Thesis (MA)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Thesis (Ph. D.)--Montana State University--Bozeman, 2004. / Title from PDF t.p. (viewed on June 12, 2006). Chairperson, Graduate Committee: Thomas McMahon. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xvi, 105 p.; also includes graphics (some col.) Includes bibliographical references (p. 95-105). Available online via OhioLINK's ETD Center

Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 40-46). Also available on the World Wide Web.

Replacement of fish meal as the primary protein source in diets for farmed carnivorous fish is a major priority for sustainability of the aquaculture industry. Three plant-based protein sources (soybeans, field peas, and canola) were investigated to compare their effects on the health and performance of rainbow trout (Oncorhynchus mykiss) and to identify significant anti-nutritional factors (ANFs). Six separate 8-week studies were conducted, over a period of one year, to assess the effects of protein source and processing level (meal versus protein concentrate) at dietary inclusion rates of 0 to 300 g kg-1. Abundance of inflammatory and immune marker transcripts including proliferating cell nuclear antigen (PCNA), immunoglobulin M (IgM), interleukin-1 beta (IL-1β), interleukin-8 (IL-8), and interleukin-10 (IL-10) was evaluated in distal intestinal tissue by quantitative PCR (qPCR) analysis. Activity of the pro-apoptotic enzyme caspase-3 was also assayed in distal intestinal tissue. Transcript abundance was highly variable and no suitable genes for the internal normalization of qPCR data could be identified. As a result, transcript copy numbers were reported per 50 ng of total RNA. At 300 g kg-1 inclusion, soybean meal (SBM) increased abundance of IL-8 and IgM, pea meal (PM) increased abundance of IL-10, and canola protein concentrate (CPC) increased abundance of IL-8. Pea protein concentrate (PPC) reduced IL-8 abundance and caspase-3 activity, while increasing abundance of IL-10. Canola meal (CM) and soy protein concentrate (SPC) did not significantly affect the transcript abundance of any assayed gene. Pearson correlation coefficients were determined between gene transcript abundance, performance parameters, protein source, inclusion level, and ANF content. Specific growth rate (SGR) was negatively correlated with the abundance of IL-1β and IgM. Dietary inclusion of SBM was positively correlated with all assayed proinflammatory markers and negatively correlated with SGR. Inclusion of PM was positively correlated with both SGR and the abundance of IL-10. The inclusion of CM was negatively correlated with average daily feed intake (ADFI) and with the abundance of both IL-8 and PCNA. Inclusion of PPC correlated positively with SGR and negatively with the activity of caspase-3. Correlation between transcript abundance and dietary content of putative ANFs suggested negative correlations between glucosinolate content, proinflammatory cytokine expression, SGR, and ADFI; whereas, isoflavone content was positively correlated with proinflammatory markers and negatively correlated with SGR. In conclusion, although high SBM and CM inclusion levels have been associated with reduced growth performance in trout, only SBM was associated with increased abundance of inflammatory marker transcripts. These contrasting responses may be mediated by CM glucosinolates, which could negatively affect palatability without inducing a pro-inflammatory response. Dietary PM was very well tolerated and may have promoted anti-inflammatory activity. Further processing of protein meals to concentrates markedly reduced any observable negative impact on performance parameters and the abundance of inflammatory marker mRNA transcripts. Interestingly, both PM and PPC were positively correlated with SGR and may contain a beneficial anti-inflammatory component.

The aim of the work will be based on the diet composition monitoring of the introduced rainbow trout and wild brown trout and grayling in defined time intervals, and assessing the degree of the food competition among the monitored species. The experiment was held in the second half of September 2010 in the river Blanice in the protected fish areas close to the village Těšovice, where several dozens of rainbow trout were stocked. In a predefined several - day interval of were caught 30 pieces of the rainbow trout (SL = 266 ? 14 mm, m = 347 ? 54 g), 33 brown trout (SL = 244 ? 19 mm, m = 219 ? 51 g), 14 graylings (SL = 256 ? 27 mm, m = 240 ? 91 g) and 12 resident rainbow trout (SL = 255 ? 13 mm, m = 280 ? 47 g) originated from the last plant. Within these groups a fish stomach content and consequently food selectivity was examined by using of the Ivlev's selectivity index (1961), a food competition was examined by using of the Schoener's index food overlap (1970) and the importance of various food components was examined by using of the index of preponderance according to Natarajan (1961). Rainbow trout began to ingest food immediately after the stocking into the stream and its occurrence was seen in all fish crop caught during the period. The insect (Insecta) was given as a preferential food of the rainbow trout and according to the index of preponderance (IP), it formed 54%, Trichoptera (IP) 24% and Ephemeroptera (IP) 19% orders belonged to the most preferable kinds of the food. According to the index of preponderance, gastropod (Gastropoda) formed (IP) 25% of a secondary food. The rainbow trout was accepting the same dietary components as well as the original sorts of salmonoid fish within the river. According to the index of a food overlap (S), the food competition between rainbow trout and brown trout was equal to the value of (S = 0.566) during the whole monitored period and the grayling was equal to (S = 0.520). Even though both of these values are slightly below the significant food overlap (S = 0.6), we can still say that the food competition is relatively high among these kinds.

Ecotoxicity testing of chemicals for environmental risk assessment is an area where a high number of vertebrates are used across a variety of industrial sectors. The application of the 3Rs in toxicity testing using fish address both the ethical and societal concerns around this issue in addition to the increasing legislative requests for the incorporation of animal alternatives. This thesis aims to highlight the potential of 3D cell culture models to "bridge the gap" between in vitro and in vivo screening procedures for testing of chemicals with the potential to persist or bioaccumulate thereby improving the predictive power of screening procedures. This thesis examines two alternative methods for their potential use as an intestinal based toxicokinetic tool for environmental risk assessment, utilising an in vitro fish cell line replacement tool (RTgutGC). In addition, for the first time a new intestinal primary cell culture based model was developed to address both intestine region specific response (pyloric, anterior, mid and posterior) and size related adaptability to toxins. Paramagnetic oximetry was used to measure oxygen content within 3D structures (spheroids) in order to better understand the microenvironment of these culture models. Using histology, immunohistochemistry, transepithelial electrical resistance (TEER), transmission electron microscopy (TEM), metabolic, fluorescence and gene expression assays, the comparability of this system to native intestinal response was established. Following exposure to carefully chosen environmental contaminants (Benzo[a]pyrene and Copper), the RTgutGC cell line demonstrated comparable responses to existing literature in terms of uptake, metabolism, DNA damage and the presence an equivalent saturable level. Primary enterocytes cultured on transwell inserts remained viable for upto six weeks, with permeability and metabolic activity comparable to native tissue (both in vitro and ex vivo). Taken in combination, these features of enterocytes represent a profile more closely representative of the intestine then the widely used "gut sac" method. With the potential advantages of incorporating complexity at differing levels (connective tissue layer, intestinal bacteria biome), the intestinal models described offer the potential to screen highly persistent toxins which may require prolonged incubation, in addition to the exploration of complex experimental designs which minimise animal usage (uptake, depuration, uptake). As a consequence, the models developed within this thesis significantly enrich the emerging fish based in vitro testing strategies.

Pharmaceutical pollutants have been aim of many recent aquatic environment studies. Due to human activities surface waters are polluted with plenty of substances, including products of personal care, which are ranked among a number of pharmaceuticals. The main goal of this work is to assess the impact of environmentally relevant concentrations of the synthetic glucocorticoid dexamethasone on aquatic organisms through biomarkers of oxidative stress. The principle of this experiment was chronic in vivo exposure model organisms (rainbow trout) to doses of this drug and the subsequent monitoring activities of antioxidant enzyme system in the liver and gill tissues. Experimental results have demonstrated a response enzymatic protection mechanism on the increased production of reactive oxygen species induced by the drug at environmentally relevant concentrations especially in gill tissue. Our results illustrate a real risk that this xenobiotic represents to aquatic organisms.

The intestine and associated bacterial microbiota have a central role the physiology and homoeostasis of the host. The understanding of how farming conditions affect the intestine and associated microbiota of fish is the high importance to counteract the potential threats to health and welfare. Thus, this thesis aims to understand the role of stressful husbandry conditions on the intestine and associated microbiota of rainbow trout and Atlantic salmon. Within this context, the role of Pediococcus acidilactici as health promoter was also investigated Chapter 3 investigated the replacement of fishmeal by different plant protein ingredients in rainbow trout. The results of this chapter revealed that the effect of P. acidilactici on the microbiota of distal intestine in rainbow trout was dependent on the ingredients of the diet. The results also showed that the FM substitution induced major changes in the intestinal microbiota. Moreover, the modulation induced by plant-based diets on the microbiota varied according to the ingredients used. Chapter 4 studied the effect of dietary oxytetracycline in the distal intestinal microbiota of rainbow trout and the role of P. acidilactici to ameliorate the impact of antibiotic therapy. Experimental groups fed the diets with oxytetracycline had substantial changes in the distal intestinal microbiota including a decrease in the bacterial diversity. P. acidilactici did not ameliorate the effect of antibiotic therapy in the intestinal microbiota. Chapter 5 used Atlantic salmon during smoltification to study the changes in the microbiota of distal intestine and the role of P. acidilactici to promote intestinal health. The results showed that bacterial communities in the mucosa differed from the digesta. Seawater transfer and P. acidilactici had significant changes in the intestinal microbiota of both mucosa and digesta. However, the modulatory effect of both factors evaluated was larger in the mucosa-associated microbiota than in the digesta-associated microbiota. Furthermore, P. acidilactici induced a significant increase in antiviral-related genes. Chapter 6 investigated the replacement of fish oil by rapeseed oil alone or combined with P. acidilactici on the intestinal health and microbiota of two intestinal regions in Atlantic salmon. Replacement of fish oil by rapeseed oil alone or in combination with P. acidilactici supplementation did not induce major changes in the intestinal health and microbiota. The bacterial communities found were significantly different between the pyloric caeca and mid-intestine. In conclusion, this thesis contributes to new knowledge regarding the effect of dietary supplementation of P. acidilactici and the impact of different potential challenging factors in the health and intestinal microbiota of farmed salmonid species.

This body of research explores the dietary application of a bioactive, solid-state fermentation (SSF) product in contemporary and lupin-containing diets of Nile tilapia (Oreochromis niltoticus) and rainbow trout (Oncorhynchus mykiss). Consequently, the work provides holistic assessment of the influences of SSF products on animal health and performance; depending upon rearing temperature, nutritional physiologies, feed formulations and extrusion conditions; alongside information on the performance of lupins in aquafeeds. The SSF product (at 0.1 % inclusion) improved growth performance of Nile tilapia fed diets containing lupins. Phosphorous retention appeared higher when the SSF product was included in a yellow lupin (Lupinus luteus) diet whilst Mg retention was significantly higher in fish fed narrow-leaf lupin (Lupinus angustifolius). The experimental ingredients did not appear to show any clear effects upon midgut macrostructure. At an ultrastructural level, the fish fed yellow lupin alone, displayed poorest brush border characteristics but those fed yellow lupin and the SSF product showed signs of amelioration since they did not differ significantly from those fed narrow-leaf lupin. Focus was then turned towards a yellow lupin-based diet in rainbow trout, with two inclusion levels of the SSF product. The SSF product significantly improved growth performance and feed efficiency at 0.5 %, with values closer to a fishmeal-based diet than the lupin control. The SSF product increased the digestibility of protein and energy and bioavailability of numerous elements. However, the digestibility and bioavailability of certain nutrients, e.g. fibre and Zn, were only increased with a 0.5 % inclusion. The SSF product influenced vertebral Ca:P ratio but no effect on vertebral morphology was identified. Fish fed yellow lupin kernel meal displayed high Mn concentrations throughout a number of tissues. The intestinal environment was explored in depth, revealing large differences dependent upon SSF product inclusion rate. Fish fed a 0.1 % inclusion exhibited deteriorated brush border characteristics and high diversity of microbes, including increased proportions of key salmonid pathogens. Those fed a 0.5 % inclusion displayed signs of increased surface area at an ultrastructural level, reduced goblet cell numbers and a low microbial diversity; with domination of one particular family, Enterobacteriaceae. Activities of alkaline phosphatase and leucine aminopeptidase within the anterior intestine also appeared to be influenced by SSF product inclusion. Variations in haemato-immunological parameters were also observed between the treatments. In the final experiment, the SSF product was applied, pre-extrusion, to a contemporary rainbow trout formulation, at 0, 0.5, 1.0 and 1.5 %. No significant improvements to performance were identified following SSF product inclusion. However, crude protein digestibility from SSF-supplemented diets was significantly higher than the control and tendencies towards elevated retention of Ca, P, and Mg were apparent. In vitro analysis of free-phosphate release was conducted upon pre- and post-extruded diets, at varying temperatures. This indicated that neither extrusion conditions (105 °C) nor an ambient temperature of 10 °C were sufficient to cease P-liberating capabilities by the SSF product; suggesting that the two conditions combined limited the product’s efficacy in practice. This research evidenced that SSF products can be an effective means of improving the available nutrient profiles of compound diets for both omnivorous, warmwater and carnivorous, temperate finfish. Lupins are a promising alternative protein source but their nutritional value can be substantially improved by SSF product application. Exploration of the effects of SSF products on the intestinal environment revealed that both negative and positive effects on intestinal health can occur, which is highly dependent upon product inclusion rate. The holistic approaches adopted within this series of studies have seldom been performed on monogastric animals and thus provide valuable, transferable information for advancing knowledge in the application of SSF products, exogenous enzymes and lupins to farm animal feeds, in general.