the third experiment, four treatments were compared: Tris including 20% egg yolk and 100 mM trehalose; and T1 with the replacement of egg yolk by LDL including 5% glycerol (T2); 100 mM trehalose (T3); and 100 mM trehalose and 5% glycerol (T4). Sperm motility and membrane integrity were higher for T2 and T3 than for the other treatments (P < 0.05), but there was no further difference among the LDL treatments (P > 0.05). Acrossome integrity did not differ across treatments (P > 0.05). Therefore, the inclusion of LDL and trehalose as cryoprotectants in extenders for frozen ram semen was associated with improvement in post-thawing sperm motility and membrane integrity.
Em ovinos, as baixas taxas de prenhez obtidas com inseminação artificial com sêmen congelado inviabilizam o seu uso como rotina. Provocando o processo de criopreservação lesões nos espermatozóides, o desenvolvimento de diluentes que incluam crioprotetores de excelência se justifica. Este trabalho testou a inclusão da lipoproteína de baixa densidade (LDL) e da trealose em diluentes para congelamento de sêmen ovino, a partir da avaliação de parâmetros de qualidade seminal pós-descongelamento. No primeiro experimento, foram comparados 3 tratamentos: Tris com 20% de gema de ovo e 5% de glicerol (T1); Tris com 100 mM de trealose (T2); e T1 com 50 mM de trealose (T3). A motilidade não diferiu entre os tratamentos (P > 0,05), mas o T2 apresentou maior proporção de gametas com membranas íntegras (P < 0,05). No segundo experimento, foram comparados 4 tratamentos: Tris com 20% de gema de ovo (T1); T1 com 5% de glicerol (T2); T1 com 100 mM de trealose (T3); e T1 com 100 mM de trealose e 5% de glicerol (T4). A motilidade e a integridade da membrana espermática do T2, T3 e T4 foram superiores a do T1 (P > 0,05), mas a integridade do acrossoma não diferiu (P > 0,05) em todos os tratamentos. No terceiro experimento, foram comparados 4 tratamentos: Tris com 20% de gema de ovo e 110 mM de trealose (T1); Tris com 8% de LDL, incluindo 5% de glicerol (T2); Tris com 8% de LDL, incluindo 110 mM de trealose (T3); Tris com 8% de LDL, incluindo 110 mM de trealose e 5% de glicerol (T4). A motilidade dos espermatozóides para o T2 e para o T3 foram superiores aos demais tratamentos (P < 0,05), mas, com relação à integridade de membrana, não houve diferença entre os tratamentos com LDL (P > 0,05). A integridade do acrossoma não diferiu entre os tratamentos (P > 0,05). Portanto, o uso de LDL e trealose como crioprotetores foi associado com melhorias na motilidade e na integridade da membrana espermática, no sêmen ovino, após o descongelamento.

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The expansion of artificial insemination (AI) programs with frozen ram semen has been limited by the need of intrauterine semen deposition and by the inconsistency of the association between the conventional methods used to evaluate semen quality and in vivo fertility. An alternative to improve the efficiency of AI programs would be the profiling of the protein content of the seminal plasma to search for fertility markers. The objective of the present study was to study the protein profile of ram seminal plasma and to evaluate its association with parameters of frozen semen quality. Ejaculates were split in two samples. The first sample was frozen in two extenders: T1 (Tris + egg yolk + glycerol); and T2 (Tris + egg yolk + threalose). The second sample was submitted to unidimensional electrophoresis to identify proteins in the seminal plasma. The presence of the identified proteins was associated with parameters of semen quality: sperm motility and membrane integrity, both prefreezing and post-thawing, and post-thawing acrosome integrity. Post-thawing sperm motility did not differ (P > 0.05) for T1 (18.6 ± 2.3) and T2 (23.4 ± 2.7). Post-thawing sperm membrane integrity was similar (P > 0.05) for T1 (12.6 ± 1.4) and T2 (15.3 ± 1.7). There was also no difference (P > 0.05) between T1 and T2 with regard to postthawing acrosome integrity (23.0 ± 1.5 e 21.3 ± 1.8, respectively). An intra-ram analysis identified 17 proteins associated with the evaluated parameters: 10 of them presented similar associations for distinct rams. In the inter-ram analysis, an 11 kDa band was associated with lower pre-freezing sperm membrane integrity, a 24 kDa band was related to reduced post-thawing sperm motility and membrane integrity, and a 45 kDa band was associated with lower pre-freezing sperm membrane integrity (P < 0.05). Thus, those three protein factors would be potential markers for ram infertility with frozen semen because their absence was associated with improved semen quality, regardless of individual ram effects.
A expansão de programas de inseminação artificial (IA) em ovinos com sêmen congelado tem sido limitada pela necessidade de deposição intrauterina do sêmen e pela associação inconsistente entre as técnicas convencionais de avaliação da qualidade do sêmen e a fertilidade in vivo. Uma das alternativas para incrementar a eficiência de programas de IA seria o mapeamento do conteúdo protéico do plasma seminal, em busca de marcadores para fertilidade. O objetivo do presente trabalho foi estudar o perfil protéico do plasma seminal de machos ovinos e avaliar a sua associação com a qualidade de amostras de sêmen congelado. Os ejaculados dos machos ovinos foram divididos em duas alíquotas. A primeira foi utilizada para criopreservação, em dois diluentes: D1 (tris+gema de ovo+glicerol); e D2 (tris+gema de ovo+trealose). A segunda alíquota foi usada para a busca de proteínas do plasma seminal através da eletroforese unidimensional. A presença ou ausência das proteínas foi associada com parâmetros de qualidade seminal: motilidade e integridade da membrana espermática, ambas pré-congelamento e pósdescongelamento, e integridade de acrossoma pós-descongelamento. A motilidade espermática pós-descongelamento não diferiu (P > 0,05) entre D1 (18,6 ± 2,3) e D2 (23,4 ± 2,7). A integridade da membrana espermática pós-descongelamento foi semelhante (P > 0,05) para D1 (12,6 ± 1,4) e D2 (15,3 ± 1,7). Também não houve diferença (P > 0,05) entre D1 e D2 quanto à integridade do acrossoma pósdescongelamento (23,0 ± 1,5 e 21,3 ± 1,8, respectivamente). A análise intra-machos identificou 17 bandas proteicas associadas com os parâmetros avaliados, sendo que 10 destas associaram-se da mesma maneira, em diferentes machos. Na análise inter-machos, a banda com 11 kDa foi associada com menor integridade da membrana espermática pré-congelamento, a banda com 24 kDa foi associada com a redução na motilidade e na integridade da membrana pós-descongelamento e a banda com 45 kDa foi associada com menor integridade da membrana précongelamento (P < 0,05). Portanto, essas três bandas protéicas seriam potenciais marcadores para infertilidade com sêmen congelado, pois sua ausência foi associada com incremento na qualidade seminal, independentemente do efeito dos machos doadores de sêmen.

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Universidade Estadual Paulista (UNESP)
Várias pesquisas foram desenvolvidas visando a melhoria da congelabilidade do sêmen ovino. Contudo, ainda não houve progressos significativos na fertilidade do sêmen congelado após a inseminação artificial cervical. Diversos sistemas de análise computadorizada do movimento espermático (CASA) têm sido propostos e aplicados na tentativa de quantificar características específicas do movimento espermático, podendo ainda determinar a presença e a cinética das subpopulações de espermatozóides. Muitos testes para avaliar a função espermática foram desenvolvidos, permitindo analisar simultaneamente diferentes aspectos da função espermática. Análise da função mitocondrial oferece uma maneira de acessar a motilidade espermática. Foram objetivos otimizar o sistema CASA na avaliação do sêmen congelado; determinar parâmetros da cinética espermática que expressem analogia com as características da avaliação das membranas plasmática, acrossomal e mitocondrial (IP, FITC-PSA e JC-1; MITO; R-123) e utilizar conjuntamente os parâmetros fornecidos pelo sistema CASA e pelas sondas fluorescentes para agrupar as amostras de maneira qualitativa. Vinte e seis amostras de sêmen congelado de diferentes carneiros foram estudadas pelo CASA obtendo-se para os parâmetros VCL, VAP, VSL, ALH, BCF, LIN, STR, ELONG dados médios e individuais para cada espermatozóide e por sondas fluorescentes para a avaliação simultânea da integridade de membrana plasmática, reação acrossomal e potencial de membrana mitocondrial. Estatisticamente aplicou-se a análise exploratória de técnicas multivariadas obtendo-se três fatoriais sendo o primeiro fator F1 positivo e alto para as variáveis VAP, VSL, STR e LIN, que é interpretado com um fator relacionado à progressividade. Para o segundo fator F2, associam as variáveis VCL, ALH e MT, que representam um fator de deslocamento...
Many researches were developed to improve the ram semen criopreservation. However, no significant advances in the fertility rates with the frozen semen were observed with cervical artificial insemination. Several computer-assisted motility assessments (CASA) systems have been considered and applied in the attempt to quantify specific characteristics of the sperm motion and still them being able to determine the spermatozoa presence and subpopulations kinematics. A large number of sperm functions evaluation had been developed, making it possible to analyze different aspects of the sperm function simultaneously. Analysis of the mitochondrial function offers a way to have access the sperm motility. The aim of this study was to optimize the CASA system in the evaluation of the ram frozen semen; determine parameters of the sperm kinematics that express analogy with the characteristics of the evaluation of plasmatic, acrossomal and mitochondrial membranes (PI, FITC-PSA and JC-1; MITO; R-123) and use CASA parameters together with the fluorescent probes to group samples in a qualitative way. Twenty and six frozen semen samples of different rams were evaluated by CASA system for mean and individual sperm motion parameters VCL, VAP, VSL, ALH, BCF, LIN, STR, ELONG and for fluorescent probes leads for the simultaneous evaluation of the integrity of plasmatic, acrossomal membranes and membrane mitochondrial potential. The statistic applied was multivariate analysis getting to three different factorials. The first factor was positive and high (F1 factor) for variables VAP, VSL, STR and LIN, that were interpreted with the forward displacement. For F2 factor, there were associate variables VCL, ALH and MT that represent the displacement. The F3 factor, whose interpretation has to do with available energy, was associated with variables BCF, MITO and ELONG...(Complete abstract, click electronic address below)

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The objectives of this study were to determine the main bacteria present in ram semen and to evaluate the efficiency of distinct antibiotics in controlling such microorganisms and on sperm viability. Ejaculates were collected from five rams and extended in TRIS-egg yolk. In Experiment 1, the following treatments were tested: control, with no antibiotics; GTLS, with 500 μg/ml of gentamicin, 100 μg/ml of tylosin, 300 μg/ml of lincomycin and 600 μg/ml of spectinomycin; PENSTREP, with 500 μg/ml of penicillin and 100 μg/ml of streptomycin; CEFT, with 50 μg/ml of sodium ceftiofur; and ENRO, with 1000 μg/ml of enrofloxacin. For bacteria identification, samples were collected: the preputial ostium; the artificial vagina; the ejaculates; the semen pool from the five rams; after treatments allocation and stabilization at 5°C; and after thawing. In Experiment 2, variation in the antibiotics concentrations used in Experiment 1 were tested: 25% higher (GTLS+25, PENSTREP+25, CEFT+25 and ENRO+25); 50% higher (GTLS+50, PENSTREP+50, CEFT+50 and ENRO+50); 25% lower (GTLS-25, PENSTREP-25, CEFT-25 and ENRO-25); and 50% lower (GTLS-50, PENSTREP-50, CEFT-50 and ENRO-50). Samples of ram semen were used for counting colony forming units (CFU). The isolated microorganisms were Staphylococcus sp., Klebsiella sp., Corynebacterium sp. and Bacillus sp. The ENRO treatment presented the lowest sperm motility for semen cooled at 5°C and after thawing (P < 0.05), although the post-thawing motility did not differ from that observed for the CEFT and GTLS treatments (P > 0.05). No differences were observed among treatments regarding sperm membrane, acrosome and sperm DNA integrity (P > 0.05). The post-thawing proportion of sperm having DNA integrity was greater for the PENSTREP than for the GTLS treatment (P < 0.05), although sperm DNA integrity may have been impaired by the freezing-thawing process. In experiment 2, the GTLS+50, PENSTREP, ENRO+25 and ENRO+50 treatments presented reduced sperm motility (P < 0.05). All treatments reduced the number of CFU in comparison with the control treatment (P < 0.05). The CFU for the GTLS+50, ENRO+25 and ENRO+50 treatments and the concentrations tested for both those treatments did not differ (P < 0.05), although both were more effective than the PENSTREP and CEFT treatments (P < 0.05). However, the ENRO treatment was associated with reduced sperm motility, especially with high antibiotic concentrations. The treatments did not influence sperm morphology and the integrity of sperm membrane, acrosome and DNA.
Este estudo tem como objetivos determinar os principais gêneros bacterianos presentes no sêmen ovino e avaliar a ação de diferentes antibióticos no controle destes microrganismos e o seu impacto sobre a viabilidade espermática. Todos os ejaculados foram coletados de cinco machos ovinos e diluídos em Tris-gema de ovo. No experimento 1 foram testados os seguintes tratamentos: Controle sem antibiótico; GTLS, com 500 μg/ml de gentamicina, 100 μg/ml de tilosina, 300 μg/ml de lincomicina e 600 μg/ml de espectinomicina; PENSTREP, com 500 μg/ml de penicilina e 100 μg/ml de estreptomicina; CEFT, com 50 μg/ml de ceftiofur sódico; e ENRO, com 1000 μg/ml de enrofloxacina. Para a identificação dos gêneros bacterianos presentes, amostras foram colhidas do óstio prepucial, da vagina artificial, dos ejaculados carneiros, do pool de sêmen dos cinco carneiros e após a inclusão dos tratamentos e estabilização a 5 °C e após o descongelamento. No experimento 2, foram testadas variações nas concentrações de antibióticos avaliadas no Experimento 1: 25% acima (GTLS+25; PENSTREP+25; CEFT+25; ENRO+25), 50% acima (GTLS+50; PENSTREP+50; CEFT+50; ENRO+50), 25% abaixo (GTLS-25; PENSTREP-25; CEFT-25; ENRO-25) e 50% abaixo (GTLS-50; PENSTREP-50; CEFT-50; ENRO-50). Amostras de sêmen dos carneiros foram utilizadas para contagem das unidades formadoras de colônias (UFC). Os microrganismos isolados foram Staphylococcus sp., Klebsiella sp., Corynebacterium sp. e Bacillus sp. O tratamento com enrofloxacina apresentou a motilidade espermática mais baixa para o sêmen refrigerado a 5 °C e após o descongelamento (P<0,05), no entanto a motilidade pósdescongelamento não diferiu (P>0,05) dos tratamentos CEFT e GTLS. Não foram observadas diferenças entre os tratamentos quanto à integridade da membrana plasmática, do acrossoma e do DNA espermático (P>0,05). Após o descongelamento, o percentual de espermatozóides com DNA íntegro foi maior (P<0,05) no tratamento PENSTREP em relação ao GTLS, apesar de que este parâmetro pode ter sido afetado pelo processo de criopreservação. No experimento 2, GTLS+50, PENSTREP, ENRO+25 e ENRO+50 foram os tratamentos que apresentaram redução na motilidade espermática (P<0,05). Todos os tratamentos reduziram consideravelmente o número de UFC em relação ao grupo controle. A contagem de UFC para os tratamentos GTLS+50, ENRO+25 e ENRO+50 e as variações de concentração para ambos não foram diferentes entre si (P>0,05), porém ambos foram mais efetivos que os tratamentos PENSTREP e CEFT (P<0,05). Porém, o tratamento com enrofloxacina prejudicou a motilidade espermática, especialmente quando se elevou a concentração Os tratamentos não influenciaram a morfologia, integridade da membrana plasmática, integridade de acrossoma e integridade de DNA dos espermatozóides.

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Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq
The use of frozen semen is a practice still little spread among the sheep producers and shows pregnancy results unsatisfactory. Because this the addition of antioxidant in the extenders to improve the sperm quality after your handing is the goal of many researchers. Therefore, the aim of this study was to analyze in vitro effects of different concentrations of cysteine anti-oxidant in extenders on the sheep sperm storage fresh, cooled and frozen. After sperm sampling and the individual analysis, the semen of six-sheep were pooled and divided in equal aliquots to be diluted in PBS (fresh semen), Equimix® (cooled semen) or Bovimix® (frozen semen). For each of these groups were added cysteine in different concentrations 0, 2.5, 5.0 and 7.5 mM, so were made the respective experimental groups Control, Cys2.5, Cys5.0 and Cys7.5. The fresh semen was stored in room temperature during two hours, the cooled semen was stored among four to eight hours in 16ºC and the frozen semen was stored into liquid nitrogen. The analyzed variables to the fresh and cooled semen were motility, vigor, viability and mitochondrial potential of the spermatozoids. To the frozen semen, the parameters evaluated were the plasmatic and acrossomal membranes integrity, kinect by the computer system (CASA) and the mitochondrial potential. The group Cys7.5 showed the highest values of motility in the fresh semen (73.50%), however there was not different (p>0.05) than the group Cys5.0 in the cooled semen after four (68.00 vs 66.50%) and eight hours (57.00 vs 55.00%). For the frozen semen, the addition of 7.5 mM cysteine promoted the highest percentage of spermatozoids with integrate plasmatic membrane (23.2%), followed by the Cys5.0 group (20.0%), however there was not statistic different (p<0.05) among the group in the acrosomal integrity. The addition of cysteine in the frozen extender promoted increased in the capacity of mobility to the spermatozoa. There was not difference (p>0.05) among the groups Cys5.0 and Cys7.5 mM for the variables average-path (VAP), straight-line (VSL) and curvilinear velocity (VCL), however the concentration of 7.5 mM cysteine was more efficient in the protection of plasmatic membrane and increased the beat/cross frequency (BCF).
O uso de sêmen conservado é uma prática pouco difundida na ovinocultura nacional, e ainda apresenta resultados insatisfatórios de prenhez. Por isso, a adição de antioxidantes aos diluidores para preservar a qualidade seminal após sua manipulação tem sido alvo de muitas pesquisas. Sendo assim, objetivou-se avaliar o efeito in vitro da adição de diferentes concentrações do antioxidante cisteína ao meio diluente no sêmen ovino mantido fresco, refrigerado e congelado. Após a colheita e análise individual, o sêmen de seis carneiros formaram um pool, iguais alíquotas deste foram diluídas em PBS (sêmen fresco), Equimix® (sêmen refrigerado) ou Bovimix® (sêmen congelado), e adicionado 0, 2.5, 5.0 e 7.5 mM de cisteína para formar os respectivos grupos experimentais: Controle, Cys2.5, Cys5.0 e Cys7.5. O sêmen fresco foi mantido em temperatura ambiente durante duas horas, o sêmen refrigerado permaneceu durante quatro e oito horas à 16°C e o sêmen congelado foi armazenado em nitrogênio líquido. As variáveis analisadas do sêmen fresco e refrigerado foram motilidade, vigor, viabilidade e potencial mitocondrial dos espermatozoides. Avaliaram-se no sêmen congelado a integridade de membrana plasmática e acrossomal, cinética espermática computadorizada e potencial mitocondrial. O grupo Cys7.5 apresentou os maiores valores de motilidade no sêmen fresco (73,50%), porém não diferiu (p>0,05) com o grupo Cys5.0 no sêmen refrigerado após quatro (68,00 vs 66,50%) e oito horas (57,00 vs 55,00%). No sêmen congelado, a adição de 7.5 mM de cisteína promoveu maior porcentagem de espermatozoides com a membrana plasmática íntegra (23,2%), seguido pelo grupo Cys5.0 (20,0%), más não houve diferença estatística (p>0,05) entre os grupos na integridade acrossomal. A adição de cisteína ao diluente de congelação promoveu aumento na capacidade de deslocamento dos espermatozoides. Não houve diferença (p>0,05) entre os grupos Cys5.0 e Cys7.5 para as variáveis velocidade de trajeto (VAP), velocidade retilínea (VSL) e velocidade curvilínea (VCL), porém a concentração de 7.5 mM de cisteína foi mais eficiente na proteção da membrana plasmática e promoveu maior frequência de batimento flagelar (BCF).

Rift Valley fever (RVF) is an arthropod-borne viral disease of significant importance in both livestock and humans. Epidemics occur periodically in domestic ruminants, typically after heavy rains, which encourage rapid multiplication of mosquito vectors. Clinical symptoms in livestock vary from inapparent infection to abortions and peracute deaths. The disease has significant zoonotic potential. People in contact with infected livestock may develop disease that varies from mild flu-like symptoms to severe neurological and haemorrhagic disorders and death. An important way of controlling the disease is through vaccination of susceptible livestock. Rift Valley fever virus (RVFV) clone 13 is a relatively new livestock vaccine against RVF that is derived from an avirulent natural mutant strain of RVFV. This vaccine has been shown in previous studies to confer protective immunity against infection with live virus. The effect of this vaccine on semen quality in male animals has never been tested. The purpose of the current trial was to determine whether RVFV clone 13 vaccine had any effect on semen quality in rams. The hypothesis tested was that animals vaccinated with RVFV clone 13 vaccine would not experience a reduction in semen quality (measured by evaluating the percentage progressively motile and percentage morphologically normal spermatozoa in successive ejaculates) relative to unvaccinated control animals. A test/control model was used to evaluate the effect of this vaccine on semen quality. A group of peripubertal ram lambs were tested for antibodies to RVFV using a serum neutralisation test (SNT). Animals without detectable antibodies (n=23) were then randomly allocated to either a test group (n=12) or a control group (n=11). Daily rectal temperature measurements were taken and weekly semen evaluations were conducted. Blood samples were drawn weekly to assess serum antibody titres. Seven animals were subsequently eliminated from the statistical analysis because of potential confounding factors. Of these seven, five animals had extremely poor semen quality at the start of the trial, one animal was found to have a persistent febrile response commencing at the start of the trial, and one animal had seroconverted to Rift Valley fever virus in the period between the initial screening and onset of the trial. Logistic regression analysis was performed on data gathered from the remaining animals to determine whether an association existed between animal group, rectal temperature and semen quality parameters. It was found that no correlation existed between treatment group and values obtained for the semen quality parameters measured. There was no statistically significant post-vaccination temporal decline in the percentage of live morphologically normal spermatozoa, or the percentage of progressively motile spermatozoa, either when assessed amongst all animals or when assessed within individual groups. Based on the data from this trial, the hypothesis was not rejected. Despite this finding, it should be stated that the elimination of animals from the analysis had some effect on the statistical power of the study. A repeat of the trial with a larger sample size and a more comprehensive pre-screening process to avoid the inclusion of animals with poor semen quality may be indicated.
Dissertation (MMedVet)--University of Pretoria, 2014.
tm2015
Production Animal Studies
MMedVet
Unrestricted

L'objectif principal de mon travail de thèse est de comprendre les processus qui agissent sur la poussière pendant le couplage entre le milieu interstellaire galactique et le milieu intra-amas. Ce processus est d'intérêt particulier dans les phénomènes violents comme les interactions galaxie-galaxie ou le "Ram Pressure Stripping" causé par la chute d'une galaxie vers le centre de l'amas.Initialement, je me suis concentré sur le problème de la destruction de la poussière et le processus de chauffage, en re-visitant les modèles présents en littérature. J'ai particulièrement insisté sur les cas des environnements extrêmes comme le gaz chaud de type coronale (e.g., IGM, ICM, HIM) et les chocs interstellaires générés par les supernovae. Sous ces conditions les petits grains sont détruits rapidement et les gros grains sont chauffés par les collisions avec les électrons énergétiques, en rendent la distribution spectral d'énergie de la poussière très différente de ce qu'on observe dans le milieu interstellaire diffus.Pour tester nos modèles j'ai les appliqués au cas d'une galaxie en interaction, NGC 4438. Les données Herschel de cette galaxie indiquent la présence de la poussière avec une température plus élevée de ce qu'on s'attendait.Avec une analyse à plusieurs longueurs d'onde on montre que cette poussière chaude semble être dans un gaz ionisé et chaud et donc subir à la fois le chauffage collisionnel et la destruction des petits grains.De plus, je me suis focalisé sur l'énigme de longue date à propos de la différence entre les échelles de temps de destruction et formation de la poussière dans la Voie Lactée. Basées sur l'efficacité de destruction de la poussière dans les chocs interstellaires, les estimations précédentes portent à une durée de vie de la poussière plus courte que l'échelle de temps typique de sa formation dans les étoiles AGB. En utilisant un modèle de poussière récent et les dernières estimations pour l'évolution de la poussière, on a réévalué la durée de vie de la poussière dans notre Galaxie. Finalement, j'ai tourné mon attention au phénomène de "Ram Pressure Stripping''. La galaxie ESO 137-001 représente un des meilleurs cas pour étudier cet effet. Sa longue queue H2 intégrée dans une queue de gaz chaud et ionisé soulève des questions sur son possible arrachement de la galaxie ou sa formation en aval dans la queue. Basé sur des récentes simulations numériques, j'ai montré que la formation des molécules de H2 sur la surface des grains dans la queue est un scénario viable
The main goal of my PhD study is to understand the dust processing that occurs during the mixing between the galactic interstellar medium and the intracluster medium. This process is of particular interest in violent phenomena such as galaxy-galaxy interactions or the "Ram Pressure Stripping'' due to the infalling of a galaxy towards the cluster centre.Initially, I focus my attention to the problem of dust destruction and heating processes, re-visiting the available models in literature. I particularly stress on the cases of extreme environments such as a hot coronal-type gas (e.g., IGM, ICM, HIM) and supernova-generated interstellar shocks. Under these conditions small grains are destroyed on short timescales and large grains are heated by the collisions with fast electrons making the dust spectral energy distribution very different from what observed in the diffuse ISM.In order to test our models I apply them to the case of an interacting galaxy, NGC 4438. Herschel data of this galaxy indicates the presence of dust with a higher-than-expected temperature.With a multi-wavelength analysis on a pixel-by-pixel basis we show that this hot dust seems to be embedded in a hot ionised gas therefore undergoing both collisional heating and small grain destruction.Furthermore, I focus on the long-standing conundrum about the dust destruction and dust formation timescales in the Milky Way. Based on the destruction efficiency in interstellar shocks, previous estimates led to a dust lifetime shorter than the typical timescale for dust formation in AGB stars. Using a recent dust model and an updated dust processing model we re-evaluate the dust lifetime in our Galaxy. Finally, I turn my attention to the phenomenon of "Ram Pressure Stripping''. The galaxy ESO 137-001 represents one of the best cases to study this effect. Its long H2 tail embedded in a hot and ionised tail raises questions about its possible stripping from the galaxy or formation downstream in the tail. Based on recent hydrodynamical numerical simulations, I show that the formation of H2 molecules on the surface of dust grains in the tail is a viable scenario

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O presente estudo avaliou os efeitos da inclusÃo de 13% de farelo de castanha de caju (CNM) na dieta de carneiros Morada Nova nos parÃmetros seminais e proteÃnas do plasma seminal. Vinte carneiros foram distribuÃdos em dois grupos iguais: grupo castanha de caju (CNG) e grupo controle (COG). O CNG e COG receberam 13% e 0% de CNM na dieta durante 90 dias. Os grupos foram comparados para peso corporal, circunferÃncia escrotal, parÃmetros seminais e proteÃnas do plasma seminal, utilizando o MÃtodo de Shotgun Proteomics. Peso corporal e circunferÃncia escrotal aumentaram durante os 90 dias do perÃodo experimental em ambos grupos, porÃm nÃo existiu diferenÃa significativa entre CNG e COG. Contudo, apÃs 60 dias, o consumo de matÃria seca foi inferior no CNG (2,9 Â 0,1; aos 90 dias) em relaÃÃo ao COG (3,4 Â 0,1; aos 90 dias). Os parÃmetros seminais nÃo diferiram entre CNG e COG. A anÃlise shotgun proteomics identificou isoaspartil peptidase L asparaginase, gliceraldeÃdo 3 fosfato desidrogenase e prostaglandina H2 D isomerase com alta expressÃo no CNG quando comparado COG. Ao mesmo tempo, 12 proteÃnas do plasma seminal apresentaram baixa expressÃo no CNG, identificadas como beta galactosidase, caltrina, beta mannosidase, glutationa peroxidase, sorbitol desidrogenase, clusterina, albumina and serotransferrina. AlÃm disso, 20 proteÃnas detectadas no plasma seminal do COG foram ausentes no CNG e cinco proteÃnas foram presentes somente nos carneiros que receberam dieta hiperlipÃdica. Em conclusÃo, o presente estudo descreveu, pela primeira vez, os efeitos de uma dieta hiperlipÃdica nos parÃmetros do sÃmen fresco e do plasma seminal de carneiros. Enquanto os critÃrios seminais nÃo foram afetados, proteÃnas especÃficas do fluido seminal foram alteradas em decorrÃncia da dieta com castanha de caju. Visto os aspectos multifuncionais dessas proteÃnas, nÃs sugerimos que certos aspectos da fertilidades de carneiros podem ser alteradas se a castanha de caju for fornecida.
The present study was carried out to evaluate the effects of 13% of cashew nut meal (CNM) inclusion in the diet of Morada Nova rams on semen parameters and seminal plasma proteins. Twenty rams were distributed in two equal groups: cashew nut group (CNG) and control group (COG). The CNG and COG received 13% and 0% of CNM in the diet for 90 days. The groups were compared for live weight, scrotal circumference, seminal parameters and seminal plasma proteins, using shotgun proteomics. Body weight and scrotal circumference increased during the 90-day experimental period in both cashew nut-fed and control groups but with no differences between CNG and COG. However, after 60 days until 90 days, percentage of dry matter intake (% live weight) was inferior in CNG (2,9 Â 0,1; at 90 days) group than COG (3,4 Â 0,1; at 90 days). The sperm quality parameters were not different between CNG and COG.The shotgun proteomics analysis identified isoaspartyl peptidase L asparaginase, glyceraldehyde 3 phosphate dehydrogenase and prostaglandin H2 D isomerase with higher expression in the cashew nut group as compared to animals receiving the control diet. Conversely, 12 seminal plasma proteins had lower expression in the cashew nut group, identified as beta galactosidase, caltrin, beta mannosidase, glutathione peroxidase, sorbitol dehydrogenase, clusterin, albumin and serotransferrin. Moreover, 20 proteins detected in the seminal plasma of the control animals were absent in the cashew nut fed animals and five were present only in the rams receiving the high fat diet. In conclusion, the present study describes, for the first time, the effects of a high fat diet on parameters of the fresh semen and seminal plasma proteins of rams. While sperm criteria were not affected, specific seminal fluid proteins did change as the result of cashew nut feeding. Given the multifunctional aspects of such proteins, we suggest that certain aspects of the ram fertility can be altered if cashew nut is provided.

Orientador: Wilma de Grava Kempinas
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Vários estudos têm demonstrado que a proteína espermática de membrana SP22 (22-28 kDa) é altamente correlacionada com a fertilidade. A localização desta proteína no segmento equatorial da cabeça espermática em várias espécies mamíferas, e a inibição de fertilização in vivo e in vitro, obtida com a incubação de espermatozóides com anticorpos anti-rSP22, sugerem sua participação na interação espermatozóide-oócito e sua importância para a capacidade reprodutiva masculina. Assim, os objetivos do presente estudo foram avaliar a presença da proteína SP22 em espermatozóides de carneiros adultos (raças Dorper e Santa Inês), identificando sua localização; quantificar os níveis desta proteína mediante as técnicas de ELISA e imunomarcação fluorescente utilizando anticorpo anti-rSP22; e correlacionar os seus níveis com os parâmetros espermáticos de morfologia, integridade de membrana avaliada por sondas fluorescentes (diacetato de carboxifluoresceína e iodeto de propídio), e cinética obtida por sistema computadorizado (CASA). A SP22 foi especificamente localizada no segmento equatorial da cabeça e no colo do espermatozóide ovino. As técnicas de ELISA e imunomarcação fluorescente mostraram-se eficientes para a quantificação da SP22 nos espermatozóides ovinos e altamente correlacionadas (R2 = 0,70). Não houve diferença significativa (P > 0,05) nos níveis de SP22 obtidos por estes dois métodos entre as raças estudadas. Para o estudo da cinética espermática foi realizada análise estatística multivariada, considerando os valores individuais obtidos pelo sistema CASA (1 ejaculado por carneiro, n = 3847 espermatozóides). Através da análise de agrupamento foi possível estabelecer um número de 3 sub-populações espermáticas coexistentes no ejaculado dos carneiros. A sub-população 1 foi a mais ativa e progressiva. A subpopulação 3 teve movimento pouco progressivo e não-linear, e a sub-população 2 teve um padrão de cinética intermediário. Houve diferença significativa (qui-quadrado = 824,39; grau de liberdade = 34; P < 0,0001) na distribuição das 3 sub-populações nos carneiros. No entanto, não houve correlação significativa (P > 0,05) entre a proporção de cada sub-população nos carneiros e os níveis de SP22 avaliados por ELISA e imunomarcação fluorescente. A integridade de membrana espermática foi positivamente correlacionada (R2 = 0,48; P = 0,02) com a proporção de espermatozóides da sub população 1 e negativamente correlacionada (R2 = 0,30; P = 0,02) com a sub-população 3. Porém, este parâmetro espermático não foi correlacionado (P > 0,05) com a proporção de espermatozóides da sub-população 2. Além disso, a morfologia espermática não foi correlacionada (P > 0,05) às proporções das três sub-populações cinéticas encontradas. Os níveis de SP22 obtidos por ELISA foram negativa e positivamente correlacionados (R2 para as 3 variáveis = 0,47) com as porcentagens de espermatozóides morfologicamente anormais e de espermatozóides com membrana plasmática íntegra, respectivamente. A quantificação de SP22 por análise de imunomarcação fluorescente não foi correlacionada com nenhum dos parâmetros espermáticos avaliados. Estes resultados demonstraram que o estudo da proteína SP22 em espermatozóides ovinos pode trazer importantes informações a respeito da capacidade reprodutiva destes animais. Entretanto, para a validação desta proteína como um biomarcador de fertilidade nesta espécie são necessários estudos que correlacionem os níveis da SP22 com as taxas de fertilidade obtidas in vivo e/ou in vitro
Abstract: Several studies have shown that the SP22 sperm membrane protein (22-28 kDa) is highly correlated with fertility. The location of this protein on the equatorial segment of the sperm head in several mammallian species, and the inhibition of in vivo and in vitro fertilization, obtained by the incubation of spermatozoa with anti-rSP22 antibodies, suggest its paticipation in spermatozoon-oocyte interaction and its importance for the male reproductive capacity. Thus, the goals of the present study were to evaluate the presence of the SP22 protein on spermatozoa from adult rams (Dorper and Santa Inês breeds), identifying its location; to quantify the levels of this protein by ELISA and FITC immunostaning analysis using anti-rSP22 Ig; and to correlate its levels with sperm parameters of morphology, membrane integrity evaluated by fluorescent probes (carboxyfluorescein diacetate and propidium iodide) and kinetics by computer-assisted sperm analysis (CASA). The SP22 was specifically located on the equatorial segment of the head and neck of the ram spermatozoon. The ELISA and FITC immunostaning techniques appear to be efficient for the SP22 quantification in ovine spermatozoa and were significantly correlated (R2 = 0.70). There was no significant difference (P > 0.05) in SP22 levels assessed by these two methods between the studied breeds. For the study of the sperm kinetics, multivariate statistical analysis was performed, considering the individual values obtained by CASA system (1 ejaculate per ram, n = 3847 spermatozoa). Through clustering analysis, it was possible to set up a number of 3 sperm subpopulations coexistent within ejaculate from rams. The subpopulation 1 was the most active and progressive. The subpopulation 3 had little progressive and non-linear movement and the subpopulation 2 had an intermediate kinetic pattern. There was significant difference (chi-square = 824.39; degrees of freedom = 34; P < 0.0001) in the distribution of the 3 subpopulations among the rams. However, there was no significant correlation (P > 0.05) between the proportion of each subpopulation in the rams and the SP22 levels evaluated by ELISA and FITC immunostaining analysis. The membrane integrity was positively correlated (R2 = 0.48; P = 0.02) with the proportion of spermatozoa in subpopulation 1 and it was negatively correlated (R2 = 0.30; p = 0.02) with subpopulation 3. However, this sperm parameter was not significantly correlated (P > 0.05) with proportion of spermatozoa in subpopulation 2. Moreover, sperm morphology was not significantly correlated (P > 0.05) with the proportions of the three kinematics subpopulations found within ejaculate semen. The SP22 levels obtained by ELISA were negatively and positively correlated (R2 = 0.47 for 3 variable) with percentages of spermatozoa morphologically abnormal and of spermatozoa with intact plasma membrane, respectively. The SP22 quantification by FITC immunostaning analysis was no correlated with any of the sperm parameters. These results showed that study of the SP22 protein in ram spermatozoa can bring important information about the reproductive capacity of these animals. Nevertheless, studies that correlate the SP22 levels with in vivo and/or in vitro fertility rate are necessary for validation of this protein as a fertility biomarker in this species
Mestrado
Biologia Celular
Mestre em Biologia Celular e Estrutural

In mammals, millions of spermatozoa are deposited in the posterior female reproductive tract but only a few hundred reach the oviducts and from these only one will fertilise an oocyte in a mono-ovulatory species. Investigating why so few spermatozoa reach the oviduct and what was so special about these spermatozoa was the central theme to the studies reported in this thesis. The studies were conducted in the facilities of the Biomedical and Tropical Veterinary Science precinct, James Cook University and used Merino sheep as the model. In initial studies, semen was collected from rams by electroejaculation and it was demonstrated that not only did ram spermatozoa in undiluted semen have a limited life span but that were differences between rams. Some rams had spermatozoa that survived for less than six hours whereas in others, spermatozoa survived for 30 hours. These differences between rams were not present in semen diluted in Tyroide’s-albumin-lactate-pyruvate (TALP) medium. Nearly all spermatozoa in freshly ejaculated semen were uncapacitated but after 12 hours incubation in Hepessynthetic oviduct fluid (HSOF), 70% were capacitated. Baseline information on the detailed motility and movement characteristics was determined with a computer-aided semen analyzer (CASA). The results demonstrated that there is a heterogenous population of spermatozoa in a semen sample and that some rams had spermatozoa that had a significantly larger head area than others. This result was supported in later studies by manual measurements of the width and length of heads of spermatozoa. Semen was collected each month for 13 months from a group of six rams. A range of measurements of the semen was made including volume, colour and velocity and movement characteristics of spermatozoa as determined by CASA. These data were correlated with meteorological data. The quality of semen was significantly influenced by the mean daily maximum temperature and hours of bright sunshine with the months January to March being the times when ram semen was of poorest quality. Samples of spermatozoa were collected from a range of sites in the reproductive tract of naturally-mated ewes at 3, 6 and 24 hours after mating. The spermatozoa were examined for detailed velocity and movement characteristics, capacitation status and dimensions of spermatozoa. A surprising result was the low and variable percentage (range 2%-22%) of motile spermatozoa in the uterus particularly at 3 and 6 hours after mating declining to a mean of 2.7% 24 hours after mating. No difference in the velocity and movement characteristics of spermatozoa between the anterior and posterior reproductive tract could be identified. However, two important and interesting results were found. There was evidence that the ovary bearing the pre-ovulatory follicle or corpus haemorrhagicum influenced the distribution of spermatozoa as significantly more spermatozoa were found in the mid and anterior ipsilateral uterine horn and oviducts than the contralateral side 24 hours after mating. The same occurred 6 hours after mating except there was only a non significant trend for the uterine horns. The second important finding was that the spermatozoa in the oviducts had a significantly smaller head than elsewhere in the reproductive tract. Analysis of the capacitation status of spermatozoa demonstrated that spermatozoa can undergo capacitation and the acrosome reaction in all sites of the reproductive tract and by 24 hours after mating most spermatozoa were capacitated and acrosome-reacted.

Thesis (M.Sc. (Animal Production)) -- University of Limpopo, 2018
The study was conducted to determine the macroscopic and microscopic raw semen characteristics of Bapedi rams, to evaluate the effect of different egg yolk (EY) concentrations in Tris-based extenders on cryopreservation of Bapedi ram semen and to determine the effect of supplementing different phosphatidylcholine (PC) concentrations in Tris-based extenders with or without egg yolk on cryopreservation of Bapedi ram semen. Semen ejaculates were collected from four matured Bapedi rams aged 2-4 years using artificial vagina (AV) and pooled to eliminate individual differences. The first experiment was performed to characterise Bapedi ram semen parameters immediately after semen collection. The macroscopic semen parameters such as volume, pH and concentration and microscopic semen parameters such as motility, viability and morphology, membrane integrity and acrosome integrity were evaluated. The experiment was replicated 8 times and the data was subjected to descriptive statistics. The second experiment evaluated the effect of Tris-based extenders with five different EY concentrations (0, 5, 10, 15 and 20 %) on the microscopic quality of cryopreserved Bapedi ram semen. The treatments were subjected to a Completely Randomized Design (CRD) and replicated 4 times. The third experiment evaluated the effects of different PC concentrations supplemented to Tris-based extenders with or without 10% EY and the PC was added as liposomes. The experiment was a 2 x 4 factorial design in a CRD with two concentrations of EY: 0 and 10 %, and four concentrations of PC: 0, 0.25, 0.50, 0.75 mg/ml in Tris-based extenders. Pooled semen samples were divided into 5 and 8 aliquots to comply with objective 2 and objective 3, respectively. The semen aliquots were diluted with Tris-based extenders and equilibrated in a refrigerator at 5°C for another 4 hours. The semen was frozen using a programmable freezer and plunged into liquid nitrogen tank (-196°C).The volume, sperm concentration and pH of Bapedi ram semen ranged between 0.4-1.5 ml, 0.52-8.84 × 109 sperm/ml, and 5-7, respectively. The average total motility (TM), progressive motility (PM) and rapid motility (RM) characteristics were 85.95±2.58 %, 29.33±2.11 % and 39.47±4.99 %, respectively. The results for the mean percentage live spermatozoa, abnormalities, intact membrane and intact acrosome were 70.19±2.29 %, 2.50±1.34 %, 72.39±1.71 % and 75.37±5.39 %, respectively. There was a general decrease trends in frozen-thawed motility characteristics such as TM, PM and RM as compared to raw semen (p<0.05). The frozen-thawed semen in Tris-based extenders with 10, 15 and 20% EY concentrations resulted in significantly (p<0.05) higher TM, PM and RM motility characteristics compared to 0 and 5%. The percentage of live spermatozoa, membrane and acrosome integrities were found higher in raw semen than in frozen–thawed semen of respective extenders (p<0.05). The supplementation of PC in extenders either with or without EY did not improve the TM, PM and RM parameters (p>0.05). The membrane integrity in extenders either with or without EY were not influenced by the supplementation PC after freezing and thawing (p>0.05). The supplementation of PC in treatments with EY did not improve the acrosome integrity (p>0.05). Interestingly, the supplementation 0.75 mg/ml PC resulted in acrosome integrity that was not significantly different (P>0.05) to treatments with EY. In conclusion, the macroscopic and microscopic semen parameters of raw Bapedi ram semen were characterized. The use of 10% EY concentration resulted in higher motility parameters and membrane integrity of frozen-thawed Bapedi ram semen. However, 20% EY resulted in higher acrosome integrity of frozen-thawed Bapedi ram semen. The supplementation of PC in extenders in extenders with or without EY did not improve the motility parameters, percentage live spermatozoa and membrane integrity. However, the acrosome integrity was improved in extenders without EY supplemented with 0.75 mg/ml PC
Agricultural research council professional development programme (ARC-PDP)

Pretendemos determinar o efeito da melatonina em características reprodutivas de carneiros das raças Merina Preta (MP) e Campaniça (C). Carneiros MP não receberam qualquer tratamento (GPC; n=6) ou foram tratados com três implantes sub-cutâneos de melatonina (GPT; n=6) tal como um grupo de carneiros C (GCT; n=7). A aplicação da melatonina foi realizada em Março. Num primeiro ensaio, durante treze semanas, avaliaram-se em todos os carneiros o perímetro testicular e características seminais (volume, concentração, mobilidade individual, coloração vital, teste de endosmose e anomalias espermáticas). Não se encontraram diferenças significativas entre animais tratados e não-tratados e entre as duas raças. Notaram-se diferenças significativas (p<0,001) entre algumas semanas no perímetro testicular, concentração, mobilidade individual e coloração vital. Na avaliação do sémen a correlação positiva encontrada entre a mobilidade individual e a coloração vital foi a mais elevada (r=0,64; p<0,001). Num segundo ensaio congelou-se sémen em dois períodos (antes a após o efeito esperado da melatonina). As provas para avaliação (em fresco e à descongelação) foram a mobilidade individual, a coloração vital e a endosmose. Não houve diferenças significativas na fertilidade obtida entre carneiros tratados ou não e entre raças. Os resultados da coloração vital do sémen em fresco foram os únicos significativos (p<0,05). Houve diferenças significativas (p<0,05) na fertilidade dos carneiros MP entre o primeiro e segundo período de congelação diagnosticada por ecografia (42,6% vs. 14,4%).Com base nos resultados concluímos que o tratamento com melatonina a carneiros das raças MP e C não conduziu a alterações nas características reprodutivas avaliadas, possivelmente por estas raças serem pouco sazonais na latitude do Sul de Portugal. ############################################################## The aim of this study was to evaluate the effect of melatonin in reproductive characteristics of Merino Preto (MP) and Campaniça (C) breed rams. MP rams were either not treated (GPC; n=6), or treated (GPT; n=6) with melatonin implants (three per ram). A group of C rams (GCT; n=7) received also melatonin implants. The administration of melatonin was performed in March. Ram’s scrotal circumference and sperm characteristics (volume, concentration, individual motility, supravital stain, HOST-test and sperm morphology) were evaluated in a first study over a thirteen weeks period. We observed no differences between treated and not-treated rams and between breeds. There were statistical differences (p<0,001) in scrotal circumference, concentration, sperm motility and supravital stain test among week periods. Semen evaluation data revealed a positive correlation between individual motility and vital staining (r=0,64; p<0,001). In a second study, semen was frozen in two distinct periods (before and after the expected effect of melatonin). The evaluation tests performed (in fresh semen and after thawing) were sperm motility, vital staining and HOST-test. No differences were found in fertility rates in treated or control rams and between breeds. Only the supravital stain test was significant (p<0,05). There were differences in MP ram fertility (ultrasound pregnancy diagnosis) between the first and the second freezing period (p<0,05; 42,6% vs. 14.4%). These data suggest that melatonin administration to MP and C rams did not affect the reproductive characteristics evaluated. This maybe due to the lower seasonality exhibited by these breeds at the latitude of Southern Portugal.

Includes bibliographical references (leaves 232-257).
xv, 257 leaves : ill. ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Aims to determine if the compatible solutes (proline, glycine betaine, and trehalose), the epididymal compounds (taurine, hypotaurine and inositol) or the antioxidants (carnosine and ascorbic acid) in tris-citrate based diluents could improve the post-thaw survival and/or fertility of ram spermatazoa.
Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Science, 1996?