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.

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 …

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, …

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 …

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) …