In vitro production of embryos from oocytes recovered by laparoscopic oocyte pick-up (LOPU) offers great potential for the propagation of genetically valuable animals. In turn, the application of these techniques to prepubertal animals presents added benefits in that prepubertal animals may supply a greater number of oocytes than adult animals. The aim of the present study was to evaluate the developmental competence of prepubertal and adult goat oocytes cultured in semi-defined media. The follicular response and recovery of oocytes via LOPU from hormonally stimulated prepubertal and adult goats were also assessed. / Oocytes were collected over a 15-wk period from prepubertal goats, ranging in age between 3--7 mo, and adult controls, ranging in age between 2--4 yr, randomly divided into 10 collection groups. Oocytes from six of the ten collections were matured for 26 h. Four collections were not completed due to technical difficulties. Following insemination, zygotes were cultured for 4 d in G1.2 followed by 4 d in G2.2. Morulae and blastocysts were scored via light microscopy on Days 7 and 9, followed by fluorescent staining on Day 9 for cell counts. (Abstract shortened by UMI.)

Acquisition of full developmental competence of oocytes not only occurs during growth stage, and the final preparation during oocyte maturation is also critical. Previous studies have shown that nuclear maturation can occur spontaneously following culture in vitro; however, there may be some insufficiency in cytoplasmic maturation of the in vitro matured oocytes. But till now, the differences of the events of cytoplasmic maturation between in vitro and in vivo matured oocytes are still not clear. Ovarian stimulation by gonadotrophins is used to permits the growth and development of follicles, to time the initiation of pre-ovulatory oocyte maturation, and to increase the numbers of oocytes ovulated. It is one of the foundations of current treatments of human infertility. The success of clinical IVF has been depending on generation of matured oocytes at high frequency. However, ovarian stimulation with gonadotropins is associated with side effects and complications. / In order to illuminate mechanisms which affect the developmental competence of oocytes produced in vitro, in the present study, we have compared the difference of the quality of oocytes produced in vitro with that of the oocytes produced in vivo using mouse model. In order to understand the relationship between oocyte competence and ovarian responses to stimulation in the mouse, we also have compared difference of the quality of oocytes produced in vitro or in vivo from gonadotrophns stimulated ovaries with that of from natural cycling ovaries. / In-vitro matured oocytes were collected from (1) naturally ovulated mice and (2) superovulated (PMSG + hCG) mice. Immature oocytes were retrieved from (3) naturally cycling mice, and (4) from mice primed with PMSG. The results indicate that the percentages of cleavage and blastocyst formation are significantly different (P<0.05) between in-vivo and in-vitro matured oocytes. Blastocyst formation rate is significantly higher (P<0.05) in immature oocytes derived from PMSG primed mice compared to immature oocytes derived from naturally cycling mice. The percentages of oocytes with comet tails and the length of comet tails are significantly higher and longer respectively in in-vitro matured oocytes compared to in-vivo matured oocytes. Total cell numbers of blastocyst are also significantly different (P<0.05) between in-vivo and in-vitro matured oocytes. However, there are no differences in ratio of trophectoderm (TE)/inner cell mass (ICM) between in-vivo and in-vitro matured oocytes. In conclusion, in-vivo matured mouse oocytes are more competent than those of matured in-vitro, suggesting that it may be due to its less damage of DNA. Embryonic development capacity of in-vivo matured oocytes is not promoted by ovarian stimulation. Gonadotropin priming prior to immature mouse oocyte retrieval is beneficial to subsequent embryonic development. / Keywords. mouse oocyte, IVM, IVF, gonadotropin, development

Microsporidia are a group of obligate intracellular fungal parasites that infect a wide range of vertebrates and invertebrates, and are of economic and academic interest. Some areas of their economic impact are in aquaculture where they can infect salmon and other fish species. In agriculture they have been considered as control agents for insect pests, but more importantly as likely contributing to colony collapse disorder of bees. As an academic topic, microsporidia are fascinating because they are the smallest and simplest eukaryotic cells and require eukaryotic host cells in order to complete their life cycle. Therefore one research avenue that moves forward both economic and academic interests is to use cultures of animal cells to support the growth and development of the microsporidia life cycle, including the production of spores. Although the use of animal cell cultures for studying the microsporidia of insect and mammals has a fairly large literature, fish cell cultures have been employed less often but have had some successes as reviewed in this thesis. Very short-term primary cultures have been used to show how microsporidia spores can modulate the activities of phagocytes. The most successful microsporidia/fish cell culture system has been relatively long-term primary cultures of salmonid leukocytes for culturing Nucleospora salmonis. Surprisingly, this system can also support the development of Enterocytozoon bienusi, which is of mammalian origin. Some modest success has been achieved in growing Pseudoloma neurophilia on several different fish cell lines. The eel cell line, EP-1, appears to be the only published example of any fish cell line being permanently infected with microsporidia, in this case Heterosporis anguillarum. These cell culture approaches promise to be valuable for describing the growth and development of the microsporidia and for documenting the responses of fish cells to infection. In this thesis, cell lines from warm water fish, goldfish, fathead minnow and zebrafish, and a coldwater species, rainbow trout, were explored as potential cellular hosts of two microsporidia species that have never been grown or associated with fish before. One is Anncaliia algerae, which is an aquatic microsporidium that most commonly infects mosquitoes. This microsporidia is one of the easiest species to grow in mammalian cells, with the rabbit kidney cell line, RK 13, being the most documented culture system. The other is Nosema apis, which is a pathogen of bees and for which few cell culture systems exist. The ability of warm water fish cell lines to support the life cycle of A. algerae was investigated first. Spores were purified from RK-13 cultures and added to cell lines from three warm water species as well as to an insect cell line. The cell lines were GFSK-S1 and GFB3C- W1 from goldfish skin and brain respectively, ZEB2J from zebrafish embryos, FHMT-W1 from fathead minnow testis, and Sf9 from ovaries of a fall armyworm moth. All cultures were maintained at 27 °C. Infection was judged to have taken place by the appearance of sporonts and/or spores in cells and occurred in all cell lines. Spores were also isolated from ZEB2J cultures and used to successfully infect new cultures of ZEB2J, RK-13 and Sf9. These results suggest that cells of a wide range of vertebrates support A. algerae growth in vitro and fish cells can produce spores infectious to cells of mammals, fish and insects. As ZEB2J was the most characterized of the fish cell lines and supported good A. algerae growth, this cell line was used in further studies described below to compare the efficacy of antimicrosporidial drugs and to test whether fish cells could support N. apis growth, but first A. algerae growth at lower temperatures was explored with cell lines from a coldwater fish. Cultures of cell lines from rainbow trout gill, RTgill-W1, and brain, RTbrain-W1, at 9, 18 and 21°C were evaluated for their ability to support the development of A. algerae. For up to 8 days after the addition of spores, living and DAPI stained cultures were examined by phase-contrast microscopy, allowing the identification of the meront, sporont, and spore stages in cultures at 18 and 21 °C. Meronts and sporonts were both spindle-shaped, but relative to meronts, sporonts were darker under phase contrast and brighter after DAPI staining. Spores were egg-shaped, phase- bright and intensely DAPI stained. These stages could not be identified conclusively in cultures at 9 °C, but their appearance at 18 °C sets a new low temperature for the growth of this species. The growth of A. algerae at room temperature allowed living cultures to be observed conveniently and videoed with a proprietary instrument, the Riveal microscope (www.quorumtechnologies.com). With this microscope, the development of A. algerae life cycle stages at room temperature was confirmed plus for the first time meront division and intracellular germination were captured on video. Spore germination in the absence of host cells and in response to 3 percent hydrogen peroxide was also observed by Riveal microscopy and for first time an abnormal germination phenomenon was clearly documented: polar tubes were extruded but the spore bodies retained the nuclei. ZEB2J cultures that had been infected with Anncaliia algerae spores were used as an in vitro test system to evaluate the curative actions of albendazole, fumagillin, and three fluoroquinolones; ciprofloxacin, norfloxacin, and ofloxacin. For each drug at concentrations above 50 µg/ml, the viability of ZEB2J cell declined sharply so concentrations of 10 and 20 µg/ml were studied. At these concentrations the drugs had little effect on the morphology and germination A. algerae spores. Each of the fluoroquinolones failed to prevent A. algerae from infecting ZEB2J cells and from growing to the same extent as in untreated ZEB2J cultures. Adding albendazole or fumagillin to cultures did not prevent A. algerae from infecting ZEB2J cells but impeded the growth and accumulation of A. algerae life-cycle stages. However, albendazole treatments caused a significant fraction of the ZEB2J cells to have nuclear abnormalities. Fumagillin reduced the intensity of infections within a ZEB2J cell, although the number of infected cells in a culture was not reduced. Over 5 days of infection with A. algerae the accumulation of ZEB2J cells in cultures was reduced but fumagillin treatment restored the accumulation to control levels. These results suggest that fumagillin has some potential as a treatment for A. algerae infections. ZEB2J was exposed to Nosema apis spores from the western honey bee (Apis mellifera). Bees were collected from hives that had been naturally infected and confirmed polymerase chain reaction (PCR) to have N. apis. Frozen bees were crushed in water to yield a mixture of bee parts, pollen grains, yeast, and microsporidial spores. The mixture was filtered and then centrifuged through Percoll to produce a pellet of spores that was resuspended in L-15 with 10 percent fetal bovine serum (FBS). Aliquots of this were added to ZEB2J cultures. Cultures were observed periodically for up to 24 days with a combination of phase contrast microscopy and of fluorescence microscopy, usually after staining with 4’,6-diamidino-2-phenylindole (DAPI). Although earlier life cycle stages were not observed, structures that were concluded to be either sporonts, sporoblasts and/or spores were seen, but these were in less than 5 percent of the fish cells. These N. apis life cycle stages had grown in ZEB2J because some appeared to be inside the cells and often they were arranged around the nucleus of the host cell rather than being randomly distributed in cultures. Despite repeated rinsing over a three week period, all cultures were ultimately lost due to yeast from the original spore preparations over growing the fish cell cultures. The overarching observation of this thesis is that fish cells in culture have been shown for the first time to support the growth A. algerae, and possibly N. apis. This suggests that the cells of vertebrates might support the growth of a wide range of microsporidia species that normally are associated with insects. In turn this suggests restriction of a microsporidial species to a particular animal group is unlikely accomplished at the cellular level but through physiological systems expressed at the organismal level and disturbances in these systems might lead to infections in new groups of animal hosts. The overarching observation of this thesis has two general implications for future studies. Firstly, for studying the expression of antimicrosporidia mechanisms in fish cells, the ZEB2J/A. algerae co-culture system promises to be useful. Secondly, for microsporidia species that are difficult to grow in culture, cell lines from a wide range of vertebrate and invertebrate species should be explored and one possibility for N. apis is fish cells.

No description available.

Thesis (MScMed)--University of Stellenbosch, 2005. / Bibliography. Also available via the Internet.

Freie Universiẗat, Diss., 2006--Berlin. / Dateiformat: zip, Dateien im PDF-Format. Erscheinungsjahr an der Haupttitelstelle: 2005.

Zugl.: Berlin, Freie Universiẗat, Diss., 2007.

Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 2004. / "December 2003" Includes bibliographical references (leaves 183-224).

Diss. (sammanfattning) Alnarp : Sveriges lantbruksuniversitet, 2006. / Härtill 5 uppsatser.

Univ., Diss.--München, 1999.