<p><i>In vitro</i> production of embryos allows efficient management of herd genetics, reduction of disease impact, and if used in combination with other reproductive technologies it could aid in preserving the threatened genetic diversity of swine. <i>In vitro</i> maturation (IVM) is identified as a deficient step in porcine in vitro production (IVP) of embryo systems, which decreases the overall success of IVP. There are problems encountered in each step of IVP; chromosomal abnormalities and decreased cell numbers in blastocysts during <i>in vitro</i> culturing (IVC), and low monospermic fertilization rates during <i>in vitro</i> fertilization (IVF) may be a result of insufficient IVM. As an addition to maturation media, porcine follicular fluid (pFF) can affect IVM. Estrogen can be found in high concentrations in pFF; possibly contributing to the effects seen when pFF is added to IVM. The objective of this thesis was to investigate the effects of estrogen supplementation during IVM on IVP of porcine embryos.</p> <p>The first objective was to evaluate the <i>in vitro</i> maturation rates of porcine oocytes in two maturation media: protein-free and 10% pFF supplemented. Nuclear maturation of oocytes was evaluated using Lamin/Dapi staining of oocytes matured in protein-free and 10% pFF maturation media to ensure the efficiency of the protein-free media. Protein-free and 10% pFF media mature oocytes at similar rates (91% and 89% respectively).</p> <p>The transcripts within the oocyte can be altered based on the <i>in vitro</i> maturation environment, so the second objective was to observe the expression of four chosen maternal effect genes: Basonuclin-1 (<i>BNC1</i>), Nucleoplasmin 2 (<i>NPM2</i>), Zygote arrest 1 (<i>ZAR1</i>), and Tripartite-motif protein-24 (<i>TRIM24</i>), using oocytes matured in 50 ng/ml, 100 ng/ml, or 1000 ng/ml of estradiol 17-&beta; (E₂), 10% pFF, or protein-free maturation media. Expression of maternal effect genes, was shown by the &Delta;Ct (cycle threshold) values, obtained from the difference between the Ct values of the normalizing gene (<i>GAPDH</i>) and the genes of interest evaluated through QRT-PCR. Values of &Delta;Ct were analyzed in place of fold change to avoid data manipulation. The &Delta;Ct expression of <i>TRIM24</i> in 0 ng/ml E₂ maturation medium and the 10% pFF maturation medium were significantly different (p<0.05) from the non-matured control, the other maternal determinant genes did not differ in their expression under any treatment.</p> <p>We hypothesized that estradiol's effects on IVM would be evident when analyzing cleavage and blastocyst formation rates. Cleavage and blastocyst formation rates were examined following <i>in vitro</i> fertilization of oocytes matured in 100 ng/ml E₂, 10% pFF, or a protein-free maturation medium to investigate the effect of estradiol on IVP embryos. Cleavage rates for the E₂ (n= 252; 60.2%) or 10% pFF (n= 256; 55.7%) additions to the maturation media did not differ (p>0.05) when compared to the protein-free maturation media (n=264; 54.9%). Both 10% pFF and E<syb>2</sub> groups had significantly higher blastocyst formation rates (p&le;0.05) than the protein-free maturation media (n=264; 3.5%), although no statistical difference was observed between the blastocyst formation rates of the 10% pFF (n=256; 12.4%) and E2 (n=252; 14.6%) groups.</o> <p>As a final study, the global gene expression of oocytes matured in a control protein-free media and the protein-free media supplemented with 100 ng/ml E₂ or 10% pFF was investigated using microarray analysis. Genes were not differentially expressed among the matured groups with the outlined threshold values of -2 &ge; log2(fold change) &ge; 2, and adjusted p-value &le;0.05. A total of 16 differentially expressed genes between the non-matured and all matured groups exceeded this threshold. Of these genes, 6 are novel transcribed regions with evidence of being an embryonic EST, and 1 is a novel protein-coding gene. The other genes are FBJ murine osteosarcoma viral oncogene homolog (<i>FOS</i>), Vimentin (<i>VIM</i>), Capthesin C (<i>CTSC</i>), Selenium binding protein 1 (<i>SELENBP1</i>), Poly(A) binding protein cytoplasmic 1 (<i>PABPC1</i>), Tissue factor pathway inhibitor 2 (<i>TFPI2</i>), Cysteine-rich, angiogenic inducer 61 (<i>CYR61</i>), Acyl-CoA synthetase long-chain family member 6 (<i>ACSL6</i>), and Phospholipase A2 group VII (<i>PLA2G7</i>).</p> <p>In conclusion, successful nuclear maturation of oocytes derived of prepubertal gilt abbatoire derived ovaries can be achieved without pFF or hormone supplementation. The expression of maternal determinant genes is not affected in a dose dependant manner, and removal of E₂ or supplementation of pFF during maturation may alter the expression of <i>TRIM24</i> from the non-matured control; where no other maternal effect gene changes through maturation. Estradiol has a similar effect as pFF during <i>in vitro</i> maturation of porcine oocytes as seen by cleavage and blastocyst formation rates. And media does not affect the global gene expression of porcine oocytes, though there is a temporal control of gene expression through maturation.</p>

The pharmacological development of non-steroidal contraceptives has yet to be achieved. Arresting oocyte maturation without blocking ovulation has been evaluated using different inhibitors of the phosphodiesterase 3A (PDE3A). Unfortunately, PDE3A is also expressed in the heart and blood vessels, and inhibition of PDE3A in oocytes can produce cardiovascular side effects. We reviewed the literature on available PDE3 inhibitors and selected cilostazol (CLZ), which is an FDA approved therapeutic. CLZ has the ability to decrease cellular adenosine uptake and consequently antagonizes side effects of PDE3A inhibition in vital organs. CLZ inhibited oocyte meiotic maturation in vitro. CLZ has more degenerative impact on arrested oocytes than matured oocytes, indicating that prolonged meiotic arrest of oocytes is harmful. Administration of CLZ any time from 9h before the ovulatory stimulus to 4h after the stimulus resulted in ovulation of immature oocytes. Controlling CLZ dose, time of CLZ administration, and time of oocyte collection resulted in ovulation of oocytes at different meiotic stages. Oral administrations of CLZ in naturally cycling mice were also observed to block pregnancy whereas remating of those previously treated females resulted in normal offspring and litter sizes. Therefore, CLZ does not only have a wide margin of contraception but also is reversible. Ovulated immature oocytes were observed to have higher rates of advanced chromatin configuration and cortical granule distribution, normal spindle and chromosomal organization, maturation, and in vitro fertilization (IVF) than ovarian immature oocytes. Ovulated metaphase I oocytes that were matured in vitro or in vivo had higher IVF rates than ovulated mature oocytes. Ovulated germinal vesicle (GV) oocytes that were in vitro matured also showed higher IVF rates but when in vivo matured, they had lower IVF rates than ovulated mature oocytes because of the high degeneration and low fertilization rates associated with in vivo maturation of GV oocytes. In summary, CLZ merits further evaluation as a non-steroidal contraceptive and is capable of producing oocytes of various meiotic stages with advanced developmental features.

In der Broschüre steht die praxisorientierte Anwendung der Humusbilanzierung im Mittelpunkt. Für ein breites Standortspektrum werden Humusbilanzen für einzelne Fruchtarten und Fruchtfolgen von Marktfrucht- und Futterbaubetrieben unterschiedlicher Intensität berechnet.

In Gefäß- und Feldversuchen wurde die Wirkung der Unterfußdüngung und der Injektionsdüngung von Phosphor auf den Ertrag und die P-Aufnahme verschiedener Kulturarten untersucht. Ebenfalls untersucht wurde die P-Düngewirkung von Schlacke aus der Hochtemperatur-Schmelzbehandlung von Klärschlamm nach dem Mephrec®-Verfahren. Die Ergebnisse zeigen, dass bei niedrigen pflanzenverfügbaren P-Gehalten (Versorgungsstufe A, B) eine entzugsorientierte Düngung nicht ausreicht, um optimale Erträge zu erreichen. Unterfußdüngung und P-Injektion steigern die P-Effizienz und dienen dem Gewässerschutz. Die Schlacke sollte gemahlen eingesetzt werden, weil sich granulierte Varianten als wenig wirksam erwiesen.

<p><i>In vitro</i> production of embryos allows efficient management of herd genetics, reduction of disease impact, and if used in combination with other reproductive technologies it could aid in preserving the threatened genetic diversity of swine. <i>In vitro</i> maturation (IVM) is identified as a deficient step in porcine in vitro production (IVP) of embryo systems, which decreases the overall success of IVP. There are problems encountered in each step of IVP; chromosomal abnormalities and decreased cell numbers in blastocysts during <i>in vitro</i> culturing (IVC), and low monospermic fertilization rates during <i>in vitro</i> fertilization (IVF) may be a result of insufficient IVM. As an addition to maturation media, porcine follicular fluid (pFF) can affect IVM. Estrogen can be found in high concentrations in pFF; possibly contributing to the effects seen when pFF is added to IVM. The objective of this thesis was to investigate the effects of estrogen supplementation during IVM on IVP of porcine embryos.</p> <p>The first objective was to evaluate the <i>in vitro</i> maturation rates of porcine oocytes in two maturation media: protein-free and 10% pFF supplemented. Nuclear maturation of oocytes was evaluated using Lamin/Dapi staining of oocytes matured in protein-free and 10% pFF maturation media to ensure the efficiency of the protein-free media. Protein-free and 10% pFF media mature oocytes at similar rates (91% and 89% respectively).</p> <p>The transcripts within the oocyte can be altered based on the <i>in vitro</i> maturation environment, so the second objective was to observe the expression of four chosen maternal effect genes: Basonuclin-1 (<i>BNC1</i>), Nucleoplasmin 2 (<i>NPM2</i>), Zygote arrest 1 (<i>ZAR1</i>), and Tripartite-motif protein-24 (<i>TRIM24</i>), using oocytes matured in 50 ng/ml, 100 ng/ml, or 1000 ng/ml of estradiol 17-&beta; (E₂), 10% pFF, or protein-free maturation media. Expression of maternal effect genes, was shown by the &Delta;Ct (cycle threshold) values, obtained from the difference between the Ct values of the normalizing gene (<i>GAPDH</i>) and the genes of interest evaluated through QRT-PCR. Values of &Delta;Ct were analyzed in place of fold change to avoid data manipulation. The &Delta;Ct expression of <i>TRIM24</i> in 0 ng/ml E₂ maturation medium and the 10% pFF maturation medium were significantly different (p<0.05) from the non-matured control, the other maternal determinant genes did not differ in their expression under any treatment.</p> <p>We hypothesized that estradiol's effects on IVM would be evident when analyzing cleavage and blastocyst formation rates. Cleavage and blastocyst formation rates were examined following <i>in vitro</i> fertilization of oocytes matured in 100 ng/ml E₂, 10% pFF, or a protein-free maturation medium to investigate the effect of estradiol on IVP embryos. Cleavage rates for the E₂ (n= 252; 60.2%) or 10% pFF (n= 256; 55.7%) additions to the maturation media did not differ (p>0.05) when compared to the protein-free maturation media (n=264; 54.9%). Both 10% pFF and E<syb>2</sub> groups had significantly higher blastocyst formation rates (p&le;0.05) than the protein-free maturation media (n=264; 3.5%), although no statistical difference was observed between the blastocyst formation rates of the 10% pFF (n=256; 12.4%) and E2 (n=252; 14.6%) groups.</o> <p>As a final study, the global gene expression of oocytes matured in a control protein-free media and the protein-free media supplemented with 100 ng/ml E₂ or 10% pFF was investigated using microarray analysis. Genes were not differentially expressed among the matured groups with the outlined threshold values of -2 &ge; log2(fold change) &ge; 2, and adjusted p-value &le;0.05. A total of 16 differentially expressed genes between the non-matured and all matured groups exceeded this threshold. Of these genes, 6 are novel transcribed regions with evidence of being an embryonic EST, and 1 is a novel protein-coding gene. The other genes are FBJ murine osteosarcoma viral oncogene homolog (<i>FOS</i>), Vimentin (<i>VIM</i>), Capthesin C (<i>CTSC</i>), Selenium binding protein 1 (<i>SELENBP1</i>), Poly(A) binding protein cytoplasmic 1 (<i>PABPC1</i>), Tissue factor pathway inhibitor 2 (<i>TFPI2</i>), Cysteine-rich, angiogenic inducer 61 (<i>CYR61</i>), Acyl-CoA synthetase long-chain family member 6 (<i>ACSL6</i>), and Phospholipase A2 group VII (<i>PLA2G7</i>).</p> <p>In conclusion, successful nuclear maturation of oocytes derived of prepubertal gilt abbatoire derived ovaries can be achieved without pFF or hormone supplementation. The expression of maternal determinant genes is not affected in a dose dependant manner, and removal of E₂ or supplementation of pFF during maturation may alter the expression of <i>TRIM24</i> from the non-matured control; where no other maternal effect gene changes through maturation. Estradiol has a similar effect as pFF during <i>in vitro</i> maturation of porcine oocytes as seen by cleavage and blastocyst formation rates. And media does not affect the global gene expression of porcine oocytes, though there is a temporal control of gene expression through maturation.</p>

The focus of my thesis research is on the interplay between individual behavior, population dynamics and community-level processes within zooplankton communities in coastal Maine. The target organisms of my thesis work are marine copepods. Copepods are small (1-10 mm) crustaceans that perform the essential ecosystem function of consuming and assimilating primary production (phytoplankton) making it available to higher trophic levels such as commercially important fishes. Therefore, copepod population growth is of critical importance to marine food webs. Fertilization limitation has been suggested as a constraint on copepod population growth but field surveys describing the prevalence of fertilization limitation are lacking. During my doctoral research, I explored the in situ fertilization success of two marine copepod species, Temora longicornis and Eurytemora herdmani in coastal Maine. I collected monthly zooplankton samples and analyzed clutches from field-caught females using an egg-staining technique. My results indicate that both species exhibit fertilization limitation in nature and the factors correlated with their fertilization span population, community and ecosystem level factors. To determine a causal relationship between predator density and copepod mating success, I conducted laboratory experiments to assess the effects of a common mysid shrimp predator, Neomysis americana on Eurytemora herdmani mating success. I subjected males and females to predators or predator cues. I found that the presence of a mysid predator, or only a predator cue, reduced copulation frequency and spermatophore transfer leading to a 38-61% decrease in E. herdmani nauplii production. These results suggest that mysid predators can constrain copepod population growth through non-consumptive processes. To determine the effects that resources can impose on copepod behavior, I explored the behavioral and fitness consequences of Temora longicornis ingesting Alexandrium fundyense, a phytoplankton species that forms harmful algal blooms in coastal Maine. My results suggest that ingesting A. fundyense causes copepods to swim faster and with more directional persistence compared to control algae. Temora longicornis increased their average swimming velocity by 24%, which leads to a 24-54% increase in their theoretical encounter rate with predators. Therefore, these findings suggest behaviorally mediated copepod-algal interactions may have significant impacts on harmful algal bloom dynamics and the fate of toxins in marine food webs.

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (M.S.)--University of North Carolina Wilmington, 2009. / Title from PDF title page (February 17, 2010) Includes bibliographical references (50-54)

Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D.)--Mississippi State University. Department of Plant and Soil Sciences. / Title from title screen. Includes bibliographical references.