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: Sony Dimas Bicudo / Banca: Paulo Henrique Franceschini / Banca: Marciane da Silva Maia / Banca: Maria Denise Lopes / Banca: Cezinande de Meira / Resumo: Objetivou-se estudar as implicações da redução na proporção de gema de ovo no meio diluidor Glicina-Gema-Leite e testar a prevenção do estresse oxidativo utilizando os antioxidantes Trolox®, Catalase ou Glutationa no processo de criopreservação de sêmen ovino. No Experimento 1, determinou-se o efeito da redução na proporção da gema de ovo no meio diluidor Glicina-Gema-Leite, utilizando-se quatro ejaculados de cada carneiro das raças Santa Inês (n=4) e Dorper (n=4), colhidos por meio de vagina artificial. Após as avaliações macro e microscópicas, o sêmen foi mantido sob temperatura de 32oC e diluído para atingir-se concentração de 400 x 106 espermatozóides/mL no meio Glicina- Gema-Leite contendo 20% (GGL20), 15% (GGL15), 10% (GGL10) ou 5% (GGL5) (v/v) de gema de ovo. O sêmen foi envasado em palhetas de 0,25 mL, e em seguida, as amostras foram refrigeradas e congeladas em sistema com controle automatizado. A descongelação foi feita em banho-maria à 40oC/20 segundos. A cinética espermática foi determinada em sistema computadorizado de análise de sêmen (CASA) e a integridade total das membranas espermáticas pela combinação dos fluorocromos diacetato de carboxifluoresceína (DIC) e iodeto de propídio (IP). Na análise pósdescongelação, as motilidades total (MT) e progressiva (MP) nos tratamentos GGL10 e GGL5 foram maiores (P<0,05) do que no GGL20. A VAP foi menor em GGL20 dos demais meios (P<0,05) e a VSL foi maior em GGL5 do que em GGL20 (P<0,01). A VCL e o ALH apresentaram maiores valores no GGL10 e GGL5 do que no GGL15 e GGL20 (P<0,01). O BCF no GGL5 foi maior (P<0,05) do que nos demais diluidores e o sêmen criopreservado nos meios GGL10 e GGL5 apresentaram maior percentual de espermatozóides com membranas íntegras do que no meio GGL20 (P<0,05). Frente aos resultados obtidos, o GGL5 foi utilizado como meio base no experimento seguinte. No Experimento 2, ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The objective of the study was the implications of the reduction in the proportion of egg yolk in the extender Glycine-Yolk-Milk and to test the prevention of oxidative stress using antioxidants Trolox, Catalase or glutathione in the process of cryopreservation of ovine semen. In Experiment 1, we determined the effect of reduction in the proportion of egg yolk in the extender Glycine-yolk-milk, using four ejaculates from each ovine of Santa Inês breed (n=4) and Doper breed (n=4), collected using artificial vagina. After the assessment, macro and microscopic, semen was kept at a temperature of 32oC and diluted to achieve concentrations up to 400 x 106 sperm / mL in the extender Glycine-yolk-milk containing 20% (GGL20), 15% (GGL15), 10% (GGL10) ou 5% (GGL5) (v/v) of egg yolk. The semen was stored in straws of 0.25 mL, and then, samples were refrigerated and frozen in system with automated control. Thawing was done in a water bath to 40oC/20 seconds. The kinetics was determined in sperm computerized semen analysis (CASA) and the overall integrity of sperm membranes by the combination of fluorochromes carboxyfluorescein diacetate (DIC) and propidium iodide (IP). In analyzing post-thawing, motilities total (MT) and progressive (MP) in the treatments GGL10 and GGL5 were higher (P <0.05) than in GGL20. The VAP was lower in GGL20 of other means (P <0.05) and VSL was higher in GGL20 than in GGL20 (P <0.01). The VCL and ALH showed higher values in GGL10 and GGL5 than in GGL15 and GGL20 (P <0.01). The BCF in GGL5 was higher (P <0.05) than in the other extenders and cryopreserved semen in the environments GGL10 and GGL5 showed higher percentages of spermatozoon with intact membranes than in the extender GGL20 (P <0.05). Given our results, the GGL5 was used as a base extender in the experiment following. In Experiment 2, ...(Complete abstract click electronic access below) / Doutor

Safe and effective oocyte cryopreservation will have a considerable impact on clinical practice in assisted reproduction. Great improvements have been made in recent years to oocyte vitrification procedures, although further controlled trials are necessary to ensure safety, and it is necessary to know more about pregnancy and live birth outcomes. This study aims to validate various methods of oocyte vitrification as assessed by comparative target gene analysis, hence contributing to information available to clinicians advising women about fertility preservation options before cancer treatment. Target genes investigated were: the maternal effect genes Deleted in Azoospermia Like (DAZL), Maternal Antigen That Embryos Require (MATER/NLRP5) and Zygote Arrest 1 (ZAR1); three genes involved in cell cycle progression and cell death, tumour suppressor protein 53 (p53), B-cell lymphoma 2 (BCL2), BCL2-Associated X Protein (BAX); three genes known to affect spindle and chromatin structure, oocyte-specific histone 1 (H1FOO), kinesin family member 11 (KIF11) and mitotic arrest deficient 2 (MAD2); together with Factor In the GermLine, Alpha (FIGLα) which regulates zona pellucida proteins, octamer-binding transcription factor 4 (OCT4/POU5F1) which is associated with pluripotency and oocyte developmental competence, and superoxide dismutase 2, mitochondrial (SOD2) which responds to oxidative stress in the mitochondria. These genes may be useful indicators of oocyte quality following vitrification. Lysis, complementary DNA (cDNA) amplification, polyadenylic acid polymerase chain reaction (polyA PCR) and quantitative polymerase chain reaction (QPCR) were used to investigate gene expression patterns in failed-to-fertilize non-vitrified, vitrified and slow frozen human MII oocytes. Comparative gene analyses included oocytes vitrified using closed and open carriers, and two different media. Results indicate that the impact of vitrification varies by gene and oocyte variability, highlighting the importance of studies based on single oocytes and the need for caution in interpretation of generalised findings. OCT4 and also β-actin were significantly affected by all methods investigated, while FIGLα, MAD2, ZAR1 and DAZL were affected by some methods. Oocyte survival rate after thawing and the number of genes expressed by individual oocytes were higher with media incorporating dimethyl sulfoxide (DMSO) and Dextran Serum Supplement (DSS) and first-step warming in a larger volume. All methods led to altered expression of target genes, most noticeably when the second media was used. Further quantitative studies of the impact of OCT4, FIGLα and β-actin should be conducted, together with clinical comparisons between media and a longitudinal multi-centre study regarding outcomes arising from different vitrification methods.

Ph.D. (Zoology) / This project involves a detailed study of three South African antelope species, springbok, impala and blesbok. The study investigates the origins of sperm in terms of testicular histology and subsequently the major storage organ, the cauda epididymis. Sperm of these species were characterized in terms of their quality (morphology, motility, vitality characteristics among others and their physiology: when exposed to different media and cryopreservation protocols. Finally sperm fertilization biology of the three species and evaluation of fertilization and developmental success when using homologous and heterologous oocytes (relative comparison) were assessed. Cauda epididymal spermatozoa was recovered post-mortem from the testes of culled springbok (n =38); impala (n =26) and blesbok (n =42) during winter months in South Africa and cryopreserved in a Tris-fructose-citric acid extender (Biladyl) supplemented with 20% egg yolk and 7% glycerol under field conditions. Longevity of sperm was assessed in Tris and Citrate extenders and modified Tyrode lactate in vitro fertilization (IVF) media. Oocytes were collected from the ovaries of domestic cows (n =165), springbok (n = 72) and blesbok (n = 42) and matured in domestic cattle M199 maturation media supplemented with 10% FCS, 10IJg/mi LH, 10IJg/mi FSH and antibiotics. Heterologous (zona intact and zona free) and homologous fertilization was carried using a domestic cattle IVF protocol. Results were analysed using SPSS version 18.0 (Statcon, South Africa). Interspecies comparisons were made using parametric tests: paired t-test for the freezing effect, one-way analysis of variance (ANOVA), Mixed between-within subjects ANOVA for longevity, Non Parametric test for motility characteristics and least squares ANOVA for...

Orientador: Gilson Hélio Toniollo / Coorientador: Maricy Apparício Ferreira / Banca: Fabricio Singaretti de Oliveira / Banca: Erika da Silva Carvalho Morani / Banca: Geórgia Modé Magalhães / Banca: Joaquim Mansano Garcia / Resumo: Esta pesquisa teve por justificativa ampliar os estudos acerca da criopreservação de tecido gonadal e células espermáticas de gatos domésticos, podendo servir de modelo experimental para felinos selvagens. Objetivou-se comparar os efeitos da criopreservação sobre diferentes tamanhos de fragmentos testiculares, empregando dois crioprotetores. Utilizaram-se os testículos de 31 gatos domésticos, submetidos à orquiectomia eletiva. Os testículos foram pesados e apenas os que apresentarem pesos entre 1 e 2 g foram empregados. O tecido testicular foi dissecado e cortado em fragmentos de tamanhos pré-determinados (0,3cm3 e 0,5cm3). Uma amostra foi direcionada para as avaliações a fresco: integridade de membrana e do DNA; histopatologia; incidência de apoptoses por imuno-histoquímica; e índice de peroxidação lipídica - TBARS. A criopreservação foi feita em meio Tris-gema Equex STM suplementado com 3% de crioprotetores (glicerol e propanediol) através da técnica congelação rápida. Após a descongelação, foram realizados os mesmos testes iniciais. Os primeiros resultados obtidos com os 12 gatos iniciais apontaram na avaliação histomorfológica e pelo teste de lipoperoxidação lipídica que o glicerol foi mais eficiente que o propanediol na criopreservação de fragmentos testiculares de gatos domésticos de 0,5cm3 (Capítulo 2). No experimento seguinte, avaliaram-se os fragmentos testiculares de outros 31 gatos domésticos, seccionados nos mesmos tamanhos e criopreservados nas mesmas condições d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This research had the justification to broaden the studies about the cryopreservation of gonadal tissue and sperm cells of domestic cats, and could serve as an experimental model for wild cats. The objective of this study was to compare the effects of cryopreservation on different sizes of testicular fragments using two cryoprotectants. The testicles of 31 domestic cats submitted to elective orchiectomy were used. The testes were weighed and only those weighing between 1 and 2 g were used. The testicular tissue was dissected and cut into fragments of predetermined sizes (0.3cm3 and 0.5cm3). A sample was directed to fresh evaluations: membrane integrity and DNA; histopathology; incidence of apoptosis by immunohistochemistry; and lipid peroxidation index - TBARS. Cryopreservation was done on Tris-egg yolk Equex STM medium supplemented with 3% cryoprotectants (glycerol and propanediol) by the rapid freezing technique. After thawing, the same initial tests were performed. The first results obtained with the 12 initial cats showed that glycerol was more efficient than propanediol in the cryopreservation of testicular fragments of domestic cats of 0.5 cm3 (Chapter 2). In the following experiment, the testicular fragments from 31 other domestic cats, sectioned in the same sizes and cryopreserved under the same conditions as in the previous experiment, were evaluated using 3% glycerol or 3% propanediol in the Tris- egg yolk Equex STM medium, using rapid freezing technique, followed b... (Complete abstract click electronic access below) / Doutor

The aim of this study was to test whether or not the equilibration time of two different cryodiluents influences the post thaw qualities of epididymal African buffalo (Syncerus caffer') sperm. Diluents and equilibration times were compared by assessing the post thaw spermatozoal motility, longevity and the acrosomal integrity. African buffaloes belong to Africa's "Big Five" and are, therefore, popular animals amongst game farmers, hunters and tourists. They are also asymptomatic carriers of foot-and-mouth-disease (FMD) and considered to be a wildlife reservoir for this plague. Other diseases, that are carried and can be transmitted from the African buffalo (Syncerus caffer') to livestock include tuberculosis, brucellosis and theileriosis or corridor disease (CD). Therefore, the transportation of African buffaloes is highly regulated. Disease-free buffalo populations are currently derived from a small genetic 8 pool and are smaller in their trophy size than the free-ranging animals from the diseased areas of the Kruger National Park (KNP) and the Hluhluwe/Umfolozi National Park. Hence there is a special interest in bringing new genetic material into the disease-free populations. Epididymal sperm from 11 mature African buffalo bulls was collected, diluted with two different semen extenders (TriiadylTM [Tris egg yolk extender] and AndroMed® [synthetic extender, i.e. fully defined medium]) and frozen. Pre-freezing equilibration times of 2 and 9 hours were tested. Total and progressive motilities, longevities and acrosomal integrity were measured and compared. Results show that there were no differences in post-thaw sperm quality when equilibration times between 2 and 9 hr were used. The use of the egg yolk containing extender (TriiadlyTM) resulted in higher percentage of post-thaw motilities than the use of the synthetic AndroMed®. Because a high percentage of progressive motile spermatozoa is one of the prerequisites for successful AI it must be concluded that TriladylTM is superior to AndroMed®. As I believe the advantages of higher motility to be bigger than the hygiene risks of a yolk containing extender I conclude that epididymal buffalo sperm should rather be frozen with TriiadylTM than with AndroMed®. / Dissertation (MSc (Production Animal Science))--University of Pretoria, 2003. / Production Animal Studies / unrestricted

In vitro production and transfer of embryos has become a common practice within the dairy industry to efficiently breed superior animals and meet the consumption demand of the growing population. Cyropreservation is necessary for the application of commercialized embryo transfer, however, in vitro-produced embryos show morphological and physiological defects which negatively impact their ability to withstand cryopreservation in comparison to their in vivo counterparts. These artifacts result from culture conditions that cause stress to the embryo during development, leading to an accumulation of intracellular lipids, mitochondrial dysfunction, and ultimately poor ability to withstand freezing and thawing. The objective of these studies was to examine the effects of various metabolic regulators on the viability and cryotolerance of in vitro-produced embryos. Pilot studies revealed that evaluating early (stage 6) versus late (stage 7) blastocysts did not affect the trend seen in results, nor did culturing embryos in continuous versus sequential media. From the main experiment performed, it was concluded that a combination of metabolic regulators decreased lipid content, improved cryopreservation survival, and lowered the percentage of apoptotic cells present after thawing. Conditioned media increased the blastocyst percentage, but did not produce superior quality embryos as measured by cryotolerance. Research concerning the metabolic needs of the preimplantation embryo must continue to determine more relevant markers of embryo quality in vitro.

With emerging trends of new cellular therapies, the need for quick access to cellular components is necessary. For most applications genetically compatible biological components are essential to prevent adverse immune responses and graft-versus host disease (GVHD). Since these biological components have a limited window to be used, techniques for long-term storage are needed. Cryopreservation is essential for this in the field of biobanking and regenerative medicine to allow for long-term storage of cell products. During this process, ice recrystallization is the major contributor to cell death and decreased cell viability post-thaw. Due to this, controlling ice growth and recrystallization is imperative to increasing cell survival and function. The Ben lab is focused on the synthesis of small molecule, carbohydrate-based cryoprotectants that function as ice recrystallization inhibitors (IRIs). Previously, many IRIs have been synthesized showing varying degrees of ice recrystallization inhibition (IRI). Through the structure-function work, a delicate balance between hydrophobic and hydrophilic portions on the same molecule must be met. These compounds are believed to disrupt hydrogen bonding networks present in the formation of ice, and control ice growth. While numerous types of functional groups on carbohydrate derivatives have been explored, many highly solvated functional groups (amines, sulfates, phosphates, etc.) have not been thoroughly investigated. Highly solvated functional groups should disrupt hydrogen bond networks due to their solvation and in theory, should illicit an IRI response. Sulfate groups have not previously been studied, but are present in several different biological processes, such as immune response and blood coagulation. This suggests that sulfated carbohydrates should be well tolerated biologically. Sulfate groups can also be easily installed on existing IRI active molecules through orthogonal protecting group chemistry. The first part of this thesis is focused on the synthesis and IRI activity of sulfated carbohydrates based upon previously synthesized, IRI active pyranose derivatives. When compared to their parent compounds, most of the sulfated derivatives were less active, but all compounds were incredibly soluble in aqueous media. These derivatives did not show much promise as new IRIs due to the length of their synthesis and reduced IRI activity compared to their parent compounds. The Ben lab has also developed a new class of IRI active carbohydrates: aldonamide derivatives. These compounds are open-chain carbohydrates with an amide bond, arising from the ring opening of a carbohydrate lactone with a substituted amine. While many of these compounds displayed high degrees of IRI activity, many were incredibly insoluble in aqueous systems (many with solubility limits under 50 mM). Since sulfate groups were able to greatly increase solubility with some derivatives retaining IRI activity, installing sulfate groups on existing aldonamide-based IRIs should increase their solubility. Additionally, since many of these derivatives display high degrees of IRI activity, a reduction in IRI activity can be tolerated. Similarly, to the sulfated pyranose derivatives, the presence of a sulfate group reduced the IRI activity compared to the parent compounds in most derivatives. Though some sulfated derivatives possessed a higher degree of IRI activity, all the derivatives experienced a drastic increase in solubility (over 200 mM in PBS). Some of the sulfated aldonamide derivatives were assessed for their ability to protect red blood cells (RBCs) during freezing with reduced glycerol concentrations (15% glycerol), although none of thew tested derivatives showed an improvement over existing IRIs explored by the Ben lab. Since the introduction of sulfate groups to existing IRIs drastically increased solubility in aqueous systems, but resulted in reduced IRI activity in most compounds, focus was switched to the addition of different hydrophilic functional groups. Amino functional groups were briefly explored with galactose-based pyranose IRIs, aldonamide derivatives had not been explored. Amino groups are present on many biological carbohydrates and should be well tolerated biologically. The addition of amino groups to aldonamide derivatives should increase solubility, with the amino derivatives ideally retaining some IRI activity. The amino aldonamide derivatives synthesized had high solubilities (>500 mM in PBS), but did possess lower degrees of IRI activity. Due to the high solubility these derivatives were initially assessed in the cryopreservation of RBCs with reduced glycerol concentrations. Initial experiments showed improvements over current IRIs, and the compounds were assessed in a number of other biological cryopreservation scenarios including articular cartilage, platelets, and hematopoietic stem/progenitor cells (HSPCs). While the compounds showed toxicity in these cell types, more studies need to be conducted for the cryopreservation of RBCs.

No description available.

Cryopreservation of biological materials has many useful applications and is currently the most effective long term storage method used across a variety of fields. The success of freezing products or biological materials, however, varies because of the process' complexity and related cryo-injuries. One of the primary issues is the ice recrystallization induced development of extracellular and intracellular ice throughout the freezing and thawing process. Ice recrystallization is a significant contributor to freezing damage, ultimately reducing post-thaw viability and function. To address this issue, the Ben laboratory has developed and synthesized a variety of classes of small molecule carbohydrate-based ice recrystallization inhibitors (IRIs). These compounds act as supplements or alternatives to current cryoprotectants, such as trehalose, DMSO, or glycerol, which do not address ice recrystallization and can be cytotoxic. This thesis focuses on the comprehensive chemical property assessment of N-Aryl-β-D-aldonamides and N-Benzyl-β-D-gluconamides, as well as optimization of biopreservation protocols for tissue products and freeze-dried proteins. Utilizing a 5-minute modified splat cooling assay, dose-response curves of five N-Benzyl-β-D-gluconamides were generated. All compounds produced ice recrystallization inhibition active IC50 values comparable to previously investigated active compounds such as, N-Octyl-β-D-gluconamide and N-4-Bromophenyl-β-D-glucopyranoside. Furthermore, validation that the dose-response curves follow a 4-parameter logistic (4PL) or 5PL sigmodal trend depending on symmetry was obtained. In addition, all tested compounds had lower cytotoxicity than N-4-Bromophenyl-β-D-glucopyranoside and higher solubility than N-Octyl-β-D-gluconamide. Overall, N-Benzyl-β-D-gluconamides proved to be a promising class of compounds with the para derivatives being the most IRI active. The second part of this work involved the examination of IRIs' ability to cryopreserve two different biological materials using different biopreservation protocols. The first being proteins and master mix (enzymes and oligonucleotides) during RT-qPCR after the freeze-drying process. The data showed that the IRIs did not interfere and were effective during both the lyophilization and qPCR processes. When compared to most effective concentration of the current industry standard, N-4-Bromophenyl-β-D-glucopyranoside increased the protein activity by ~30%, reducing the number of cycles to reach threshold value. The most significant contribution of this work was the discovery that carbohydrate-based small molecules may be working in more than one mechanism, as both cryoprotectants and lyoprotectants. In addition to proteins, the ability of IRIs to cryopreserve tissue products was investigated. Cell media supplemented with IRIs indicated that they can increase viability and reduce mortality in both cell suspension and single dermal sheets. With N-4-Methylbenzyl-β-D-gluconamide and N-Octyl-β-D-gluconamide being the most effective at reducing the damage associated with freezing and increasing recovery of the cells within the system of a simple one cell type thin tissue matrix.