Thesis (Ph. D.)--University of Texas at Austin, 2005. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ludwig-Maximilians-Universität München, 2000. / Title from PDF t.p. (viewed on June 4, 2006). Includes bibliographical references (p. 65-76).

Orientador: Fabíola Freitas de Paula Lopes / Resumo: A função do espermatozoide pode ser comprometida por condições ambientais adversas. Estudos já demonstraram que a exposição in vivo e in vitro de espermatozoides bovinos a temperatura elevada induz a morte celular, reduz a motilidade espermática e o potencial fertilizante do espermatozoide. No entanto, as alterações celulares induzidas pelo choque térmico em espermatozoides bovinos ainda são controversas. Portanto, o objetivo deste estudo foi determinar o efeito do choque térmico em espermatozoides de touros holandeses na motilidade espermática, produção de espécies reativas de oxigênio (EROs), atividade mitocondrial, atividade de caspase, potencial de fertilização, cinética e desenvolvimento embrionário pré-implantacional. As amostras de sêmen foram descongeladas e submetidas ao gradiente de Percoll. Na amostra controle (sem incubação) os espermatozoides foram avaliados imediatamente após o gradiente de Percoll. Posteriormente, as amostras foram incubadas à 35 °C (Controle da temperatura testicular), 38,5 °C (temperatura corporal) e 41 °C (choque térmico) por 4 horas. O choque térmico de 41 °C reduziu a motilidade espermática após 2 h de incubação em comparação com 35 e 38,5 °C. A exposição de espermatozoides a diferentes temperaturas aumentou a produção de EROs, sendo este efeito mais acentuado no grupo 41°C em relação aos demais tratamentos. O aumento das EROs em espermatozoides submetidos ao choque térmico foi seguido da redução na atividade mitocondrial espermática e au... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

Purpose: Sperm nuclear proteins and DNA integrity have been implicated in infertility and treatment failures. High stallion to stallion variability is observed in sperm cryopreservation protocols. The cells are destroyed with harsh chemicals prior to using biochemical assays to test sperm DNA quality. The feasibility of using Raman spectrometry in combination with a laser trap for non-destructive micromanipulation and characterization of DNA damage in motile stallion and human sperm is experimentally investigated in this thesis. Methods: Live stallion sperms were subjected to controlled cellular damage: (a) four grades of chemically induced oxidative stress using Xanthine – Xanthine Oxidase (b) three grades of osmotic stress using PBS and (c) membrane damage using thermal shock. Live human sperm DNA disintegration with time and oxidative stress were explored on fresh, cryopreserved and swim-up categories. The specimens ranged from sub-fertile patients to fertile donors in a limited study. Post-treatment sperms resuspended in sperm media, placed on a quartz coverslip were trapped with a 785 nm, 25 mW laser, using a 1.4 NA, 60X, water immersion microscope objective. A Raman spectrum of a trapped cell was acquired for 20 – 30 seconds. The spectra from 20 – 40 cells from each specimen were analysed in the 630 cm-1 – 1630 cm-1 region using statistical variance and PCA. Results: The Raman spectra from trapped motile sperm head contain intense peaks that did not require smoothing prior to analysis. PCA of the Raman spectra could not resolve the different grades of applied osmotic and oxidative stress in stallion cells. PCA showed high variability between specimens from the same stallion and between stallions, with distinct clustering by ejaculate. Membrane damage study and spectra from extended trapping also showed distinct specimen to specimen difference within and between stallions. Specimen to specimen variability is observed in motility and viability tests on 1000s of stallion cells using CASA and flow cytometry. Human sperms showed some clustering by category, time, stress and motility and appeared more sensitive to the tests than stallion sperms. Conclusions: Raman spectra originate from the dense region of the trapped sperm head and resemble the fingerprint of dense calf thymus DNA. The cells show species specific response to the applied stress/damage. Stallion sperms show high variability between ejaculates that could not be differentiated by stallions. Human cells appear more sensitive to the applied processes. LTRS of live sperms needs further detailed research, cross correlated with other established complementary techniques, to identify spectral bands that are most sensitive to the various grades of induced DNA and membrane damage.

Fetal exposure to excess glucocorticoid is associated with low birth weight and increased cardiovascular disease risk in first generation offspring. Such phenotypes can be produced experimentally through the administration of the synthetic glucocorticoid dexamethasone (Dex) to pregnant rats during the last week of gestation. These ‘programmed effects’ can be transmitted to a second generation through both maternal and paternal lines. The overall hypothesis for this thesis was that the transmission of programmed effects through the male line may result from alterations in fetal germ cells, which form sperm in adulthood. Epigenetic reprogramming of germ cells is characterised by the genome-wide erasure and subsequent re-establishment of 5-methylcytosine (5mC), however this process has not previously been described for the rat. Furthermore, the involvement of more recently identified cytosine modifications; 5-hydroxymethylcytosine (5hmC), 5- formylcytosine (5fC) and 5-carboxylcytosine (5caC), has not been characterised during germ cell ontogeny. Using immunofluorescence to study DNA modifications during late gestation I identified that 5hmC, 5fC and 5caC were present between e14.5 and e16.5 but absent thereafter. In contrast, 5mC was absent during this time but remethylation was noted from e19.5 onwards. Prenatal Dex exposure was associated with the presence of significantly more 5mC-positive germ cells at e19.5 relative to controls. This difference did not persist at e20.5 suggesting that Dex exposure promotes premature global remethylation. The mechanisms for this are unclear since there were no differences between groups in the localisation of the DNA methyltransferases DNMT3a and 3b, or in markers of normal testis maturation. To enable the study of gene-specific changes in DNA methylation in the germline a colony of Germ Cell Specific-Enhanced Green Fluorescent Protein (GCS-EGFP) rats was established and characterised. GCS-EGFP rats had a transgenerational decrease in pup weight with Dex exposure, as in Wistar rats. The expression of both established and novel candidate genes was compared between strains. Multiple genes across different pathways had altered expression, with some affected in both Wistar and GCS-EGFP rats, whilst other differences were strain-specific. Enhanced Reduced Representation Bisulfite Sequencing was performed on liver and fetal germ cells from males exposed to Dex in utero to explore effects on DNA methylation. These studies confirm that epigenetic reprogramming occurs in the rat and that this process may be susceptible to modification by prenatal Dex exposure. GCS-EGFP rats also exhibited a Dex programming phenotype, with decreased pup weight and altered liver gene expression. The use of this unique strain of rats will permit dissection of the mechanisms for the transmission of programmed phenotypes across generations.

In the present research several aspects of the reproductive biology of the marine hermaphroditic bryozoan, Celleporell hyalin (Linnaeus, 1767) were investigated. First (see Chapter 2), aliquots, of different ages from a stock of allosperm suspension were used to fertilize a series of virgin ramets, so characterizing the decay in fertility of released sperm and any effects of sperm ageing on subsequent embryogenesis and larval metamorphosis. The effect of temperature on the above variables was also investigated. The fertile half-life of C. hvalina sperm was about 1-2 h, although significant decay in fertility occurred within a few minutes after release. Sperm ageing showed no deleterious effects on embryogenesis, larval viability, or metamorphosis. No clear effects of temperature on sperm ageing and fertilization success were found. Allosperm. storage was studied in colonies of C. hyalina. (L. ) under several experimental conditions (see Chapter 3). Recipient virgin colonies were exposed to sexually compatible allosperm suspension and the appearance of the last newly ovulated oocytes in the coelorn. was used to assess duration of sperm storage. The same experiments examined continuation of brooding cycle and brooding success throughout the period of allosperm storage. Similar obserVations were conducted on wild colonies of C. hyalina taken from the field and kept in reproductive isolation in the laboratory. Production of progeny in females zooids budded beyond the original colonial growing edge was taken as evidence of sperm movement. The results of the present study show that recipient colonies continue producing coelomic oocytes up to 5 weeks after exposure to allosperm suspension. Moreover, the progeny were produced not only by female zooids present at the moment of allosperm dosage but also by female zooids, budded later, beyond the limit of the original growing edge. Since oocytes were not present in control colonies exposed to selfsperm, the results of the present study indicate that recipient colonies store sexually compatible allosperm and transport them within the colony in order to produce viable progeny. The effect of water flow on both sperm release and fertilization success in colonies of C. hyalin (L. ) was studied (see Chapter 4). Maximum numbers of released sperm were found at low and zero water velocities. Moreover, protruded male lophophores were observed only under those conditions. Fertilization success was studied in virgin colonies of C. hyalina (L. ) exposed to compatible allosperm suspensions under different feeding activity and water flow conditions. Fertilization success was higher in colonies with more active feeding autozooids than in those with fewer feeding autozooids. High water flow conditions induced reduction in the proportion of protruded lophophores, and reduced the frequency of ovicells bearing progeny. Moreover, in all the experiments offspring were concentrated in areas of the colony bearing active feeding autozooids. The results of this study suggest that sperm release take place under similar conditions that enhance cross fertilization, with a possible role of feeding activity in bringing sperm to the proximity of receiver colonies. Sperm competition and female choice was investigated in virgin colonies of C. hyalin (L. ) exposed to sexually compatible allosperm cocktails (see Chapter 5). A microsatellite-based. genotyping system was used to determine paternity. Progeny were mainly the product of outcrossing. In a few cases, a small proportion of progeny was attributed to selffertilization. These results suggest that outcrossing is the main reproductive strategy in this species and that neither selective female choice nor sperm competition occur in C. hyalin . Cryptic incompatibility allowing a flexible mating strategy to produce out-crossed progeny Z) in the presence of allospenn and selfing when they were absent was not found. The effects of mating sequence and temporal interval between matings (2 or 48 h) on sperm precedence in double-mated individuals were studied (see Chapter 6). Paternity was determined by using a microsatellite-based genotyping system. Settled colonies produced after short mating showed evidence of sperm mixing and first-male precedence. However, last colonies produced after both short and long mating intervals showed evidence of first-male precedence. When analyses were conducted using all the sampled progeny, low incidence of paternity by the second sperm donor (P2) and absence of self fertilization were found. No effect of mating order on success of the second donor was found. Prevalence of outcrossing was also found. These results suggest that first-male precedence in C. hyalin may promote outcrossing under sperm limitation conditions, by acceptance of the first compatible allosperm to become available. The effects of exposure to sperm suspensions and stressors on sexual allocation in colonies were studied (see Chapter 7). In the first experiment the effect of a waterborne factor on receptor colonies was studied. Adult colonies were exposed to compatible allosperm suspensions that had been filtered through 0.45 Vrn pores potentially able to remove cellular sized particles. As a control, receptor colonies were exposed to non-filtered allosperin suspensions. Appearance and growth of oocytes occurred only in the coelorn. of the control colonies. The active factor is not a dissolved molecule, but a particle that can be removed from water by filtration through 0.45 [im pores. This result plus the absence of developing oocytes in the receptor colonies exposed to similar concentration of selfsperm, suggest the operation of self/honself recognition and an important and active role of allosperm in initiating colonial reproductive investment in C. hyalina. Prevention of colonial growth and others stressors were associated with production of basal male zooids. In other experiments, exposure to sperm suspensions of different degrees of genetic relatedness showed a virtual absence of production of progeny in those colonies exposed to closely related sperm (i. e. self and halfsibs). Finally, in experiments with sexually immature colonies, the onset of sexual reproduction was triggered by exposure to allosperm, resulting in the production of female zooids even before the appearance of male zooids. Contacts between colonies of different genetic relatedness were studied under laboratory conditions (see Chapter 8). Moreover, observations were made on colonies growing on their natural substrata. Five types of responses were observed, from total fusion to overgrowth. Maximum degree of fusion, or morphological fusion, was manifested as morphologic interconnection between the ad oining colonies (i. e. production of coalescent zooids). Fusion occurred in all contacts between colonies of the same genotype, between parental colonies and their progeny and, between full and half sib colonies. In most cases the production of coalescent zooids was found. Absence of fusion occurred in all contacts between unrelated colonies and between some of the half sibs. Observations on wild colonies growing in contact with each other failed to reveal any incidences of coalescence. Non-aggressive overgrowth was confined to dead areas of one colony overgrown by zooids of the other healthy colony, independent of the genetic relatedness of the pairs. Differences in the fusibility between isocontact and allocontact suggest that colony specificity exists in C. hyalin , as has been found in other sessile colonial marine organisms.

Made available in DSpace on 2014-06-11T19:29:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-12-10Bitstream added on 2014-06-13T18:39:04Z : No. of bitstreams: 1 jaskulski_rw_me_botfmvz.pdf: 1847334 bytes, checksum: 1f11eb107cc8e8df0c448ac1e5134fc8 (MD5) / Universidade Estadual Paulista (UNESP) / No presente estudo foram correlacionados os resultados das contagem de perfurações espermáticas na membrana perivitelínica externa de ovos incubáveis com as taxas de eclosão e fertilidade, objetivando-se analisar a possibilidade do uso dessa técnica como alternativa aos métodos tradicionais de avaliação da fertilidade, executados no incubatório. As avaliações foram realizadas a cada três semanas durante todo o período reprodutivo de um lote comercial de matrizes de corte da linhagem Avian Cobb 48, submetidas à monta natural a partir da 25a semana de idade. Foram realizadas 10 avaliações, sendo utilizados 30 ovos em cada avaliação. Foi realizada a extração, fixação e coloração da membrana perivitelínica externa para possibilitar a visualização das perfurações ao microscópio óptico. A idade da ave influenciou negativamente o número de perfurações espermáticas na membrana perivitelínica externa, bem como a taxa de fertilidade e de eclosão. Houve correlação positiva entre o número de perfurações espermáticas na membrana perivitelínica e a taxa de fertilidade (r= 0,885; P<0,05), assim como com a taxa de eclosão (r= 0,800; P<0,05). É possível inferir que a contagem de perfurações espermáticas pode ser utilizada como ferramenta de avaliação da fertilidade de ovos incubáveis / In this study we correlated the results of counting sperm penetration in the outer perivitelline layer of hatching eggs to the fertility and hatching rates, aiming to examine the possibility of using this technique as an alternative to traditional methods of fertility evaluation, performed in the hatchery. The evaluations were performed every three weeks throughout the reproductive period of a flock of commercial broiler strain Cobb Avian 48, submitted to natural mating from the 25th week of age. Were performed 10 evaluations, 30 eggs were used in each assessment. Was extracted, fixed and stained outer perivitelline layer to enable viewing of holes in an optical microscope. The bird's age negatively influenced the number of sperm holes in the outer perivitelline layer and the fertility rate and hatching. A positive correlation between the number of sperm holes in the perivitelline layer and the fertility rate (r = 0.885, P <0.05), as well as the hatching rate (r = 0.800, P <0.05). It can infer that the sperm count of holes can be used as a tool for assessing the fertility of eggs hatching

Made available in DSpace on 2014-06-11T19:29:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-02-22Bitstream added on 2014-06-13T18:48:33Z : No. of bitstreams: 1 monteiro_ga_me_botfmvz.pdf: 8347367 bytes, checksum: 82c2cef3b1dcf4f8284dbffecca4034d (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A recuperação de espermatozóides da cauda do epidídimo e sua criopreservação pode ser a última chance para recuperação do material genético quando ocorre morte súbita ou lesão grave em garanhões de alto valor genético. Sendo assim, o objetivo do experimento I foi comparar os parâmetros espermáticos dos espermatozóides da cauda do epidídimo recuperados imediatamente após orquiectomia e em diferentes momentos após armazenamento a 5°C e a temperatura ambiente. Já o experimento II, teve o objetivo de comparar os parâmetros espermáticos e fertilidade dos espermatozóides do ejaculado e do epidídimo recuperados logo depois da orquiectomia e após armazenamento por 24 horas a 5°C. No experimento I, 48 garanhões foram submetidos à orquiectomia bilateral, sendo que em oito a colheita dos espermatozóides do epidídimo foi realizada imediatamente após orquiectomia (grupo controle). Nos outros 40 garanhões, um epidídimo foi armazenado a temperatura ambiente e o contralateral armazenado a 5°C por 6, 12, 18, 24 e 30 horas, de acordo com o grupo. No experimento II, dois ejaculados de oito garanhões foram colhidos com vagina artificial e congelados (grupo EJ-0h). Uma semana após, os garanhões foram submetidos à orquiectomia bilateral, sendo que os espermatozóides de um epidídimo foram congelados imediatamente após orquiectomia (grupo EP-0h) e o epidídimo contralateral foi previamente armazenado por 24 horas a 5°C (EP-24h). O teste de fertilidade demonstrou que o grupo EP-0h (92,3%; n=13) tendem a ser maior que os grupos EJ-0h (61,5%; n=13) e EP-24h (61,5%; n=13). Conclui-se que o armazenamento dos testículos-epidídimos a 5°C proporcionou melhor preservação espermática e que, independente da temperatura, a motilidade progressiva é o parâmetro espermático mais sensível ao tempo de armazenamento. / Cauda epididymis sperm recovery and cryopreservation may be the last chance to obtain genetical material when sudden death or serious injury occur in valuable stallions. Thus, the aim of experiment I was to compare sperm parameters of epididymal sperm recovered immediately after orchiectomy and at different moments after storage at 5°C and at room temperature. Experiment II aimed to compare sperm parameters and fertility of ejaculated sperm and epididymal sperm recovered immediately after orchiectomy and after storage for 24h at 5°C. In experiment I, 48 stallions were submitted to bilateral orchiectomy, in eight of those the sperm collection were done immediately after orchiectomy (control group). In the other 40 stallions, one epididymis was stored at room temperature and the other was stored at 5°C for 6, 12, 18, 24 and 30 hours accorded to groups. In experiment II, two ejaculated from eight stallions were obtained with artificial vagina and cryopreserved (group EJ-0h). One week later, the stallions were submitted to bilateral orchiectomy, and the sperm of one epididymal were frozen immediately after orchiectomy (group EP-0h) and the contralateral epididymal was previously storage for 24h at 5°C (EP-24h). Fertility test showed that group EP-0h (92,3%; n=13) tended to be higher than groups EJ-0h (61,5%; n=13) and EP-24h (61,5%; n=13). These results allowed concluding that storage of the testis-epididymis complex at 5°C is more efficient in preserving sperm parameters than room temperature storage and that progressive motility is the parameter that is more affected by storage period, regardless of the temperature.

Orientador: Frederico Ozanam Papa / Banca: Rubens Paes de Arruda / Banca: Fernanda da Cruz Landim / Banca: Maria Denise Lopes / Banca: Andre Maciel Crespilho / Resumo: As biotécnicas de refrigeração e congelação podem causar danos irreversíveis à célula espermática devido ao estresse gerado pela queda de temperatura ou, no caso da criopreservação, pela formação de cristais de gelo intracelulares. Estas alterações resultam na diminuição da longevidade do espermatozóide no trato reprodutor da fêmea e, conseqüentemente, na queda dos índices de fertilidade. Sabendo-se que as alterações das características espermática afetam a viabilidade do sêmen sobre as diferentes formas de preservação seminal e que garanhões de diferentes taxas de fertilidade nem sempre apresentam diferenças significativas nas análises seminais padrões, faz-se necessário uma análise mais específica da células espermática, como a avaliação de vários parametros funcionais em células individuais, o que possivelmente reduziria as incertezas inerentes a previsão da fertilidade na avaliação in vitro do sêmen. Para comprovação desta hipótese três experimentos foram realizados. No experimento 1 o objetivo foi identificar e avaliar as principais modificações ocorridas nas características morfofuncionais do sêmen equino estocado a temperatura ambiente (18-22ºC) por um período de 12 horas. No experimento 2 o objetivo foi verificar a possibilidade de modular estas alterações através de duas temperaturas de refrigeração (5ºC e 15ºC), e, no experimento 3, o objetivo foi avaliar e verificar a relação destas alterações com os índices de fertilidade em sêmen congelado. Para os experimentos 1 e 2, três ejaculados de seis diferentes garanhões foram avaliados quanto a cinética espermática através da análise computadorizada do movimento espermático; para a avaliação morfofuncional foram avaliadas a integridade das membranas plasmática... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cooling and freezing can cause irreversible damage to sperm cells due to the stress generated by the decrease in temperature or, in the case of cryopreservation, the formation of intracellular ice crystals. These changes result in decreased longevity of sperm in the female reproductive tract and, consequently, a decrease in fertility rates. The changes in sperm characteristics affect the viability of the semen, and these changes vary based on the different preservation techniques used to preserve the semen from different stallions. In addition, fertility rates do not always show significant differences in semen analysis patterns. Therefore, a more specific analysis of sperm cells is necessary. This analysis should include a functional assessment of multiple parameters in individual cells, which might reduce uncertainties in the prediction of fertility based on the in vitro evaluation of semen. To test this hypothesis, three experiments were performed. In experiment 1, the objective was to identify and assess the main characteristic morph functional changes in equine semen that is stored at room temperature (18-22°C) for a period of 12 hours. In experiment 2, the objective was to determine the possibility of modulating these changes using two refrigeration temperatures (5°C and 15°C). In experiment 3, the objective was to evaluate and compare the influence of these changes on fertility rates between semen at various temperatures and in frozen semen. For experiments 1 and 2, the sperm kinetics of three different ejaculates from six stallions were evaluated using a computerized analysis of sperm motility. For morphological and functional assessments, the following parameters were evaluated: plasma and acrosomal membrane integrity, mitochondrial membrane potential, rate of DNA fragmentation, caspase activation and... (Complete abstract click electronic access below) / Doutor

Orientador: José Fernando Garcia / Coorientador: Marc Roger Jean Marie Henry / Coorientador: Marcos Vinícius Gualberto Barbosa da Silva / Coorientador: Vera Fernanda Martins Hossepian de Lima / Banca: Jose Bento Sterman Ferraz / Banca: Heidge Fukumasu / Banca: Roberto Carvalheiro / Banca: Joaquim Mansano Garcia / Resumo: Estudo de Associação Genômica (GWAS - Genome Wide Association Study) foi conduzido para identificar regiões cromossômicas relacionadas à capacidade de criotolerância espermática em bovinos da raça Nelore, empregando os fenótipos: diferença entre a motilidade do sêmen fresco e descongelado (DMD) e diferença entre a motilidade do sêmen fresco e pós-teste de termo-resistência (DMT), com o intuito de compreender melhor os fenômenos que regem tal característica. O perfil de SNP (SNP - Single Nucleotide Polymorphism) de cento e vinte e cinco touros foi determinado com o auxílio de micro-arranjo de alta densidade de SNP (BovineHD Illumina®). As estimativas dos valores genéticos dos touros (EBV - Estimated Breeding Value) (Experimento 1) e as médias das medidas fenotípicas (Experimento 2) foram calculadas para as características em questão. As metodologias estatísticas GRAMMAR e EIGENSTRAT foram utilizadas para a realização do teste de associação fenótipo / genótipo após procedimento de controle de qualidade dos dados. A análise de enriquecimento funcional foi realizada com auxílio do software DAVID. Essa análise apontou a família de genes Serpina (inibidores de proteases), reguladores de cAMP e componentes do citoesqueleto celular como o grupo de genes que apresentam maior importância funcional para as características avaliadas. Todas as metodologias testadas demonstraram-se capazes de identificar regiões genômicas associadas aos fenótipos DMD e DMT, destacando-se os cromossomos 1, 16 e 19 / Abstract: Genome Wide Association Study (GWAS) was performed in order to identify chromosomal regions related to sperm cryotolerance capacity in Nellore cattle for the phenotypes: difference between fresh and post-thaw semen motility (DMD) and difference between fresh and post thermal resistance test semen motility (DMT), aiming to better understand the phenomena governing this characteristic. SNP (Single Nucleotide Polymorphism) profile of one hundred and twenty five Nellore bulls were determined using high density SNP chip (BovineHD Illumina®). Estimated Breeding Values - EBV (Experiment 1) and phenotypic measures means (Experiment 2) were calculated for the evaluated traits. The statistical methodologies GRAMMAR and EIGENSTRAT were used to test the phenotype / genotype association after data quality control. Functional enrichment analysis was performed using DAVID software. These analyses pointed out to Serpine (protease inhibitor), cAMP metabolism control and cytoeskeletal components gene families as the most important functional gene groups for the evaluated traits. All the methods tested shown to be able to identify genomic regions associated with phenotypes DMD and DMT, especially chromosomes 1, 16 and 19 / Doutor