A two-step protocol has been devised as a rapid and selective alternative to conventional differential extraction techniques with an increased recovery of DNA. The protocol involves pressure cycling with the Barocycler® NEP 2320 from Pressure Biosciences. Inc. in alkaline conditions for epithelial cell lysis and removal. This step is followed by alkaline lysis at 95º C for extraction of sperm cell DNA. At 1:1 or 2:1 female to male cell ratios, high selectivity and complete separation can be achieved. But at higher ratios, male allelic dropout is observed. This protocol has been modified to generate a clean male profile at a 20:1 cell ratio through optimization of NaOH concentration and inclusion of an additional pressure cycling step. Validation studies have been performed to assess the efficiency of this method under various conditions. An additional immunomagnetic cell capture pretreatment allowed for nearly complete separation at cell ratios of up to 200:1.

Made available in DSpace on 2016-01-26T18:55:30Z (GMT). No. of bitstreams: 1 DISSERTACAO_HUBERSON_FINAL.pdf: 232987 bytes, checksum: 845a7b338d4388c6a546aefabcc59018 (MD5) Previous issue date: 2010-06-21 / For the economy, the Brazilian industry of cattle is a very important activity because it is one area that gives the possibility of having a very big potential in terms of growth. Having this in mind, it can conclude that the artificial insemination is probably one of the most important way for contributing with the advancement of the modern animal techniques used for the production, however, it requires the use of the frozen semen for this realization. During the cryopreservation process, a decrease in the number of sperm viability happens because of the osmotic effects, temperature, and morphological changes that occur because of the organization changes, fluidity, permeability and the lipid composition of sperm membranes. The interaction presents between sperm cells and the thinner represents a crucial factor to preserve the integrity and the fertilizing ability. The thinner has as main objective to protect the sperm cells during the cold shock, and also in the freezing and thawing. Conventional parameters used in the sperm evaluation have showed that they are limited in their ability of predicting the semen potential fertility, and then tests that have different characteristics of the sperm and evaluation of many attributes can provide a better fertility estimate. To evaluate the integrity of the plasmatic membrane, it was successfully utilized one combination of fluorescent probe (IP that means propidium iodide and CFDA that means carboxyfluorescein diacetate). The system CASA (one computer program that analyzes the semen) provides real information about the individual cell movement and also the subpopulations of sperm cells realizing draw for each sperm that has as objective to represent the real path. This review has the objective of presenting the relevant information to the effect of thinners in the frozen ox semen (Bos taurus indicus). / A pecuária brasileira é uma atividade de grande importância para a economia do país por se destacar pelo seu elevado potencial e possibilidade de crescimento. Neste sentido, a inseminação artificial é um dos instrumentos mais importantes para contribuir no avanço das modernas técnicas de produção animal, entretanto, para sua realização é necessário o uso do sêmen congelado. Durante o processo de criopreservação, ocorre diminuição da viabilidade espermática devido aos efeitos osmóticos, temperatura e alterações morfológicas que ocorrem por mudanças na organização, fluidez, permeabilidade e composição lipídica das membranas espermáticas. A interação entre as células espermáticas e o meio diluidor representa um fator crucial para a preservação da integridade espermática e habilidade de fecundação. O meio diluente tem como finalidade proteger a célula espermática durante o choque térmico, na congelação e descongelação. Parâmetros convencionais utilizados na avaliação espermática têm se mostrado limitados quanto à capacidade de predizer o potencial de fertilidade do sêmen, assim testes que mensuram diferentes características do espermatozóide e avaliação de vários atributos podem fornecer uma melhor estimativa da fertilidade. Para avaliar a integridade da membrana plasmática foi utilizada, em diversos trabalhos com sucesso a associação de sondas fluorescentes como Iodeto de Propídeo (IP) e Diacetato de Carboxifluoresceína (CFDA). O CASA (sistema informatizado para análise de sêmen) oferece informações precisas do movimento individual de cada célula bem como as subpopulações de células espermáticas realizando um desenho para cada espermatozóide com a finalidade de representar a sua trajetória real. A presente revisão de literatura tem o objetivo de apresentar informações relativas ao efeito de meios diluentes sobre a viabilidade de sêmen congelado bovino (Bos taurus indicus).

Made available in DSpace on 2016-07-18T17:53:07Z (GMT). No. of bitstreams: 1 DISSERTACAO_HUBERSON_FINAL.pdf: 232987 bytes, checksum: 845a7b338d4388c6a546aefabcc59018 (MD5) Previous issue date: 2010-06-21 / For the economy, the Brazilian industry of cattle is a very important activity because it is one area that gives the possibility of having a very big potential in terms of growth. Having this in mind, it can conclude that the artificial insemination is probably one of the most important way for contributing with the advancement of the modern animal techniques used for the production, however, it requires the use of the frozen semen for this realization. During the cryopreservation process, a decrease in the number of sperm viability happens because of the osmotic effects, temperature, and morphological changes that occur because of the organization changes, fluidity, permeability and the lipid composition of sperm membranes. The interaction presents between sperm cells and the thinner represents a crucial factor to preserve the integrity and the fertilizing ability. The thinner has as main objective to protect the sperm cells during the cold shock, and also in the freezing and thawing. Conventional parameters used in the sperm evaluation have showed that they are limited in their ability of predicting the semen potential fertility, and then tests that have different characteristics of the sperm and evaluation of many attributes can provide a better fertility estimate. To evaluate the integrity of the plasmatic membrane, it was successfully utilized one combination of fluorescent probe (IP that means propidium iodide and CFDA that means carboxyfluorescein diacetate). The system CASA (one computer program that analyzes the semen) provides real information about the individual cell movement and also the subpopulations of sperm cells realizing draw for each sperm that has as objective to represent the real path. This review has the objective of presenting the relevant information to the effect of thinners in the frozen ox semen (Bos taurus indicus). / A pecuária brasileira é uma atividade de grande importância para a economia do país por se destacar pelo seu elevado potencial e possibilidade de crescimento. Neste sentido, a inseminação artificial é um dos instrumentos mais importantes para contribuir no avanço das modernas técnicas de produção animal, entretanto, para sua realização é necessário o uso do sêmen congelado. Durante o processo de criopreservação, ocorre diminuição da viabilidade espermática devido aos efeitos osmóticos, temperatura e alterações morfológicas que ocorrem por mudanças na organização, fluidez, permeabilidade e composição lipídica das membranas espermáticas. A interação entre as células espermáticas e o meio diluidor representa um fator crucial para a preservação da integridade espermática e habilidade de fecundação. O meio diluente tem como finalidade proteger a célula espermática durante o choque térmico, na congelação e descongelação. Parâmetros convencionais utilizados na avaliação espermática têm se mostrado limitados quanto à capacidade de predizer o potencial de fertilidade do sêmen, assim testes que mensuram diferentes características do espermatozóide e avaliação de vários atributos podem fornecer uma melhor estimativa da fertilidade. Para avaliar a integridade da membrana plasmática foi utilizada, em diversos trabalhos com sucesso a associação de sondas fluorescentes como Iodeto de Propídeo (IP) e Diacetato de Carboxifluoresceína (CFDA). O CASA (sistema informatizado para análise de sêmen) oferece informações precisas do movimento individual de cada célula bem como as subpopulações de células espermáticas realizando um desenho para cada espermatozóide com a finalidade de representar a sua trajetória real. A presente revisão de literatura tem o objetivo de apresentar informações relativas ao efeito de meios diluentes sobre a viabilidade de sêmen congelado bovino (Bos taurus indicus).

Depuis quelques années, une biopsie testiculaire suivie d’une congélation du tissu testiculaire est proposée aux enfants atteints de cancer avant introduction d’un traitement gonadotoxique. Cette procédure de préservation de la fertilité est proposée avec l’espoir qu’une méthode de restauration de la fertilité soit développée. Le tissu testiculaire décongelé pourrait ainsi être utilisé afin d’effectuer une maturation in vitro, évitant la réintroduction de cellules tumorales, pour produire des spermatozoïdes. Ce travail de thèse a consisté, dans un premier temps, à évaluer la mise en place de la méthylation de l’ADN au sein du tissu testiculaire prépubère de souris au cours de la spermatogenèse in vitro. La culture de tissu testiculaire frais ou décongelé de souris prépubère permet le maintien des niveaux d’expression des ADN méthyltransférases 1 et 3a dans les spermatogonies et les spermatocytes. De plus, la méthylation de l’ADN est retrouvée jusque dans les spermatozoïdes produits in vitro. Par la suite, la qualité nucléaire des spermatozoïdes ainsi obtenus a été analysée. La culture de tissu testiculaire n’a pas d’impact sur le taux d’aneuploïdie, la condensation de la chromatine et la fragmentation de l’ADN spermatique. Cependant, la congélation suivie par la culture organotypique augmente la proportion de spermatozoïdes avec un ADN oxydé. Enfin, la fonctionnalité des spermatozoïdes produits in vitro a été analysée par micro-injection ovocytaire et la dynamique de différentes marques épigénétiques a été étudiée au cours du développement préimplantatoire. Les taux de développement embryonnaire sont diminués par l’utilisation de spermatozoïdes produits in vitro. Les niveaux des histones H3K4me3, H3K27me3 et H3K9ac sont peu modifiés dans les embryons issus de spermatozoïdes générés in vitro alors que la méthylation et déméthylation de l’ADN sont plus impactées. La production de spermatozoïdes après culture de tissu prépubère frais ou décongelé dans le modèle murin a permis de mettre en évidence que cette procédure n’est pas sans impact sur l’embryon précoce bien que la qualité des spermatozoïdes produits soit peu altérée. / In recent years, testicular biopsy followed by the freezing of testicular tissue has been proposed to children with cancer before the introduction of a gonadotoxic treatment. This fertility preservation procedure is offered with the hope that a fertility restoration method will be developed. The thawed testicular tissue could thus be used to perform in vitro maturation, avoiding the reintroduction of tumor cells, to produce spermatozoa. This thesis work first consisted in assessing the establishment of DNA methylation in mouse prepubertal testicular tissue during in vitro spermatogenesis. The culture of fresh or thawed mouse testicular testicular tissue allows the expression levels of DNA methyltransferases 1 and 3a to be maintained in spermatogonia and spermatocytes. In addition, DNA methylation is found even in in vitro produced spermatozoa. The nuclear quality of these spermatozoa was then analyzed. The culture of testicular tissue has no impact on sperm aneuploidy rate, chromatin condensation and DNA fragmentation. However, freezing followed by organotypic culture increases the proportion of spermatozoa with oxidized DNA. Finally, the functionality of in vitro produced spermatozoa was analyzed by oocyte microinjection and the dynamics of different epigenetic marks was studied during preimplantation development. Embryo developmental rates are decreased when using in vitro produced spermatozoa. The levels of H3K4me3, H3K27me3 and H3K9ac are slightly modified in embryos derived from spermatozoa generated in vitro whereas DNA methylation and demethylation are more affected. The production of spermatozoa after culture of fresh or thawed prepubertal tissue in the mouse model has shown that this procedure is not without impact on the early embryo, although the quality of the spermatozoa produced is relatively unaltered.

MSCAGR (Animal Science) / Department of Animal Science / The objectives of the study were to evaluate the effect of two extenders (Triladyl® and Bioxcell®) and the removal of seminal plasma on goat buck semen. Six ejaculates were collected from six indigenous bucks by means of electro-ejaculator method, and semen was pooled, and replicated 10 times. Raw semen were randomly allocated into six groups as follows: (i) Raw non-washed, (ii) Raw washed, (iii) Triladyl®-washed, (iv) Triladyl®-non-washed, (v) Bioxcell®-washed and (vi) Bioxcell®-non-washed. All six groups were analysed for spermatozoa motility rates using computer-aided sperm analysis (CASA). The spermatozoa viability for all groups were assessed using Eosin-Nigrosin, acrosome integrity using Spermac, chromatin structure using Acridine Orange, and mitochondria using JC-1 staining solutions. Both the Triladyl® and Bioxcell® washed semen groups were diluted (1:4) with Phosphate Buffered Saline (PBS) then centrifuged at 1500 x g for ten min and seminal plasma was aspirated using 1 mL sterile plastic pipette. Semen samples were diluted (1:4) as follows: Triladyl® (washed and non-washed) or Bioxcell® (washed and non-washed) and then equilibrated at 5 ºC for 2 hours. Following equilibration, semen parameters were analysed. Thereafter, the semen samples were loaded into straws and placed 5 cm above a liquid nitrogen vapour for 10 min, and then stored at -196 ºC until use. Following one month of storage, frozen semen straws per treatment group were thawed at 37 ºC for 30 seconds, then semen parameters were analysed again. Significant differences among the mean values of semen parameters were determined by Tukey’s test using ANOVA, GLM procedure of SAS version 12.1 of 2010. Total Spermatozoa motility rate of Bioxcell® (92.5±4.6), (68.2±13.5) and Triladyl® (94.9±5.5), (63.1±15.1) were significantly reduced (P < 0.05) following equilibration and freeze-thawing process, respectively on washed semen groups. Live and normal spermatozoa percentages were drastically reduced in Bioxcell® (5.2±4.9) and Triladyl® (6.9±8.6) washed semen groups, following freeze-thawing. There was a significantly lower number of spermatozoa with high mitochondrial membrane potential in non-washed semen extended with Triladyl® (68.7±26.8) compared to non-washed semen extended with Bioxcell® (49.8±20.1) following the freeze-thawing process. In conclusion, the freezing-thawing process did reduce the indigenous buck semen parameters irrespective of removal or non-removal of seminal plasma. However, Bioxcell® extender was found to be more suitable for preserving spermatozoa during equilibration and freezing/thawing process of buck semen.

Thesis (M.Sc. (Medical Sciences)) -- University of Limpopo, 2020 / Infertility affects nearly 186 million couples globally, with male factors contributing to half of the cases. Oxidative stress is an established cause of declining semen quality. Moringa oleifera has proven antioxidants. This study aimed to investigate in vitro effects of aqueous leaf extract of M. oleifera on human sperm functions. Semen samples from donors (n = 40) and patients (n = 30) were washed with HTF-bovine serum albumin (BSA), and then incubated with various concentrations of M. oleifera (0, 0.625, 6.25, 62.5, and 625 μg/ml) at 37°C for 1 hour. Sperm motility, vitality, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), DNA fragmentation, capacitation, and acrosome reaction were assessed. Sperm motility, vitality, MMP, and capacitation were enhanced, while ROS production, and DNA fragmentation decreased after M. oleifera treatment. Uncapacitated spermatozoa increased significantly with a reduction in acrosome reaction in donors. M. oleifera antioxidant compounds suppressed excessive ROS, preserved mitochondrial membrane, DNA and acrosome integrity, while enhancing sperm motility and viability. / National Research Foundation (NRF)

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No description available.

The conservation status of the species studied in this thesis (European eel and pufferfish) is currently frail, thus the main goal of this research was to develop, improve and apply several techniques and protocols with the aim of increasing the knowledge about their sperm biology, improving their reproductive performance and even helping future breeding in captivity. The reproductive performance of the males is often assessed through the sperm motion parameters analysed by the CASA system, so first we focused on how to standardize this technique in terms of procedural and biological settings. In this respect, we laid the foundations for applying a standard method to assess sperm quality in fish, making it possible for sperm studies to be compared both intra- and inter-laboratories using the proper CASA settings. Secondly, with the aim of improving the reproductive performance of European eel males, 3 thermal regimes (two of them variable: T10 and T15; and one of them constant: T20) and 3 hormonal treatments (hCG, hCGrec and PSMG) were assessed based on different sperm quality parameters. In the case of the thermal regimes, our results demonstrated that the onset and progression of spermiation are strongly influenced by water temperature, with treatment T20 showing the best results in all the sperm quality parameters. In the case of hormonal treatments, hCGrec produced the best results in all the sperm quality parameters, becoming an economically profitable/viable treatment and an effective alternative to the standard hCG treatment often used to induce spermiation in eel species. A preliminary physiological study regarding the changes to the main ions involved in the fish sperm activation process was carried out. Our results showed that intracellular concentrations of Ca2+ and K+ increased upon eel sperm activation, while pH gradually decreased over time, thus it is likely that all of them play an important role in the initiation of sperm motility in the European eel, as with other marine and freshwater teleosts. In the second part of this thesis, which focuses on the pufferfish, an in-depth study into the sperm of this species was carried out for future application in aquacultural matters. A short-term storage method for pufferfish sperm was developed, enabling us to preserve the sperm quality parameters for a relatively long time period (7 days) compared to fresh sperm samples. Moreover, the effects of both the osmolality and the ion composition of the activation media on the sperm motion parameters were evaluated, concluding that both factors play an essential role in the initiation of sperm motility of pufferfish sperm and probably, in marine fish sperm. Finally, in vitro fertilization trials were developed to assertain how the quantity and quality of male gametes affects the fertilization and hatching rates. We demonstrated that sperm/egg ratio and sperm quality are strongly related factors, suggesting that both should be taken into account as unique interrelated elements. In addition, coefficients of correlation among all the spermatozoa motion parameters provided by a CASA system and fertilization and hatching rates were estimated for the first time in a marine species. Spermatozoa velocities showed the highest coefficients (¿0.80), suggesting that the kinetics of the spermatozoa are a key factor in the fertilization process. / Gallego Albiach, V. (2013). Sperm physiology and quality in two marine teleosts: Anguilla anguilla & Takifugu niphobles [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/34625 / Premios Extraordinarios de tesis doctorales

Die vorliegende Arbeit beschäftigt sich mit den physiologischen Veränderungen porciner Spermatozoen, die durch einen metabolischen Einbau von Fettsäuren in Spermatozoenlipide hervorgerufen werden. Ziel dieser Arbeit war die Untersuchung der metabolischen Aufnahme von Fettsäuren in die Spermatozoenlipide und die Bewertung des physiologischen Zustandes porciner Spermatozoen mit Hinblick auf die Niedrigtemperaturlagerung. Alle in den porcinen Spermatozoen vorkommenden Lipide wurden mittels GC und MALDI-TOF-MS analysiert. Hauptvertreter der polaren Lipidklassen sind Glycerophospholipide (GPC, GPE). Der Hauptvertreter der neutralen Lipidklassen ist Diacylglycerol (DAG). Die metabolische Aufnahme von Fettsäuren in die Lipide wurde durch die Supplementierung des Flüssigkonservierungsmediums mit [14C]-Octadecadiensäure radiochemisch untersucht. Anhand dieser Experimente wurde gezeigt, dass die Temperatur und die Inkubationsdauer wichtige Faktoren für die metabolische Aufnahme dieser Radiochemikalie in die Spermatozoenlipide sind. Die zugesetzten Fettsäuren werden sowohl in die neutralen (DAG) als auch in die polaren Lipide (diacyl-GPC) der Spermatozoen eingebaut. Nach Supplementierung mit 13C-markierter Octadecadiensäure wurden die Lipide mittels MALDI- und Q-TOF-MS als DAG (18:2/18:2), GPC (16:0/18:2) und GPC (18:2/18:2) charakterisiert. Die gleichen Ergebnisse wurden auch für die in den Spermatozoenlipiden vorkommenden Hexadecen-, Octadecen-, und Octadecatriensäure erhalten. Bei der Untersuchung des physiologischen Zustandes von Spermatozoen wurde gezeigt, dass insbesondere Supplementierungsvarianten mit endogen vorkommenden Fettsäuren zu einer besseren Spermatozoenvitalität und Motilität bei Niedrigtemperaturlagerung führten. Gleichzeitig wurde eine Verminderung des Auftretens von akrosomalen Schäden festgestellt. Damit stellt eine Supplementierung der Spermatozoen mit ausgewählten Fettsäuren eine effektive Maßnahme zur Lagerung von Spermatozoen bei 4 bis 6°C dar. / This study examines the metabolic incorporation of selected fatty acids into the lipids of porcine spermatozoa and evaluates the physiological state of spermatozoa subsequent to low temperature storage supplementation with selected free fatty acids. The aim was to understand the role of fatty acids in relation to the (cryo-)preservation of spermatozoa and successful reproduction in more detail. All lipids present in porcine spermatozoa were analysed using gas chromatography (GC) and mass spectrometry (MALDI-TOF-MS). The main representatives of the polar lipid classes are glycerophospholipids (in particular GPC and GPE). The main representatives of the neutral lipid classes are diacylglycerols (DAG). Metabolic incorporation of fatty acids into lipids was radiochemically monitored using [14C]-octadecadienoic acid in the supplied spermatozoa-preservation medium. Temperature and incubation time were shown to be particularly important determinants. The added fatty acids were incorporated into both the spermatozoas’ neutral (DAG) and polar lipids (diacyl-GPC). The affected lipids were characterised by means of MALDI- and Q-TOF-MS subsequent to the supplementation of uniformly 13C-labelled octadecadienoic acid. DAG (18:2/18:2), GPC (16:0/18:2) and GPC (18:2/18:2) could be identified and a de-novo biosynthesis of DAG (18:2/18:2) could be proven. The same results were obtained when spermatozoa were supplemented with hexadecenoic, octadecenoic and octadecatrienoic acids. Finally, it was shown that the physiological state of the spermatozoa, especially those supplemented with endogeneously present fatty acids, led to an enhanced vitality and motility in spermatozoa subsequent to low temperature storage. It was also observed that acrosomal damage was reduced and that hexadecenoic acid significantly stabilised all the vitality parameters. In conclusion, supplementing spermatozoa with selected fatty acids is an effective solution for the storage of spermatozoa at 4 to 6°C.