College of Agriculture & Natural Resources
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The collections in this community comprise faculty research works, as well as graduate theses and dissertations.
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Item OVARIAN STROMAL CELLS IMPROVE SURVIVAL, BUT NOT GROWTH, IN PRE- AND EARLY ANTRAL FELINE FOLLICLES(2024) Marks, Batsheva Naomi; Keefer, Carol; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ovarian stromal cells act as crucial support and regulators for in vivo folliculogenesis; however, less is known about their effect on in vitro grown follicles. The objective of this study was to investigate the impact of ovarian stromal cell co-culture or conditioned medium (CM) on survival and development of cat pre-, early, and antral follicles in vitro. Ovaries were obtained from cats older than six months (n = 3), then enzymatically digested to release stromal cells. The ovarian stromal cells were allowed to grow to confluency in a T75 flask, before being cryopreserved for long term storage in liquid nitrogen. Cells were thawed one week prior to follicular culture onset, and passaged once before CM collection. CM was subsequently removed 24 - 48 hours after feeding, and stored at -80C until used. Ovarian follicles were mechanically isolated from cats older than six months (n = 23 cats, 155 follicles), encapsulated in 0.5% alginate hydrogel. The isolated follicles were then divided into five treatment groups (control, ovarian stromal cell co-culture, 20% CM, 50% CM, and 100% CM in Endothelial Cell Growth Medium), and classified based on initial diameter as preantral (224.4 + 4.7 m), early antral (394.8 + 7.4 m), or antral (592.2 + 18.8 m). Culture subsequently lasted for 13 days, and survival and growth of the follicles were evaluated on Days 0, 4, 6, 8, 11 and 13. At the end of culture, follicles were assessed via qRT-PCR for expression of CYP19A, FSHR, and GDP9 to further quantify development. Statistical analysis was done in R software. Follicles in 100% CM had higher survival up to Day 11 of culture as compared to other treatment groups (Cox proportional hazards model, p < 0.01). Initial stage also influenced survival, with antral follicle survival significantly lower than that of pre- and early antral follicles (p < 0.0001). However, no differences in growth were detected across the treatment groups, nor across initial size classifications (Kruskal-Wallis test, p > 0.05). Post culture qRT-PCR analysis of the three selected genes showed upregulation of CYP19A in 50% CM follicles compared to the control (ANOVA, p < 0.05). However, there were no differences in CYP19A expression between the control and other treatment groups, or in GDF9 and FSHR expression among culture groups (p > 0.05). In summary, the findings demonstrated that conditioned medium collected from primary culture of ovarian stromal cells improves in vitro survival and modulates CYP19A expression of isolated cat follicles. Further research to identify paracrine factors present in conditioned medium will elucidate the roles of ovarian stromal cells pertaining to follicle survival during in vitro folliculogenesis.Item SPATIO-TEMPORAL ANALYSIS OF PHOTOTROPISM IN ARABIDOPSIS SEEDLINGS(2019) Pritchard, Candace; Murphy, Angus S; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Optimization of light capture during seedling development is a major determinant of plant fitness. As seedlings emerge from the soil, the processes of photomorphogenesis and phototropism optimize deployment of structures that capture light for photosynthesis. Photomorphogenesis produces hypocotyl thickening, cotyledon expansion, and chloroplast maturation. Concurrent phototropic responses initiated by blue light position the expanding cotyledons to maximize photosynthesis. The mechanisms underlying both processes have been explored for more than 140 years, but are still not fully understood. This dissertation seeks to provide a better understanding of phototropism by exploring the timing and localization of the constituent mechanisms downstream of the well-characterized perception of blue light by the PHOTOTROPIN photoreceptors. The experiments described herein characterize temporally and spatially distinct processes involved in asymmetric auxin accumulations that lead to differential hypocotyl elongation. To better identify the link between early perception and later auxin transport and elongation events, an open-air system was used to remove seedling hindrance and provide better spatio-temporal resolution. These experiments confirmed the more rapid bending conferred by loss of the ATP Binding Cassette class B (ABCB) 19 auxin efflux transporter and loss of differential elongation in the mid hypocotyl elongation zone in higher order pinformed mutants. However, apart from the enhancement of phototropic bending observed in abcb19 and pin4 mutants, no auxin transport mutants tested showed alterations in early phototropic responses, and no mutant exhibited a delay in the onset of phototropic bending. Recently identified CBC1 and CBC2 (CONVERGENCE OF BLUE LIGHT (BL) AND CO2 1/2) have been shown to act in downstream signaling during phot1-mediated regulation of stomatal conductance. Similarly, during phototropism cbc1cbc2 double mutants show early defects in phot1-mediated phototropism. Further, CBC1 and CBC2 have been shown to regulate S-type anion channels. Analysis of S-type anion channel mutants also reveals defects in early bending responses. These results point to blue light-dependent regulation of anion channel activity having an important role during the earliest stages of phototropism.Item TRIBUTYRIN, A BUTYRATE PRO-DRUG, AS A MUSCLE GROWTH PROMOTER IN A PORCINE MODEL(2018) Murray, Robert Len; Stahl, Chad H.; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Postnatal muscle growth is dependent on myonuclear accretion and subsequent protein synthesis and accumulation. Altering the ability of muscle resident stem cells (satellite cells, SC), which provide nuclei to the growing muscle fiber, to progress through their myogenic lineage can have lifelong effects on muscle growth and repair. The use of butyrate, a histone deacetylase (HDAC) inhibitor, has promoted SC myogenesis in vitro. In animal models, dietary butyrate has improved growth performance and has had promising results in treating myopathic conditions, but the impact of dietary butyrate on SC activity and the direct promotion of muscle growth have not been examined. In the first experiment, we investigated whether the use of a physiologically relevant concentration of butyrate (0, 0.1, 0.5, or 1mM) could enhance the myogenic potential of porcine SC. We found increased expression of myogenin, the late stage myogenic regulatory factor, and increased SC fusion in the treated groups, suggesting an increased capacity during terminal differentiation. We further tested this hypothesis by conducting an animal study to test the effects of tributyrin, a butyrate pro-drug, on in vivo SC behavior. We found that tributyrin inclusion (0.25% and 0.5%) into the milk replacer of neonatal piglets led to significant increases in muscle DNA content, suggesting increased myonuclear accumulation. The neonatal portion (21-days) of the study was repeated, and the animals were then weaned and crossed into dry nursery treatment diets. At the end of the 37-day nursery feeding, animals that received tributyrin in their milk replacer formula weighed significantly more and had increased muscle hypertrophy than the untreated animals. Also, SC from treated animals responded similarly ex vivo to those in the first experiment, suggesting an increased capacity to progress through the differentiation process. We determined in our final experiment that tributyrin supplementation altered the epigenetic landscape of SC by globally reducing the transcriptionally repressive chromatin mark H3K27me3 around key genes. In addition, we found that this is not the sole mechanism through which butyrate alters SC behavior. These findings help support the notion that tributyrin may be used to enhance muscle growth and could prove useful at treating myopathies.Item THE REGULATION OF THE INTESTINAL COPPER EXPORTER IS COORDINATED WITH SYSTEMIC COPPER HOMEOSTASIS(2017) Chun, Haarin; Kim, Byung-Eun; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Copper (Cu) plays key catalytic and regulatory roles in biochemical reactions essential for normal growth, development, and health. Defects in Cu metabolism cause Menkes and Wilson’s disease, myeloneuropathy, and cardiovascular disease and are associated with other pathophysiological states. Consequently, it is critical to understand the mechanisms by which organisms control the acquisition, distribution, and utilization of Cu. While it is well established that the enterocyte is a key regulatory point for Cu absorption into the body, how the intestine responds to systemic Cu requirements is poorly understood. Here, we demonstrate that fine-tuned Cu homeostasis is required for normal growth and development in C. elegans. Moreover, we show that CUA-1, the ATP7A/B homolog in worms, localizes to lysosome-like organelles (gut granules) in the intestine under Cu-overload conditions for Cu detoxification, while Cu-deficiency results in a redistribution of CUA-1 to basolateral membranes for Cu efflux to peripheral tissues. Defects in gut granule biogenesis exhibit result in abnormal Cu sequestration and increased susceptibility to toxic Cu levels. Our studies establish that CUA-1 is a key intestinal Cu exporter, and that its trafficking is regulated in response to systemic Cu status in worms. In addition, while the Cu transporter ATP7A plays a major role in both intestinal Cu mobilization to the periphery and prevention of Cu over-accumulation, it is unclear how regulation of ATP7A contributes to Cu homeostasis in response to systemic Cu fluctuation in mammals. Here we show, using Cu-deficient mouse models, that steady-state levels of ATP7A are lower in peripheral tissues (including the heart, spleen, and liver) under Cu deficiency and that subcutaneous administration of Cu to these animals restore normal ATP7A levels in these tissues. Importantly, ATP7A in the intestine is regulated in the opposite manner - low systemic Cu increases ATP7A while subcutaneous Cu administration decreases ATP7A suggesting that intestine-specific non-autonomous regulation of ATP7A abundance may serve as a key homeostatic control for Cu export into the circulation. Altogether, our results implicate CUA-1/ATP7A Cu exporter in the intestine as a key modulator for organismal Cu homeostasis in metazoans.Item The effect of freezing rate on striped bass (Morone saxatilis) spermatozoa(2013) Frankel, Tyler Edward; Woods, Lewis C; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Several studies have attempted to determine the optimal freezing rate for the cryopreservation of striped bass spermatozoa. In this study, the effects of freezing rate (-10, -15, -20 and -40oC/min) on sperm quality was examined utilizing Sybr-14/Propidium Iodide staining to confirm membrane integrity, a luciferin-luciferase assay to estimate ATP concentration and a Hamilton-Thorne CEROSTM system to characterize sperm motion. Males (n=12) were sampled once a week for five weeks. Samples were extended, cryo-protected using a 7.5% (v/v) dimethyl sulfoxide solution, and frozen using a controlled-rate freezer. Samples were stored in liquid nitrogen for 49 days and re-evaluated post-thaw. Sperm cryopreserved at -40oC per minute resulted in: total motility (10.06%), progressive motility (7.14%), ATP concentration (0.86 pmol/million cells), and sperm viability (56.5%); which were greater (P < 0.05) than the slower rates. Results demonstrate that -40oC/minute was the optimal freezing rate among those tested for the cryopreservation of striped bass spermatozoa.Item The comparative effects of three Sedum species on green roof stormwater retention(2013) Starry, Olyssa; Lea-Cox, John; Plant Science and Landscape Architecture (PSLA); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Green roofs are typically dominated by Sedum species because they can tolerate hot, xeric environments. However, due to their high water use efficiency, some have questioned the selection of these species for stormwater management. We investigated (1) how three common Sedum species contribute to overall stormwater retention efficiency by green roofs in the mid-Atlantic region, and (2) whether species-specific differences in water use could be explained by morphological and physiological characteristics. Water use and CO2 exchange were continuously monitored in growth chamber studies under increasing drought stress for S. album, and S. kamtschaticum, two species known to variably cycle between CAM and C3 metabolisms. Under fall temperature conditions, S. kamtschaticum had gas exchange rates akin to C3 photosynthesis and used 35% more water compared to S. album. Interestingly, S. album conserved water and had malic acid accumulation confirming CAM metabolism for the duration of the experiment, even under well-watered conditions. In field studies, sixteen replicate green roof platforms (n=4 per species) were planted with S. album, S. kamtschaticum, S. sexangulare, or left unplanted during summer 2010. The platforms were monitored intensively for canopy growth, leaf area, root biomass, substrate moisture and runoff for two years (2011 and 2012). Plant treatment effects on stormwater runoff were significant, but most discernible for small and intermediate-sized rainfall events less than 62.5mm. The two species with the greatest stormwater retention efficiencies, S. kamtschaticumand S. sexangulare, also had the highest rates of evapotranspiration (ET), and higher ET rates resulted in less total runoff. Because evapotranspiration was identified as important for predicting performance by plants in the field study, I investigated how ET data from this study, combined with environmental data collected from a weather station at the study site, could be used to improve the application of the FAO56 Penman-Monteith evapotranspiration equations to green roofs. The incorporation of specific seasonal crop coefficients were found to improve correlations between predicted and measured rates of ET and these coefficients were related to plant characteristics. The refinement of ET equations can lead to more accurate hydrologic models of green roofs and design and management tools.