Abstract Birth of offspring of a pre-determined sex using flow cytometrically sorted fresh spermatozoa was first achieved in rabbits by Johnson et al. (1989). Since then offspring have been produced using sex-sorted spermatozoa from several different species (reviewed by Johnson, 2000). Initially, efficiency of the sex-sorting technology was poor with only low numbers of spermatozoa sorted per hour. Thus, the offspring derived from flow cytometrically sorted spermatozoa were produced with the use of artificial reproductive technologies (ART) such as in vitro fertilisation (IVF) and culture (IVC), intracytoplasmic sperm injection (ICSI) and deep artificial insemination (AI) which facilitated low dose insemination of potentially compromised spermatozoa. More recently, the development of high-speed sorters (Johnson and Welch, 1999) has facilitated the production of offspring using conventional AI techniques with low dose inseminates (Seidel et al., 1999) and successful cryopreservation of sorted spermatozoa (Schenk et al., 1999; Johnson et al., 2000; Lindsey et al., 2002; Schenk and DeGrofft, 2003). Increased efficiency of sorting bull spermatozoa has evolved through significant instrumentation and biological developments which have enabled the commercialization of the sperm sexing technology in the dairy industry, although conception rates in cows after low dose AI with sexed frozen-thawed spermatozoa are still lower than after standard frozen semen AI (Seidel et al., 1999). Subsequently, over 20 000 calves of pre-determined sex have been produced from commercially available sex-sorted frozen-thawed bull spermatozoa (Seidel, 2003). However, similar developments have not been made in the sheep industry and were examined in this thesis. In this study, successful cryopreservation of sex-sorted ram spermatozoa and production of offspring of the pre-determined sex (X: 94.4 %; Y: 100 %) was achieved after low dose (2-4 x 106 total) insemination using conventional laparoscopic intrauterine (IU) AI. However, the overall pregnancy rate for ewes inseminated with sex-sorted frozen-thawed spermatozoa was low (25 %) compared to ewes inseminated with a commercial dose (140 x 106 total) of non-sorted frozen-thawed spermatozoa (54 %). Cryopreservation has been found to not only reduce the proportion of motile spermatozoa, but cause the remaining spermatozoa to undergo changes that advance membrane maturation thereby shortening their lifespan, especially after in vivo fertilisation (Gillan and Maxwell, 1999). It was found that sorting prior to cryopreservation accelerated the maturation of sperm membranes and after co-incubation with oviducal cells in vitro, sorted frozen-thawed spermatozoa were released more rapidly than non-sorted (control) frozen-thawed spermatozoa. The potentially reduced lifespan of sorted frozen-thawed spermatozoa, and practical constraints on the number of spermatozoa that can be sorted for an insemination dose, makes insemination close to the site of fertilisation and time of ovulation critical for successful fertilisation. After treatment of ewes with GnRH to increase the precision of insemination in respect to the time of ovulation, there was no difference in pregnancy rate between ewes inseminated before, during or after the assumed time of ovulation. Furthermore, there was no difference in pregnancy rate after IU AI with similar doses of sorted frozen-thawed and non-sorted frozen-thawed spermatozoa in GnRH-treated ewes. The minimum dose of sorted frozen-thawed spermatozoa required for commercially acceptable pregnancy rates determined after IU AI was high (20 x 106 motile). Consequently, alternative methods for efficiently producing large numbers of offspring of a pre-determined sex using flow cytometrically sorted ram spermatozoa were investigated. Ram spermatozoa can be stored for short periods of time in a chilled state (liquid storage) or for an indefinite period of time in a frozen state (frozen storage; Salamon and Maxwell, 2000). The fixed location of the sperm sorter requires the need for transport of semen from the point of collection to the site of sorting and processing, but also from the sperm sorter site to the recipient females under artificial conditions. In this study, ram spermatozoa liquid stored for 24 h prior to sorting were efficiently sorted, frozen, thawed and after in vitro fertilisation and culture produced a high proportion of grade 1 blastocysts. Similarly, spermatozoa stored at reduced temperatures after sorting maintained high sperm quality for up to 6 days. Furthermore, frozen-thawed spermatozoa from rams and some non-human primates were successfully prepared for sorting and efficiently sorted producing spermatozoa with high quality in vitro parameters. The quality of frozen-thawed ram spermatozoa after sorting was such that successful re-cryopreservation after sorting was possible. Low numbers of frozen-thawed sorted and re-frozen and thawed spermatozoa were optimal for IVF and a high proportion of grade 1 in vitro embryos of a pre-determined sex were produced. These embryos were either transferred immediately or vitrified prior to transfer, extending the application of the sperm sexing technology further. The birth of lambs of pre-determined sex after transfer of both fresh and vitrified embryos derived from frozen-thawed sorted spermatozoa was achieved. The findings in this thesis suggest that sorted frozen-thawed ram spermatozoa may have more advanced membrane maturation state than non-sorted frozen-thawed spermatozoa, resulting in a decreased fertilizing lifespan in the female reproductive tract. Despite this, the use of sexed ram spermatozoa in a number of physiological states (fresh, liquid, frozen) with several different ARTs is possible in producing significant numbers of offspring of a pre-determined sex. Improved efficiency in both sperm sexing and associated reproductive technologies is required for commercialization to be achieved in the sheep industry.

The Amyloodinium ocellatum was collected from cobia ( Rachycentron canadum ) gill and four tests including 4 ¢J storage, toxicity of cryoprotectant, cryopreservation and in vitro cultivation on fish cell line were conducted to establish the methods of preservation of Amyloodinium ocellatum. Survival of trophont, morphology and division of tomont and number of dinospore released were evaluated the effects of this study. The results showed that division irregulated, delayed and stopped of the tomont were found after stored at 4 ¢J over 48 hours. It was produced 1.08 x 10 4 cell/ml dinospores from 1 x 10 3 trophont at 4 ¢J, 24 hours storage group and significant higher ( p¡Õ0.0001 ) than other storage groups. For the toxicity of cryoprotectant, the concentration of DMSO 3~10¢M, Glycerol 3~10¢M, Methanol 3~10¢M, Ethanol 3~5¢M, PrOH 3~5¢M, DMAc 3~5¢M, Sucrose 3~15¢M, Trehalose 3~15¢M, Dextran 3~5¢Mand Ficoll 3~10¢Mwere safety to use on A. ocellatum trophont preservation. It was unsuccessful to cryopreserve the trophont of A. ocellatum when stored at direct liquid N2 freezing, different -20 ¢J freezing time, -1 ¢J min-1 freezing container and different cryoprotectant equilibration time contain 10¢MGlycerol and DMSO, respectively. Using the U-shaped tube of sigle and double loop could gain pure and bacteria-free dinospores. The results of in vitro cultivation of A. ocellatum showed that eel epidermis and cobia fin cell line with different culture mediums were unable to grow the trophont and tomont of A. ocellatum.

Abstract Birth of offspring of a pre-determined sex using flow cytometrically sorted fresh spermatozoa was first achieved in rabbits by Johnson et al. (1989). Since then offspring have been produced using sex-sorted spermatozoa from several different species (reviewed by Johnson, 2000). Initially, efficiency of the sex-sorting technology was poor with only low numbers of spermatozoa sorted per hour. Thus, the offspring derived from flow cytometrically sorted spermatozoa were produced with the use of artificial reproductive technologies (ART) such as in vitro fertilisation (IVF) and culture (IVC), intracytoplasmic sperm injection (ICSI) and deep artificial insemination (AI) which facilitated low dose insemination of potentially compromised spermatozoa. More recently, the development of high-speed sorters (Johnson and Welch, 1999) has facilitated the production of offspring using conventional AI techniques with low dose inseminates (Seidel et al., 1999) and successful cryopreservation of sorted spermatozoa (Schenk et al., 1999; Johnson et al., 2000; Lindsey et al., 2002; Schenk and DeGrofft, 2003). Increased efficiency of sorting bull spermatozoa has evolved through significant instrumentation and biological developments which have enabled the commercialization of the sperm sexing technology in the dairy industry, although conception rates in cows after low dose AI with sexed frozen-thawed spermatozoa are still lower than after standard frozen semen AI (Seidel et al., 1999). Subsequently, over 20 000 calves of pre-determined sex have been produced from commercially available sex-sorted frozen-thawed bull spermatozoa (Seidel, 2003). However, similar developments have not been made in the sheep industry and were examined in this thesis. In this study, successful cryopreservation of sex-sorted ram spermatozoa and production of offspring of the pre-determined sex (X: 94.4 %; Y: 100 %) was achieved after low dose (2-4 x 106 total) insemination using conventional laparoscopic intrauterine (IU) AI. However, the overall pregnancy rate for ewes inseminated with sex-sorted frozen-thawed spermatozoa was low (25 %) compared to ewes inseminated with a commercial dose (140 x 106 total) of non-sorted frozen-thawed spermatozoa (54 %). Cryopreservation has been found to not only reduce the proportion of motile spermatozoa, but cause the remaining spermatozoa to undergo changes that advance membrane maturation thereby shortening their lifespan, especially after in vivo fertilisation (Gillan and Maxwell, 1999). It was found that sorting prior to cryopreservation accelerated the maturation of sperm membranes and after co-incubation with oviducal cells in vitro, sorted frozen-thawed spermatozoa were released more rapidly than non-sorted (control) frozen-thawed spermatozoa. The potentially reduced lifespan of sorted frozen-thawed spermatozoa, and practical constraints on the number of spermatozoa that can be sorted for an insemination dose, makes insemination close to the site of fertilisation and time of ovulation critical for successful fertilisation. After treatment of ewes with GnRH to increase the precision of insemination in respect to the time of ovulation, there was no difference in pregnancy rate between ewes inseminated before, during or after the assumed time of ovulation. Furthermore, there was no difference in pregnancy rate after IU AI with similar doses of sorted frozen-thawed and non-sorted frozen-thawed spermatozoa in GnRH-treated ewes. The minimum dose of sorted frozen-thawed spermatozoa required for commercially acceptable pregnancy rates determined after IU AI was high (20 x 106 motile). Consequently, alternative methods for efficiently producing large numbers of offspring of a pre-determined sex using flow cytometrically sorted ram spermatozoa were investigated. Ram spermatozoa can be stored for short periods of time in a chilled state (liquid storage) or for an indefinite period of time in a frozen state (frozen storage; Salamon and Maxwell, 2000). The fixed location of the sperm sorter requires the need for transport of semen from the point of collection to the site of sorting and processing, but also from the sperm sorter site to the recipient females under artificial conditions. In this study, ram spermatozoa liquid stored for 24 h prior to sorting were efficiently sorted, frozen, thawed and after in vitro fertilisation and culture produced a high proportion of grade 1 blastocysts. Similarly, spermatozoa stored at reduced temperatures after sorting maintained high sperm quality for up to 6 days. Furthermore, frozen-thawed spermatozoa from rams and some non-human primates were successfully prepared for sorting and efficiently sorted producing spermatozoa with high quality in vitro parameters. The quality of frozen-thawed ram spermatozoa after sorting was such that successful re-cryopreservation after sorting was possible. Low numbers of frozen-thawed sorted and re-frozen and thawed spermatozoa were optimal for IVF and a high proportion of grade 1 in vitro embryos of a pre-determined sex were produced. These embryos were either transferred immediately or vitrified prior to transfer, extending the application of the sperm sexing technology further. The birth of lambs of pre-determined sex after transfer of both fresh and vitrified embryos derived from frozen-thawed sorted spermatozoa was achieved. The findings in this thesis suggest that sorted frozen-thawed ram spermatozoa may have more advanced membrane maturation state than non-sorted frozen-thawed spermatozoa, resulting in a decreased fertilizing lifespan in the female reproductive tract. Despite this, the use of sexed ram spermatozoa in a number of physiological states (fresh, liquid, frozen) with several different ARTs is possible in producing significant numbers of offspring of a pre-determined sex. Improved efficiency in both sperm sexing and associated reproductive technologies is required for commercialization to be achieved in the sheep industry.

No description available.

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

The mammalian ovary contains a vast reserve of oocytes, most of which are enclosed within preantral follicles. A Biopsy Pick-Up (BPU) method was tested to determine the feasibility of retrieving preantral follicles from mare ovaries in vivo. BPU fragments were collected from 5 to 21 yr old mares during the breeding season and processed using a tissue chopper, submitted to histological analysis or submitted to in vitro culture for 1 or 7 days. In conclusion, the BPU method is a reliable way to repeatedly harvest large numbers of viable, morphologically normal preantral follicles from living mares without affecting ovarian function or general reproductive health. Additionally, equine preantral follicles undergo development and growth when submitted to in vitro culture in α-MEM for 7 days, with some follicles maintaining morphological normality throughout. These technologies could be used in the future to enable the use of numerous oocytes present within the equine ovary to preserve genetic material or for large-scale embryo production.

Osteoporosis is a disease of the bone metabolism which is characterised with a decrease of bone substance. The cause of this disease is the imbalance between the creation of a new bone substance by osteoblasts and the resorption of a bone tissue by osteoclasts, in favour of the bone resorption. The risk group of the development of this disease are women after menopause, who naturally register a decline of the estrogen hormone. Estrogen operates as an inhibitor of proosteoclastic factors such as receptor activator of NFκB ligand (RANKL), interleukin (IL)-1, IL-6 or TNF-α. The imbalance of the bone metabolism can also be caused by a disbalance in the production of Prostaglandin E2 (PGE2) and 1α,25-dihydroxyvitamin D3. They are strong mediators which can both stimulate and inhibit an osteoclastogenesis in vitro in concordance with the conditions of the culture/co-culture. This thesis focuses on the examination of an influence of those mediators (PGE2 in the concentration of 10-6 M and 10-8 M; 1α,25-dihydroxyvitamin D3 in the concentration of 10-8 M and 10-9 M) on the osteoclastogenesis from the rat PBMC at the presence of osteoblasts, with or without the combination of proosteoclastic factors macrophage colony-stimulating factor (M-CSF) and RANKL. Osteoclastogenesis was stimulated if PGE2 and...

Cuscuta campestris is an obligate parasitic plant that lacks expanded leaves and roots and requires a host to complete its lifecycle. Parasite-host connections occur via an haustorium, a unique organ that acts as a bridge for the exchange of water, nutrients, macromolecules like mRNA, microRNA, and proteins, and microorganisms. Studies of Cuscuta spp. are challenging due to its dependence on the host and other host influences on the parasite. Recent research has shown intriguing aspects of Cuscuta biology like exchange genetic material with its hosts and loss of genes involved in processes such as high photosynthetic rates and defense. We developed new tools and methodologies that allow us to explore C. campestris biology in an unprecedent way. Foremost of these is an axenic method to grow C. campestris on an Artificial Host System (AHS). The AHS allows C. campestris to display its entire life cycle in vitro, including seed production. Using the AHS, we studied haustorial function, determining the role of nutrients and phytohormones on parasite haustorium development and growth, and found genes involved in haustorial function. The AHS allowed us to demonstrate the positive effect of light on C. campestris growth in the absence of a photosynthetic host and to investigate carotenoid- and ABA- related processes in the haustorial regions. We also wanted to understand how C. campestris defenses work independently from a plant host, so we studied the parasite responses to the bacterial epitope flg22 and the bacteria Peudomonas syringe. Our findings indicate that C. campestris is able to sense flg22, but its response differs from those observed in other non-parasite plants. Transcriptomic analysis revealed up-regulation of genes related to biotic and abiotic stresses, and downregulation of genes related to cuticle development. Our study contributes to understanding the C. campestris immune response in the absence of a host plant. Taken together, this research contributes novel methodologies that enable insights into C. campestris biology without the interference of a plant host on the parasite. / Doctor of Philosophy / Field dodder (Cuscuta campestris) is a parasitic plant that lacks leaves and roots and attacks a wide range of plants, such as tomato and beets. Dodders are not able to carry out full photosynthesis and thus are incapable of producing enough food or obtaining water to survive on their own, so they parasitize other plants. Dodders have developed specialized structures called haustoria that allow them to take resources directly from their hosts. Studying dodder is challenging due to the dependence of the parasite on its host, such that effects of one plant on the other are hard to disentangle. We developed new tools and methodologies that allow us to explore the biology of dodder in unprecedent ways. We developed an Artificial Host System (AHS) that allows the growth and study of dodder without involving a living host plant. Thanks to this new tool, we were able to improve understanding of the function of the haustorium, discover nutrients and growth factors that are indispensable for dodder development, and prove that dodder growth benefits from light. Using the AHS, we compared haustorial regions and shoot tips of dodder to identify genes specific to haustorial function. Additionally, we studied the responses of dodder to bacteria to understand how it reacts to microbial colonization. Our studies contribute with the development of novel methodologies that allow unprecedent discoveries into the biology of dodder. We expect that this work will promote the study of parasitic plant biology.

Tocopherols, collectively known as vitamin E, are lipophilic antioxidants, which are synthesized only by photosynthetic organisms. Due to their enormous potential to protect cells from oxidative damage, tocopherols are used e.g. as nutraceuticals and additives in pharmaceuticals. The most biologically active form of vitamin E is α-tocopherol. Most tocopherols are currently produced via chemical synthesis. Nevertheless, this always results in a racemic mixture of different and less effective stereoisomers because the natural isomer has the highest biological activity. Therefore, tocopherols synthesized in natural sources are preferred for medical purposes. The annual sunflower (Helianthus annuus L.) is a well-known source for α-tocopherol. Within the presented work, sunflower callus and suspension cultures were established growing under photomixotrophic conditions to enhance α-tocopherol yield. The most efficient callus induction was achieved with sunflower stems cultivated on solid Murashige and Skoog medium supplemented with 30 g l-1 sucrose, 0.5 mg l-1 of the auxin 1-naphthalene acetic acid and 0.5 mg l-1 of the cytokinin 6-benzylaminopurine. Photomixotrophic sunflower suspension cultures were induced by transferring previously established callus into liquid medium. The effects of light intensity, sugar concentration and culture age on growth rate and α-tocopherol synthesis rate were characterized. A considerable increase (max. 230 %) of α-tocopherol production in the cells was obtained within the photomixotrophic cell culture compared to a heterotrophic cell culture. These results will be useful for improving α-tocopherol yields of plant in vitro cultures.

INTRODUÇÃO: Os programas de preservação de fertilidade baseados na criopreservação de gametas e tecido ovariano têm sido amplamente empregados para salvaguardar o potencial reprodutivo de mulheres em idade reprodutiva. Entretanto,o risco de reintrodução de células malignas após o reimplante do tecido e a dificuldade de aplicação dos protocolos de estimulação ovariana em pacientes estrogênio dependentes, dificultam o acesso destas pacientes aos programas de preservação de fertilidade já estabelecidos. O cultivo de folículos isolados vem se mostrando uma técnica promissora capaz de atender a essas pacientes, com resultados bastante satisfatórios. A recuperação de oócitos fertilizáveis tem se mostrado bem sucedida em camundongos resultando em nascidos vivos; já em outros mamíferos de grande porte,como primatas não humanos e humanos,há escassos relatos da obtenção de oócitos em estagio MII . OBJETIVO: Este estudo teve como objetivo analisar a eficiência do cultivo tridimensional in vitro no desenvolvimento de folículos ovarianos bovinos e a sua influência sobre o metabolismo oxidativo, a geração de estresse oxidativo celular e os níveis de transcritos de genes ligados atividade mitocondrial, apoptose e crescimento e desenvolvimento oocitário. MATERIAL E MÉTODOS: Foi realizado o cultivo in vitro tridimensional de folículos secundários bovinos por 16 dias em meio ?- MEM suplementado, em atmosfera de 5% de CO2 e 20% de O2 (Grupo in vitro - Gvt). Como controles foram incluídos dois grupos: um de folículos secundários isolados frescos não submetidos a cultivo (Grupo imaturos- GI) e outro de folículos extraídos já em estágios pré-antrais mais avançados do desenvolvimentos (Grupo in vivo- Gvv). Para a avaliação do desenvolvimento folicular, foram realizadas análises morfológica e testes de viabilidade, além da expressão por RT- qPCR de genes alvos para a atividade mitocondrial e estresse oxidativo. RESULTADOS: As análises do desenvolvimento dos folículos in vitro mostraram que 81,1% dos folículos secundários apresentaram crescimento, com uma taxa de viabilidade em torno de 84%. Cerca de 50% de oócitos recuperados após o cultivo estavam viáveis pela avaliação morfológica, embora nenhum deles tenha atingido o estágio final de maturação (MII). Além disso, o cultivo não promoveu aumento da expressão de genes ligados a apoptose o que também sugere uma boa viabilidade dos folículos cultivados. O mesmo não ocorreu com o sistema Kit Ligand e c-Kit, considerados como marcadores da capacidade global de crescimento e desenvolvimento folicular, verificou-se uma menor expressão de KL nas células somáticas da parede folicular. Com relação ao metabolismo oxidativo, embora o estado redox, marcado pela produção de NAD e FAD, estivesse bastante aumentado nos folículos produzidos in vitro quando comparado com o controle imaturo fresco e maturado in vivo, não se observou aumento das espécies reativas de oxigênio em oócitos obtidos destes mesmos folículos, podendo sugerir a manutenção da capacidade de controlar o balanço oxidativo por parte destas células. ,Também não houve alteração na expressão de genes ligados à biogênese mitocondrial entre os grupos estudos. CONCLUSÃO: O método de cultivo proposto neste estudo, utilizando o sistema 3D em matriz de gel de alginato, permitiu a maturação parcial de folículos secundários isolados, mantendo a viabilidade e características do metabolismo oxidativo semelhantes a folículos maturados in vivo de mesmo tamanho. Entretanto, há algum grau de comprometimento na saúde global do folículo, especialmente em relação às células somáticas da parede folicular. / The fertility preservation programs based on oocyte and ovarian tissue cryopreservation has been widely employed to safeguard the reproductive potential of these patients. However, the risk of reintroducting malignant cells after ovarian tissue transplantation and the difficulty of applying ovarian stimulation protocols in patients with estrogen dependent diseases hinder the access of these patients to already established fertility preservation programs. Isolated follicle culture appears as a promising technique for these cases, with satisfactory results. The recovery of fertilizable oocytes has been shown to be successful in mice resulting in live births; in other large mammals, such as non-human primates and humans, there are a few reports of the obtention of oocytes in the MII stage. OBJECTIVE: This study aimed to analyze the efficiency of in vitro three-dimensional culture in the development of bovine ovarian follicles and their influence on oxidative metabolism, on cellular oxidative stress generation and on the transcript levels of genes linked to mitochondrial activity, apoptosis and oocyte growth and development. MATERIAL AND METHODS: Three-dimensional in vitro culture of bovine secondary follicles was carried out for 16 days in supplemented ?-MEM medium, in an atmosphere of 5% CO2 and 20% O2 (In vitro Group - Gvt). As controls, two groups were included: one of the fresh none cultured isolated secondary follicles (GI-immature group) and one of follicles isolated in a more advanced preantral stage (in vivo Group-Gvv). For the evaluation of the follicular development, morphological analysis and viability tests were performed, besides the expression by RT-qPCR of target gene for mitochondrial activity and oxidative stress were also evaluated. RESULTS: Analysis of the in vitro follicle development showed that 81.1% of the secondary follicles have grown, with a viability rate of 84%. About 50% of oocytes recovered after culture were viable by morphological evaluation, although none of them had reached the final maturation stage (MII). In addition, the culture did not promote increased on the expression of apoptosis-linked genes which also suggests good viability of the cultured follicles. The same was not observed with the Kit Ligand and c-Kit, considered as markers of the global capacity of follicular growth and development; there was a lower expression of KL in the somatic cells from the follicular wall. In relation to the oxidative metabolism, although the redox state, marked by the production of NAD and FAD, was greatly increased in follicles produced in vitro when compared to the immature controls and the in vivo matured group, there was no increase on the production of reactive oxygen species in the oocytes obtained from these follicles, suggesting that these cells were able to control their oxidative imbalance. Also, there was no alteration in the expression of genes linked to mitochondrial biogenesis between the studied groups. CONCLUSION: The culture method proposed in this study, using the 3D system in an alginate gel matrix, allowed the partial maturation of isolated secondary follicles, maintaining the viability and oxidative metabolism characteristics similar to follicles of the same size matured in vivo. However, there is some degree of impairment in overall health, especially in relation to the somatic cells from the follicular wall.