Developing chicken embryos have two ductus arteriosus (DA) that shunt blood away from the lungs and to the chorioallantoic membrane, the embryonic gas exchanger. In mammals, DA closure is stimulated by an increase in blood gas O2 that occurs as the animal begins to breathe with its lungs. The goal of this study was to determine the influence of O2 levels during incubation on the vascular reactivity and morphology of the O2-sensitive DA and to examine the effects of changing O2 levels during late incubation on the morphology of the DA from chicken embryos. In comparison to normoxia, hypoxia (15%) reduced venous O2 levels in day 16 and day 18 embryos and reduced aircell O2 values in day 16, day 18, and internally pipped (IP) embryos, whereas hyperoxia (30%) increased venous O2 levels and aircell O2 level in day 16, day 18, and IP embryos. In comparison to normoxia, hypoxia delayed closure of the DA, whereas hyperoxia accelerated DA closure. In comparison to the left DA from externally pipped (EP) normoxic embryos, the left DA from EP hypoxic embryos exhibited a significantly weaker contractile response to O2. The DA from day 18 hypoxic embryos exhibited a significantly weaker contractile response …

Plant estrogens have long been associated with health benefits. The potential of tissue culture techniques for the production of several secondary metabolites has been known for many years. Tissue cultures stimulate the production or induce the biosynthesis of novel compounds not found in the mature plant. Tissue culture of Morus alba, family Moraceae, is known to contain phytoestrogens, was established on plant-hormone supplemented Murashige and Skoog (MS) medium. Petiole and the stem tissue from mature trees were the best explants for initiation and proliferation of calli. The best callus proliferation was obtained on MS medium containing 1-napthalene acetic acid (1mg/ml) and benzylaminopurine (0.5mg/ml) for M. alba. Comparison of phytoestrogens of Moraceae species from in vivo and in vitro tissue isolation were carried out. The estrogenic activities of callus extracts were assayed in an estrogen-responsive yeast system expressing the human estrogen receptor alpha. Male callus extracts had higher estrogenic activity than male and female extracts from in vivo and in vitro tissues. Isolation and characterization of phytoestrogens from above tissues were carried out using solid phase extraction, high perfomance liquid chromatography and mass spectrometry techniques. Biochanin A, an isoflavonoid, was isolated as one of the compounds in male callus extracts. Biochanin …

Cryopreservation in suspension is commonplace for a variety of cell types. However, cryopreservation of adherent cells has achieved limited success. This research aimed to cryopreserve adherent nerve cell networks in vitro in a manner that preserved network morphology and physiology. Successful implementation would enable long term storage of adherent neuronal networks on microelectrode arrays and on-demand access for use in pharmacological and toxicological testing. Based upon morphological assessments, excellent post-thaw preservation was obtained and post-thaw cultures survived in a transitional medium for up to 3.5 hours. However, transitions to native culture medium post-thaw presented difficulties, ultimately resulting in necrosis. A discussion of methods to supplement the current research and increase post-thaw viability is included in the thesis.

Early development of O2 chemoreception and hypoxic responses under normoxic (150 mmHg) and chronically hypoxic (110 mmHg) conditions were investigated in Xenopus laevis from hatching to 3 weeks post fertilization. Development, growth, O2 consumption, ventilatory and cardiac performance, and branchial neuroepithelial cells (NEC) density and size were determined. At 3 days post fertilization (dpf), larvae started gill ventilation at a rate of 28 ± 4 beats/min and showed increased frequency to 60 ± 2 beats/min at a PO2 of 30 mmHg. Also at 3 dpf, NECs were identified in the gill filament buds using immunohistochemical methods. Lung ventilation began at 5 dpf and exhibited a 3-fold increase in frequency from normoxia to a PO2 of 30 mmHg. Hypoxic tachycardia developed at 5 dpf, causing an increase of 20 beats/min in heart rate, which led to a 2-fold increase in mass-specific cardiac output at a PO2 of 70 mmHg. At 10 dpf, gill ventilatory sensitivity to hypoxia increased, which was associated with the increase in NEC density, from 15 ± 1 to 29 ± 2 cells/mm of filament at 5 and 10 dpf, respectively. Unlike the elevated rate, cardiac and ventilatory volumes were independent of acute hypoxia. Despite increased cardioventilatory frequency, …

As selection pressures exist for not only adults, but for every life history stage, it is important to understand how environmental factors shape developing animals. Despite the significance placed on aquatic hypoxia as a driving force in the evolution of air breathing, this is the first known study to examine the effects of hypoxia on cardio-respiratory ontogeny of an air breathing fish. Blue gouramis are obligatory air breathing fish that possess a labyrinth-like structure that serves as the air breathing organ. Gouramis were reared for up to 90 d in normoxia or hypoxia, and morphological and physiological development was observed. Hypoxic larvae had increased lamellar and labyrinth organ surface areas. Bradycardia and increased gill ventilation rates were observed when larvae from either rearing group were briefly exposed to hypoxia. Hypoxic larvae also showed a reduced heart rate and gill ventilation rate in the absence of a hypoxic stimulus, possibly indicative of a more comprehensive, long-term respiratory plasticity. The similarity of routine oxygen consumption between rearing groups suggests that metabolic demand did not change for hypoxic larvae, but that they were more efficient at oxygen acquisition. This is further supported by increased resistance time of hypoxic gouramis to extreme hypoxia. The …

Triclosan and triclocarban are antimicrobials found in numerous consumer products, while caffeine is the most commonly consumed stimulant by humans. This study was undertaken to determine the effects of triclosan, triclocarban, and caffeine on the development and physiology of amphibian larvae. LC50 values of triclosan and triclocarban were determined after 96 hours for three North American larval species: Acris crepitans blanchardii, Bufo woodhousii woodhousii, Rana sphenocephala, and for a common amphibian developmental model: Xenopus laevis. Amphibian larvae were most sensitive to triclosan and triclocarban exposure during early development based upon 96-hour LC50 values. Heart rates for X. laevis and North American larvae exposed to triclosan were variable throughout development. However, significantly lower heart rates were observed in all larvae exposed to triclocarban. Metabolic rates of X. laevis and R. sphenocephala larvae exposed to triclosan were significantly affected in larvae exposed to ½ LC50 and the LC50 concentration. Metabolic rates of X. laevis larvae exposed to triclocarban were significantly affected by exposure to ½ LC50 concentrations in three of four stages investigated. No significant differences were observed in North American larvae exposed to triclocarban. Tissue uptake, lipid uptake, tissue bioconcentration factor (BCF) and lipid BCF of triclosan and triclocarban were investigated …

Fetal programming results from stressors during fetal development and may influence the occurrence of disease later in life. Maternal nutritional status and/or environment can affect renal development by inducing limited nephron endowment at birth, which results in diseases such as hypertension and coronary heart disease in mammals. Birds are likely to be effective models for this process because, like mammals, they have high pressure cardiovascular systems, mammalian-type nephrons and are homeothermic. This project uses the chicken embryo to explore physiological responses of disrupted hydration state thereby providing insights into renal fetal programming. Under normal conditions the chorioallantoic membrane (CAM) and developing avian kidney work in unison to ensure a proper balance of ions and water within the egg. White leghorn chicken eggs were incubated at 37.5oC±0.5oC and either <35%, 55-60% (normal) or >85% relative humidity. Amniotic fluid serves as the drinking source for the embryo late in development; its composition is important to salt and water homeostasis. High amniotic fluid osmolality increased the blood osmolality for embryos exposed to low humidity incubation thereby indirectly influencing the renal developmental program of the embryos from this group. Indeed estimated filtering capacity was doubled in the low humidity group (6.77 ± 0.43 mm3) …

Weekly collections were conducted from May to December, 2007 (153 trap nights, total) in Denton, Texas, in and around large storm drains and overpass drainage facilities in residential and non-residential areas, using Centers for Disease Control (CDC) light traps and gravid traps. A total of 1964 mosquitoes were collected, representing 24 species within 6 genera: Aedes, Anopheles, Culiseta, Culex, Psorophora, and Uranotaenia. Culex was the most abundant genus, representing 75% of all mosquitoes collected; Aedes was the second most abundant, representing 12 % of all mosquitoes collected. Cx. quinquefasciatus was the dominant species collected via gravid traps; Cx. (Melanoconion) species were the dominant species collected via CDC light traps. Data of gravid traps and light traps were analyzed separately using nonparametric correlation analysis, comparing environmental data and physical characteristics to total abundance of mosquitoes. There was no significant correlation found when comparing the three dominant species collected in light traps (unidentified Cx. (Melanoconion) sp, Cx. quinquefasciatus, and Ae. vexans) to environmental characteristics and physical characteristics. Analysis of Cx. quinquefasciatus collected in gravid traps indicated no significant correlation between abundance, environmental data, and physical characteristics. Linear regression models were analyzed to determine if either environmental variables or physical characteristics of the …

Oxygen deprivation can be pathological for many organisms, including humans. Consequently, there are several biologically and economically relevant negative impacts associated with oxygen deprivation. Developing an understanding of which genes can influence survival of oxygen deprivation will enable the formulation of more effective policies and practices. In this dissertation, genes that influence adult anoxia survival in the model metazoan system, C. elegans, are identified and characterized. Insulin-like signaling, gonad function and gender have been shown to influence longevity and stress resistance in the soil nematode, C. elegans. Thus, either of these two processes or gender may influence anoxia survival. The hypothesis that insulin-like signaling alters anoxia survival in C. elegans is tested in Aim I. The hypotheses that gonad function or gender modulates anoxia survival are tested in Aim II. Insulin-like signaling affects anoxia survival in C. elegans. Reduction of insulin-like signaling through mutation of the insulin-like receptor, DAF-2, increases anoxia survival rates in a gpd-2/3 dependent manner. The glycolytic genes gpd-2/3 are necessary for wild-type response to anoxia, and sufficient for increasing anoxia survival through overexpression. Gonad function and gender both affect anoxia survival in C. elegans. A reduction of ovulation and oocyte maturation, as measured by oocyte flux, …

Cardiac, metabolic and growth response of early-stage chicken embryos to perturbations in yolk environment was investigated. Also, effects of parental hypoxia exposure on hypoxia resistance, thermal tolerance and body length of zebrafish larvae were investigated. In the first study, thyroxine, triiodothyronine and testosterone produced differential effects on heart rate and development rate of chicken embryos during the first 4 days of development. Triiodothyronine caused a dose-dependent increase in heart rate when applied at 40 or 70 hours of age, while thyroxine caused a dose-dependent increase in heart rate when applied at 40 hours only. Testosterone and propyl-thiouracil (deiodinase antagonist) did not have an effect on heart rate. Development rate was not changed by thyroxine, triiodothyronine, testosterone or propyl-thiouracil, which suggested that heart rate changes did not result from changes in embryo maturity. In the second study, chicken embryos exposed to yolks of different bird species during early-stage embryonic development showed changes in heart rate, mass-specific oxygen consumption and body mass that scaled with the egg mass, incubation period length, and yolk triiodothyronine and testosterone levels of the species from which yolk was derived. In the third study, this phenomenon was investigated between layer and broiler chickens. Heart rate, oxygen consumption …

Raptor rehabilitation has become commonplace globally, yet studies on the survival and adaptation of great horned owls (Bubo virginianus) after release has been neglected to an appreciable extent. The primary objective of this study is to provide quantitative data on the success of rehabilitated and released great horned owls in the North Texas region. Owls (N=12) were rehabilitated and released onto the Ray Roberts Greenbelt Corridor in Denton County, Texas, and monitored using radio telemetry to evaluate home range (November 2002 - February 2005). With approximately 75% of the birds released for this study surviving until transmitter battery failure, it is believed that the rehabilitation process was successful for these birds.

This thesis describes the development of an optimized method for culturing dissociated, monolayer neuronal networks from murine frontal cortex and midbrain. It is presented as a guidebook for use by cell culture specialists and laboratory personnel who require updated and complete procedures for use with microelectrode array (MEA) recording technology. Specific cell culture protocols, contamination prevention and control, as well common problems encountered within the cell culture facility, are discussed. This volume offers value and utility to the rapidly expanding fields of MEA recording and neuronal cell culture. Due to increasing interest in determining the mechanisms underlying Parkinson's disease, the newly developed procedures for mesencephalon isolation and culture on MEAs are an important research contribution.

The chicken ductus arteriosi (DA) are two embryonic blood vessels that shunt blood away from the non-ventilated lungs and towards the body and chorioallantoic membrane. I show that prostaglandins have a diminished role in maintaining chicken DA patency and nitric oxide inhibits oxygen induced contraction of the day 19 proximal DA in a time dependent manner. The pathways governing oxygen induced contraction in the chicken DA are similar to those found in mammals and include contributions from ROS, Kv channels, L-type Ca2+ channels, and the Rho kinase pathway. Longer exposure to high oxygen generates increased oxygen induced constriction of the day 19 DA that may be mediated through the Rho kinase pathway.

The rapid development of once-rural landscapes often produces detrimental effects on surface water quality entering local reservoirs through vulnerable stream channels. This study presents a methodology that incorporates geographic information systems (GIS) and remote sensing techniques for the creation of a stream corridor evaluation mechanism, coined the water quality corridor management (WQCM) model. Specifically, the study focuses on determining the viability of the WQCM model in assessing the stream corridor conditions within a northern Denton County pilot study region. These results will aid in the prediction and evaluation of the quality of stream water entering reservoirs that serve as the primary drinking water source for local municipalities.

A bacterial challenge assay was developed utilizing the earthworm, Lumbricus terrestris, in order to assess potential immunotoxic effects from exposure to specific polychlorinated biphenyl congeners. Earthworms were inoculated with Aeromonous hydrophila, establishing a 10-day LD50. In vitro assays for effects of PCBs on phagocytosis agreed with mammalian studies, demonstrating potent suppression of phagocytosis by the non-coplanar PCB congener 138 and no suppression by the coplanar congener 126. However, when the effects of the two PCB congeners were evaluated for suppression of resistance to a whole animal infection challenge assay, coplanar PCB 126 decreased the ability of L. terrestris to withstand infection while non-coplanar PCB 138 did not.

Established interspecific metabolic allometric relationships do not adequately describe the complexity and variable physiological states of developing animals. Consequently, intraspecific allometric relationships of oxygen consumption and carbon dioxide production as a function of body mass; the respiratory quotient; the function of the silk cocoon; and body composition were investigated for each distinct developmental stage of the silkworm, Bombyx mori. Whole animal O2 consumption in Bombyx ranged from 0.00064 + 0.000047 ml O2 .hr-1 at larval instar I to 0.77 + 0.06 ml O2 .hr-1 in pre-pupal, falling to 0.21+ 0.01 ml O2 .hr-1 in the pupae. Those instars having a significant relationship between O2 consumption as a function of body mass, the slope of the line relating O2 consumption to body mass varied between 0.99 and 1.02, while across all instars the slope was 0.82. Developmental allometry should be presented for individual developmental stages because the individual allometric exponents of the stages can be significantly different from the overall allometric exponent throughout development and in some cases, the overall allometric exponent can be a statistical artifact. The first larval instar of Bombyx mori has the lowest cross sectional area of high metabolic tissue of the midgut (27%) and had one …

The black-tailed prairie dog (Cynomys ludovicianus) has experienced dramatic declines over much of its historical range due to habitat loss, plague, poisonings, and shootings. Many populations now occur as isolated genetic relicts. A multiple locus genetic profile was obtained using microsatellite analyses of six polymorphic nucleotide repeats from 319 black-tailed prairie dogs collected from 16 colonies throughout the state of Texas. This assessment revealed that existing populations have sufficient variation at all six loci to verify the usefulness of this approach as a primary genetic tool in conservation and preservation. The data reveals regional-dependent frequency patterns as well as support for founder/bottleneck effects for several of the 16 sites. Although the prairie dog population in Texas as a whole may appear genetically diverse, considerable genetic divergence has already occurred among the subpopulations (FST = 0.164). Isolation by distance is supported by genic differentiation analysis (P < 0.001) and pairwise correlation analysis between genetic distance and geographic distance (P < 0.001). Prairie dogs from six (COC, LUBA, LUBC, LUBD, LUBE, and TAR) of the original 16 sites have been relocated or exterminated, or were in the process of being relocated. Results indicated the following colonies (COT, DAL, HOW, and HUD) are …

Superior swimming stamina in adult fish is presumably passed on to their offspring, but the ontogeny of the appearance of superior stamina and the requisite enhanced cardio-respiratory support for locomotion in larval fishes has not been determined. Is the expression of the suite of parental traits enabling superior swimming stamina in their offspring dependent upon their achieving juvenile/adult morphology, or does it appear earlier in their larvae? To answer this, adults were classified into three groups based on swimming stamina, followed by measurement of length, mass, and width. Larval offspring from the two parental groups -high stamina larvae (HSL) and low stamina larvae (LSL)- were reared at 27°C in aerated water (21% O2). Routine and active heart rate, routine and active mass specific oxygen consumption were recorded through 21dpf, and cost of transport (COT) and factorial aerobic scope were derived from oxygen consumption measurements. Routine heart rate at 2dpf of LSL was 164 ± 1 b·min-1, compared to only 125 ± 2 b·min-1 for HSL. Routine heart rate subsequently peaked at 203 ± 1 b·min-1 at 5dpf in the HSL group, compared to 207 ± 1 b·min-1, at 4dpf in the LSP larvae. Active heart rate at 5 dpf of …

Proteomic techniques were used to evaluate the protein profile of the earthworm, (Lumbricus terrestris), following a bacterial challenge. One control group received no injection; a second control group received injections of phosphate buffer solution (PBS). The experimental group received injections of PBS containing (Aeromonas hydrophila). After incubation for 12 hours at 20°C, coelomic fluid was collected from each group for analysis by 2-D electrophoresis. There were significant differences in spot appearance and density between control and experimental groups. Sixteen spots showed a two-fold increase in density and 63 showed at least a two-fold decrease in density between samples from control and bacteria-challenged earthworms, respectively, suggesting up- and down-modulation of proteins potentially involved in the earthworm's response to bacterial challenge.

Dispersal of aquatic insects is difficult to measure with traditional direct trapping methodologies. However, genetic markers are an ideal surrogate to indirectly infer dispersal and gene flow. For this research, a portion of the cytochrome oxidase I gene was used to evaluate gene flow and dispersal of Epeorus pleuralis located in the northern Appalachian headwater streams of the Allegheny, Genesee, and Susquehanna watersheds. A total of 536 basepairs from 16 individual insects were used for analysis. Thirteen haplotypes were discovered, two of which were shared between the Allegheny and Genesee streams. Although no shared haplotypes were found in the Susquehanna, analysis of molecular variance results suggest that there is not a significant genetic difference between the three populations and attributes the majority of variation to within population differences.

Growth and element assimilation was investigated in post oak seedlings exposed to four different treatment combinations of fertilization and ectomycorrhizal inoculation. Element concentration in excised leaves was analyzed via particle induced X-ray emission spectrometry with a 1.8 MeV proton macrobeam. Mean growth was significantly different across the treatment groups as well as mean concentration of Mg, Al, S, K, Ca, Fe, Cu, and Zn. The data suggest that fertilization rather than mycorrhizal inoculation had a stronger influence on plant growth and nutrient uptake. A follow up study was conducted with a 3 MeV microbeam. A 850 μm2 scanned area of a post oak leaf produced topographical maps of 11 elements.

A study of the life history and ecology of Camelobaetidius variabilis was conducted in Honey Creek, OK from February 2003-April 2004. Nymph development was assessed using changes in external morphology. Laboratory reared nymphs were used to calculate number of degree days to complete development (772 degree days at 20.8° C ±.38° C), which was used to determine voltinism. Field collected nymph microhabitat distribution was used in assessing microhabitat distribution. Nymphal thermoregulation was assessed during the winter and spring by comparing nymphal numbers present in shaded and un-shaded habitats. Camelobaetidius variabilis nymphs showed preference for algal microhabitats during the spring and leaf packs in the winter. Nymphs inhabited leaf packs to increase metabolic rate during the winter. Increased temperatures aid in development of nymphs. Camelobaetidius variabilis exhibited a multivoltine life cycle with six overlapping generations.

The development of cholinergic neurons in mouse frontal cortex was studied both in vivo and in vitro by immunocytochemistry with an antibody to choline acetyltransferase (ChAT), the enzyme responsible for acetylcholine synthesis. While cortical cholinergic neurons have previously been characterized in rat cortex, up until very recently, intrinsic cortical cholinergic neurons were considered to be absent in mouse, and little is known about their development or phenotypic characteristics. The present study found no ChAT-positive neurons in mouse frontal cortex on postnatal day 0 (P0, the day of birth). On P7 there were few, faintly stained, ChAT-positive neurons. The numerical density of ChAT-positive neurons increased substantially with age, from none on P0, to 9.2 + 1.4 on P7, to 14.8 + 0.9 on P16, and 41.6 + 3.9 in adulthood. Considering that the numerical density of total neurons decreases during this postnatal period, the data represent a marked developmental increase in the percentage of cholinergic neurons. The development of cholinergic neurons showed very similar timelines in rat and mouse frontal cortex. Cultures prepared from mouse frontal cortex on embryonic day 16 were maintained for 25, 76, or 100 days in vitro (div). The percentage of ChAT-positive neurons was considerably higher than …

Primary cilia are non-motile organelles having 9+0 microtubules that project from the basal body of the cell. While the main purpose of motile cilia in mammalian cells is to move fluid or mucus over the cell surface, the purpose of primary cilia has remained elusive for the most part. Primary cilia are shortened in the kidney tubules of Tg737orpk mice, which have polycystic kidney disease due to ciliary defects. The product of the Tg737 gene is polaris, which is directly involved in a microtubule-dependent transport process called intraflagellar transport (IFT). In order to determine the importance of polaris in the development of neuronal cilia, cilium length and numerical density of cilia were quantitatively assessed in six different brain regions on postnatal days 14 and 31 in Tg737orpk mutant and wildtype mice. Our results indicate that the polaris mutation leads to shortening of cilia as well as decreased percentage of ciliated neurons in all brain regions that were quantitatively assessed. Maintainance of cilia was especially affected in the ventromedial nucleus of the hypothalamus. Furthermore, the polaris mutation curtailed cilium length more severely on postnatal day 31 than postnatal day 14. These data suggests that even after ciliogenesis, intraflagellar transport is necessary …