Thesis / Master of Science (MS)

This thesis examined the effects of exhaustive exercise and post-exercise recovery on metabolism and respiratory gas exchange in juvenile rainbow trout, in relation to the classical oxygen (O₂) debt hypothesis (Hill & Lupton, 1923). The initial study was a detailed quantitative analysis of the metabolic cost of post-exercise recovery in terms of O₂ and ATP equivalents. A 5 min bout of exhaustive exercise resulted in about a 2.0-2.5 fold increase in O₂ consumption (MO₂), a 6-8 fold increase in whole-body lactate (LAC) levels and a near depletion in whole-body glycogen (GLY), adenosine triphosphate (ATP) and creatine phosphate (CP) stores. Recovery of MO₂, LAC and GLY was usually complete by 6 h, though GLY did not always return to resting levels. Recovery of resting whole-body ATP required 1.0-1.5 h, whereas restoration of CP required only 5 min. Quantitative budgets of the cost of recovery were prepared, based on two opposite assumptions. The first scenario (A) assumed that all the GLY resynthesized came from LAC, and that the remaining 25% of the total LAC cleared was oxidized. This scenario accounted for 48% of the excess post-exercise O₂ consumption (EPOC) and 94% of the ATP budget. The alternative scenario (B), attributed 100% of the EPOC to LAC oxidation (64% of the total LAC cleared), while the remaining LAC (36%) was resynthesized into GLY. As scenario B accounted for only 35% of the ATP budget, scenario (A) appeared more probable. Subsequent experiments attempted to experimentally dissociate LAC disappearance from EPOC, via repetitive exercise bouts and prior GLY depletion. A 2nd bout of exhaustive exercise, given 6 h after the 1st, significantly reduced EPOC by 40%, whereas metabolite status (LAC disappearance and GLY, ATP, and CP restoration) remained essentially unchanged. In contrast, prior GLY depletion by 5 days starvation significantly lowered the LAC burden while EPOC remained unchanged. Neither the two scenarios of the theoretical analysis could adequately explain the relationships between EPOC and the various metabolites. It is likely that some blend of the two approaches may be more realistic. Nevertheless, these approaches all point to the conclusion that the EPOC is not directly related to the metabolism of LAC after exercise in the rainbow trout. Thus, in contrast to the classical O₂ debt hypothesis, LAC disappearance does not determine the magnitude EPOC after exhaustive exercise in rainbow trout. / Thesis / Master of Science (MS)

Juvenile rainbow trout were chronically exposed to cadmium in hard water, soft water, and in calcium-supplemented soft water in order to understand the effects of long term cadmium exposure in freshwater fish. A particular goal was to characterize changes in gill cadmium burden and the cadmium-binding properties of the gills during chronic sublethal exposures, so as to examine the applicability of the acute gill surface metal binding model or Biotic Ligand Model to trout chronically exposed to cadmium. Trout were exposed for 30 days to sublethal concentrations of cadmium in: a) moderately hard, Hamilton tap water (Ca = 1000 μM), b) synthetic soft water (Ca = 130 μM), or c) calcium-supplemented soft water (Ca = 260, 470, 770, and 1200 μM Ca). For both the hard and soft water cadmium exposures, no effects were observed on growth, swimming performance, and whole body ions. Growth and whole body and plasma Ca²⁺ concentrations were similar for all treatments in the calcium-supplemented soft water experiment; however, swimming performance was significantly reduced for the 470 μM Ca + Cd exposed fish. Acclimation to cadmium occurred in the hard water and lower concentrations of calcium-supplemented exposures but not in the soft water exposure. Cadmium accumulation was greatest in kidneys and gills and was directly related to cadmium exposure concentration. Tissue metal burdens were reduced with increased water calcium concentrations. Affinity of the gill for cadmium and the number of binding sites for cadmium decreased at higher water calcium concentrations. Affinity of the gill for Cd decreased with chronic cadmium exposure but binding site numbers increased with chronic Cd exposure. The acute gill binding model or Biotic Ligand Model, originally developed in soft water, was successfully applied to fish in both hard and soft water; however, complications arose when extending the model to fish chronically exposed to cadmium at various water calcium concentrations. / Thesis / Master of Science (MS)

Thesis / Master of Science (MS)

The expression of the estradiol-responsive genes in the liver of rainbow trout, Salmo gairdnerii, has been studied as a system which may permit quantitative measure of adverse sublethal effects of various xenobiotics. Upon administration of 17 β-estradiol to male or immature female rainbow trout, the expression of two genes are markedly enhanced. One of these genes codes for the egg yolk precursor protein, vitellogenin, whereas the other codes for another, as yet unidentified, protein. Complementary DNA (cDNA) genes of these two proteins have been employed as probes to characterize the dose-response relationship as well as the time course induction of these two classes of transcripts in liver both in vivo and in vitro. The maximal net transcription of pRTC 2 occurs at 20 ug β -estradiol per 100 gram fish body weight whereas those encoded for by pRTC 5 display maximal transcription at 5 μg β-estradiol per 100 gram fish body weight. With doses exceeding the dose required for maximal induction of transcripts, both classes of transcripts reach their maximal levels between 4 and 8 hours after primary and secondary induction. Transcripts homologous to pRTC 5 return to control levels by 16 hours and 2 days after primary and secondary induction respectively. Those homologous to pRTC 2 however, were not observed to recover to normal levels in the primary induction even 16 days after stimulation with estradiol. However, upon secondary induction pRTC 2 transcripts returned to control levels 8 days post-inoculation with estradiol. The effect of various xenobiotics on the induction of these two estradiol-responsive genes was investigated. Pretreatment with β-naphthoflavone resulted in neither the induction or repression of both classes of transcripts. Administration of Kepone prior to a subsequent stimulation with estradiol, resulted in a decrease in the accumulation of pRTC 2 transcripts only. In contrast, an isomer of DDT, p,p'-DDT, was found to enhance the expression of pRTC 5 transcripts in fish subsequently administered β-estradiol. Oncogenes have been found to be activated in transformed cells or cells treated with various xenobiotics. In this study, I have shown that the cellular oncogene, pRTC-myc 1-81, is expressed in the liver at an elevated level in response to estradiol treatment. The two classes of polyadenylated transcripts were detected (2.5 kb and 5.6 kb). The three estradiol-responsive genes mentioned above (pRTC 2, pRTC 5, and pRTC-myc 1-81) were also shown to be transcribed in an established rainbow trout hepatoma cell line. Transcriptional activities of all three genes, as well as the translational activity of vitellogenin, were diminished upon growth in serum stripped of endogenous hormones. Further characterization of this rainbow trout hepatoma cell line is warranted so that it may be employed in a sensitive and quantitative bioassay for assessing the pathobiochemical effects of environmental xenobiotics on fish reproduction. Such an index may be beneficial for establishing adequate guidelines for water contaminants that potentially could affect aquatic and human reproductive success. / Thesis / Master of Science (MS)

Trout genetically engineered to possess extra functioning growth homone genes were simulated, using injections of fish and bovine growth hormone. This was done to test potential and ecological impact of such genetic engineering on trout. Through analysis of growth and dry-mass budgets, it was determined that elevated levels of growth hormone resulted in increased growth and consumption. Potential constraints pertaining to respiration, density and evolutionary history were considered. Surprisingly, bovine growth hormone appeared to act as a super-normal stimuli, being more potent than the natural trout hormone. In fact the natural hormone inhibited growth at high dosages. Juvenile growth rates were less sensitive to elevated levels of growth hormone than those in more mature fish. Thus, improved growth was achieved by altering its normal ontogeny. This suggests that duplicating copies of growth hormone gene in an organism is not equivalent to a speciation event. Evidently, other manipulations would be required to increase the intrinsic metabolic power. Growth rate apparently is used as a cue for determining a particular ontogenetic trajectory (Stearns and Crandall, 1984). The fact that various species conform to one of at least four plastic developmental trajectories that are shaped by natural constraints and mortality patterns, means that growth hormone may elicit different responses in different species. The findings and implications of this study underscore the importance of using the holistic scope of ecology to achieve effective and efficient genetic engineering of target species. / Thesis / Master of Science (MS)

With World Health Organization estimation of population increase to over 9 billion by 2050, malnutrition and food insecurity are predicated to be two of the main challenges facing the world in the future. Fish is a source of protein that can have a significant contribution for solving the global food crisis. Fish for human consumption is provided by both the aquaculture industry and the capture fisheries, however, the flat-lined production of seafood from capture fisheries means aquaculture production should double in number to provide enough protein by 2050. Culturing fish at higher densities is being used as a way to increase the global aquaculture production. Modern aquaculture intensified practices expose the fish to various stressors that can negatively impact their welfare. Good nutrition can improve the health status of aquacultured fish while accumulation of specific nutrient can add value to the fillet. In line with that, the studies described in this dissertation aimed to examine the vital role of fish in human health and evaluate strategies to improve the sustainable aquaculture production of fish using novel aquafeeds. This was completed by conducting a survey of the essential minerals and heavy metals of commercially available catfish, salmon, and tilapia fillets in the United States marketplace and three fish nutritional trials to evaluate the impacts of novel aquafeeds on production and health of fish and the food quality of their respective fillets. Selenium with the established health benefit for human and fish was the trace mineral of interest in this dissertation. This element is the main component of a group of selenium containing proteins called selenoproteins that are antioxidant and maintain oxidative homeostasis of fish and human. Additionally, selenium is an antagonist for many heavy metals such as mercury. The market study revealed that catfish, salmon and tilapia samples available in United States marketplace are good source of selenium, zinc and phosphorous. The concentration of heavy metals in all the samples were lower than safety standards. Nile tilapia (Oreochromis niloticus) and rainbow trout (Oncorhynchus mykiss), which are two economically important aquacultured fish, were used as animal models in the fish nutritional trials. Different forms of selenium supplemented to Nile tilapia diet. The form of Dietary Se form did not have any significant (p>0.05) effect on growth, and plasma and hepatic GPX activity of Nile tilapia. However, organic selenium supplement showed significantly (p<0.05) higher bioavailability and accumulated in the fillets at the significantly higher concentration. In the second fish trial selenoneine, a novel selenium containing compound, was supplemented at various concentrations to rainbow trout diet. Other than growth parameters multiple other health biomarkers (lysozyme, glutathione peroxidase, superoxide dismutase activities) were examined to determine whether selenoneine supplementation had a positive impact on fish health. Selenoneine enriched diet significantly(p<0.05) increased the weight gain, lysozyme and oxidative enzymes activities of rainbow trout. Lastly rainbow trout feed was supplemented with commercially available probiotic (Actisaf®), prebiotic (Safmmann®), and their mixture (synbiotic). These dietary supplements did not have any significant effect on fish production but led to a significant increase in activity of oxidative enzymes in plasma when compared to control diet. These results suggest that feed supplements used in this dissertation can be used to promote the fish health raised in recirculating aquaculture systems and support an economically and environmentally sustainable aquaculture. / PHD / Fish contributes significantly to human’s diet in terms of high quality, easily digestible animal protein that helps fight malnutrition and food insecurity. More than half of the fish for human consumption is produced by the aquaculture industry and the growth of the industry is very critical for meeting the future needs for protein. Other than production of high-quality protein the aquaculture industry has made a great contribution to people’s livelihood, poverty alleviation, income generation, employment and, trade globally. To continue contributing effectively to global food security aquaculture industry should overcome various obstacles such as disease and sustainable production of high-quality feed. In this dissertation, important roles of fish in human diet was examined by measuring the mineral composition of commercially available catfish, salmon and tilapia in the United States market place. Also diets of Nile tilapia and rainbow trout were supplemented with various forms of selenium, prebiotics, and/or probiotics which have positive effect on health of both human and fish. These supplementations led to production of valued-added fish fillets enriched in selenium and improved the fish growth and health.

The effects of oxygenation and aeration on water quality, rainbow trout (Oncorhynchus mykiss) production, and economics were compared at the Wytheville State Fish Hatchery (WSFH) for 270 days. Mean dissolved oxygen (DO) concentrations and delta DO were significantly higher (P < 0.001) in the oxygenated raceways (9.5 and 2.75 mg/L, respectively) compared to aerated raceways (7.4 and 0.57 mg/L). Total settleable solids loads were significantly greater (P < 0.001) in aerated raceways (10.3 g/L/day) than in oxygenated raceways (8.8 g/L/day). Dissolved nitrogen (%), total gas pressure, and other water quality parameters (CO2, nitrite nitrogen, alkalinity, pH, and TAN) did not differ significantly between the treatments (P > 0.05). Raceway trout production (kg/day), trout growth rates (grams), feed conversion rate (FCR), and fish survival were not significantly different between treatments (P > 0.05). Blood hematocrit (Hct) and percent visceral mass were significantly elevated (P < 0.001) in oxygenated raceways compared to aerated raceways at 46 and 14.4% and 44 and 13%, respectively. Carrying capacity estimates derived from fish loading trials were significantly different (P < 0.001) at 3,355 and 2,217 kg/raceway in oxygenated and aerated raceways, respectively. Estimates of carrying capacity calculated using a fish loading (Ld) equation were also significantly different (P < 0.001) at 1,530 and 990 kg for oxygenated and aerated raceways, respectively. Oxygen injection increased the cost of production by $0.20/kg, however, net present value analysis (NPV) of oxygenated and aerated raceways over 5 years at a 10% discount rate yielded estimates of $50,666.51 and $32,742.15, respectively. Oxygen injection is an effective means of increasing DO concentrations, reducing effluent solids loading, and increasing raceway carrying capacity. / Master of Science

Steelhead and rainbow trout are common names for marine-migratory (anadromous) and freshwater-resident forms of Oncorhynchus mykiss, a partially migratory salmonid fish. Anadromous and resident forms are sympatric and can produce offspring with a life history different from their own (i.e., steelhead parents can produce rainbow trout offspring and vice versa). The expression of these alternative life histories is a plastic response to individual patterns of energy acquisition, assimilation, and allocation during juvenile life stages. Individual performance during early stream life is of particular interest because of potential carry-over effects on subsequent growth and developmental trajectories. In a series of experiments in laboratory streams, I determined the influence of individual variation in energy metabolism on behavior, growth, and life-history expression in O. mykiss. Individual variation in energy metabolism was a strong predictor of feeding territory acquisition by juvenile fish during the transition from dependence on maternal provisioning of egg yolk reserves to independent feeding. Feeding territory acquisition was positively associated with standard metabolic rate (SMR) under conditions of an abundant and predictable food supply. When the density of intraspecific competitors was manipulated, the association between SMR and territory acquisition was strongest at intermediate stocking densities, moderate at the highest stocking densities, and weakest at the lowest stocking densities. However, reducing the spatial predictability of food resources within streams reversed the influence of SMR on competitive outcomes. These experiments determined that variation in ecological conditions during early life stages imposes different selection regimes on juvenile O. mykiss and results in physiological divergence among cohorts. Subsequent rearing experiments determined that behavioral dominance influences rates of anadromy and freshwater maturation, most likely through the association between SMR and territory acquisition. In addition to the effects of behavioral dominance, I observed a significant influence of sex, rearing temperature, and individual growth trajectories on the expression of anadromy and freshwater maturation. Partially migratory populations of O. mykiss maintain an exceptionally diverse portfolio of life-history strategies. Results from this work lend insight into a suite of behavioral and physiological processes influencing individual life histories. / Graduation date: 2013

The research assessed the potential of developing a selective breeding programme for the UK rainbow trout industry. Levels of genetic variation at 12 microsatellite loci were first compared in seven different commercial strains. The Observed heterozygosity ranged from Ho = 48.1% in a gold rainbow trout strain (GTR) to Ho = 66.4% in a newly derived broodstock population constructed from a number of different sources (GIT). The Expected Heterozygosity (He) was highest in GIM1 (He= 79.5%) and lowest in the GTR strain (He = 56.9%). The Effective number of alleles (Mae) showed that the GIM1, GIM2, GIM3, and GIT strain (5.4; 5.2; 4.8; 4.2) were significantly more variable than the other strains and that GTR strain had the lowest value (2.5). There appears to be substantial genetic variability within the commercial United Kingdom rainbow trout strains surveyed in this study. This appears to be the case despite very different management histories and levels of record keeping. The strains appear to be genetically distinct (based on population genetic analyses), though the reasons for this remain unclear (and possibly unanswerable given the poor records kept by the different companies). The Glenwyllin farm strains (GIM) were chosen to form the base population for the project because of their high genetic variability, disease free status and because the farm produced around 20 million ova per year, so any genetic gains would have a widespread impact. The farm has an early (Strain A) and a late spawning (Strain B) and these were mated in a partial factorial design, 20 females and 20 neomales per strain (A & B) were chosen on the basis of maturity and gamete quality in November 2002 so that each male was crossed to 4 females (2 in the same strain and 2 in the other), a total of 160 families were created. All broodstock were biopsied to enable them to be genotyped. The families were reared separately up to the eyed stage at which point the eggs from each family were divided into three to generate three communal replicate populations. One of these was sent to a fingerling producer (Iwerne Spring) for ongrowing to fingerling size and formed the basis of a commercial production trial at Test Valley Trout farm (TVT) in Hampshire. When the fish reached an average weight of 5 g they were transferred from Iwerne Spring to TVT and 1500 were randomly selected, PIT tagged and biopsied to enable them to be assigned to their family using 11 multiplexed microsatellite loci. Parental assignment was based on exclusion (FAP) but the results were compared with another parental assignment based on likelihood (PAPA). Of the 1500 offspring (OIM) PIT tagged 1242 82.8% could be assigned to a single family utilizing different combinations of more than 6 loci (6 to 11). The growth of the 1500 OIM fish was tracked throughout the grow out period before they were finally harvested and fully processed. The results of OIM strain at the end of the trial period were mean weight of 415.5 g, and a mean length of 314.5 mm. The visual measurement of colour gave a mean flesh colour values of 26.01 on the 20-34 scale (SalmoFan™), and 11.0 with the colotimetry evaluation of colour (a*). The heritability results for the IOM strain were 43 ± 9% for weight, 42 ± 9% for gutted, and 28 ± 8% for length. The heritability estimates for the visual colour variables were 19 ± 7% and when using the colorimeter, the red chromaticity (a*) heritability was 14 ± 6%. Therefore, the heritability results of the IOM strain indicate that there are opportunities of substantial and rapid improvement of the growth rate and flesh colour traits. Also no line effects were observed or indications of non-additive genetic variation. In contrast to these last results, the overall survival of the GIM strain from the time of the physical tagging with PIT until harvest was 52.8%, and survival heritability was extremely low, 3 ± 2%, hardly significant.