An experiment was conducted to determine the effect of addition of antioxidants or encapsulation of linseed oil on the oxidative stability of linseed oil and the effect on growth and fatty acid composition of rainbow trout fed these products. Four diets differing only in their lipid sources were prepared by cold extrusion: 1) fish oil (FO), 2) linseed oil (LO), 3) linseed oil (980 g/kg) stabilized with vitamin E (7.5 g/kg) and butylated hydroxytoluene (BHT) (12.5 g/kg) (stabilized linseed oil; SLO) and 4) linseed oil (350 g/kg) containing vitamin E (7.5 g/kg), BHT (12.5 g/kg) and encapsulated in a coating material primarily consisting of hydrogenated palm oil (630 g/kg) (encapsulated linseed oil; ELO). Diets were fed twice daily to rainbow trout to apparent satiation (n=22 / replicate; 7 replicates per treatment) during a 168 day growth trial. Following the growth trial, the fish were humanely euthanized by a sharp blow to the cranium and analyzed for fatty acid composition, thiobarbituraric reactive substances (TBARS), fillet colour and sensory attributes (trained and consumer panels). There were no significant differences between treatments on any of the growth parameters investigated or TBARS levels of fish fillets. Omega-3 polyunsaturated fatty acids of trout fed LO were significantly higher than those fed FO (35.5% of total fatty acids vs. 27.6%) and ELO (28.9%) (P < 0.05). EPA and DHA levels were not significantly different between treatments. Diet samples were stored for 168 days at room temperature in sealed plastic containers. Following storage, the oxidative stability index (OSI) of the FO and LO diets were reduced to 0.00 hours while that of the SLO diet 9.20 hours and the ELO diet was 11.40 hours. Trained panelists determined fish fed FO had a significantly higher aroma intensity and significantly lower aroma desirability and overall acceptability than those fed SLO. The rancid aroma and flavour of the FO-fed fish was significantly higher than fish fed the other treatments (P < 0.05). Consumer panelists found no significant differences between the sensory attributes of fish fed the four experimental diets and exhibited no preference between treatments (P > 0.05). Fillets from fish fed FO had significantly higher values than the other three treatments for redness (3.59 vs values between 1.86 and 2.07) and yellowness (25.35 vs values between 20.51 and 21.22) (P < 0.05). Addition of antioxidants to linseed oil improves its oxidative stability during storage and processing and results in fish fillets with fatty acid composition and consumer acceptance equal or superior to fish fed fish oil.

For ras gene mutation analysis in the rainbow trout (Oncorhynchus mykiss) model system, a partial trout ras sequence was identified using the polymerase chain reaction (PCR). Two synthetic oligonucleotides based on rat K-ras gene sequence were used as primers for the PCR procedure. A 90 base pair (bp) sequence, referred to as the trout K-ras, was amplified from trout genomic DNA and cDNA. Cloned 90 by PCR products from several normal liver tissues were sequenced resulting in the same sequence. Large-sized PCR products, 111 and 237 bp, were also cloned and sequenced indicating that these fragments included the 90 by sequence information expressed in mRNA. This 5'-terminal partial trout K-ras nucleotide sequence was 88% homologous to that of the goldfish ras gene, and less homologous to those of mammalian ras genes. Based on the partial sequence information of two trout ras genes, K-ras and H-ras, DNA from trout tumors induced by chemical carcinogens, aflatoxin B1 (AFB1) and N-methyl-N'-nitro-N-nitrosoguanidene (MNNG), were analyzed for the presence of point mutations. Using the PCR and oligonucleotide hybridization methods, a high proportion (10/14) of the AFB1-initiated liver tumor DNA indicated evidence for ras point mutations. Of the 10 mutant ras genotypes, seven were probed as G to T transversions at the second position of codon 12, two were G to T transversions at the second position of codon 13, and one was a G to A transition at the first position of codon 12. Nucleotide sequence analysis of cloned PCR products from four of these tumor DNAs provided definitive mutation evidence in each case, which seemed to occur in only a fraction of the neoplastic cells. However, no mutations were detected in exon 1 of the trout K-ras gene, nor in DNA from trout normal livers. Results indicated that the hepatocarcinogen AFB1 induced similar ras gene mutations in trout as in rat liver tumors. By comparison, the mutation specificity of MNNG in trout liver tumors was for G to A transitions, but no ras mutations were detected in trout kidney tumors. This investigation was the initial study of experimentally induced ras gene point mutations in a lower vertebrate fish model. / Graduation date: 1991

Graduation date: 1992

Lake trout (Salvelinus namaycush), chinook salmon (Oncorhynchus tschawytscha), coho salmon (Oncorhynchus kisutch), steelhead trout (Salmo gairdneri), and brown trout (Salmo trutta were collected from April 30 to November 18, 1970 with gill nets at selected sites in Lake Michigan near Gary, Burns Ditch, and Michigan City, Indiana. Catch-per-uniteffort was law and most catches occurred when nets were set in water temperatures in the 50 F (10.0 C) range or lower. Sea lamprey (Petrcnyzon marinas) scars and wounds were found on 25.4% of 71 lake trout captured and fresh wounds occurred on 6.0% of the fish. Scars and wounds were restricted to lake trout 20.0 in. (50.6 cm) or greater in total length and 32.7% of the 55 fish in this size group carried scars or wounds.Food items in stomachs of 39 lake trout, 34 coho salmon, and 29 chinook salmon were expressed as percent frequency of occurrence, numerical percent, and volumetric percent. The alewife (Alosa pseudoharenp_us) was the predominate food consumed by all three species. The crustaceans, Mysis sp. and Pontoporeia affinis, were consumed only by lake trout less than 19.0 in. (48.1 cm) total length.The age and growth of 69 known age fin clipped lake trout were examined. The body-scale relationship was TL = 6.2001 + 0.1238 Sc. The mean calculated total lengths for the first 5 years of growth were 5.3, 10.7, 16.3, 21.0, and 24.0 in. (13.5, 27.2, 41.4, 53.2, and 60.8 cm). Annual growth is considerably greater than recorded for Lake Michigan lake trout prior to alewife introduction and dominance. The length-weight relationship was log W = -2.5087 + 3.2338 log L which agrees closely with previous length-weight analyses of Lake Michigan lake trout.Ball State UniversityMuncie, IN 47306

The central objective of this thesis is to better understand early life history dynamics of salmonids in large regulated rivers. I studied spawning, incubating, and age-0 life stages of rainbow trout in the Lee’s Ferry reach of the Colorado River below Glen Canyon Dam, AZ. My first objective was to evaluate the effects of hourly fluctuations in flow on nearshore habitat use and growth of age-0 trout. Catch rates in nearshore areas were at least 2- to 4-fold higher at the daily minimum flow compared to the daily maximum and indicated that most age-0 trout do not maintain their position within immediate shoreline areas during the day when flows are high. Otolith growth increased by 25% on Sundays in one year of study, because it was the only day of the week when flows did not fluctuate. My second objective was to evaluate the effects of flow fluctuations on survival from fertilization to a few months from emergence (early survival). Fluctuations were predicted to result in incubation mortality rates of 24% in 2003 and 50% in 2004, when flow was experimentally manipulated to reduce trout abundance, compared to 5% in 2006 and 11% in 2007 under normal operations. Early survival increased by over 6-fold in 2006 when egg deposition decreased by at least 10-fold. Because of this strong compensatory dynamic, flow-dependent incubation mortality in experimental years was likely not large enough to reduce the abundance of age-0 trout. My final objective was to determine how flow, fish size and density effects habitat use, growth, and survival of age-0 trout. Apparent survival rates from July to November were 0.18 (2004), 0.19 (2006), and 0.32 (2007). A stock synthesis model was developed to jointly estimate parameters describing early life history dynamics, and indicated that early survival was lower for cohorts fertilized during the first half of the spawning period and was negatively correlated with egg deposition, that movement of age-0 trout from low- to high-angle shorelines increased with fish size, and that survival varied by habitat type and over time in response to flow changes from Glen Canyon Dam.

Major Histocompatibility Complex (MHC) receptors serve a critical role in self/non-self recognition through the presentation of peptide antigen to circulating T lymphocytes and are also believed to play a role in mate selection. Through the development of antibodies to MHC homologues in trout, this report demonstrates the presence of MHC expression in germ cells, as well as a soluble form in seminal fluid. What role these immune molecules may perform in reproduction and mate selection is discussed. In addition, as ectotherms, fish are often subjected to low temperatures. Previous data indicates that the expression of these genes is abolished by low temperatures. Employing these same antibodies, this report further demonstrates that trout maintain the expression of MH I and its critical light chain component, beta-2-microglobulin when subjected to 2oC for 10 days. Expression of the MH II receptor sub-units however, was sensitive to both confinement stress and low-temperature in vivo, as well as to factors secreted from a known fungal pathogen in cultured macrophage. As the cause of "winter kill", Saprolegniales cultures induced homotypic aggregation and pro-inflammatory gene expression in the macrophage cell line, RTS11 as well as down-regulation of MH II. Though no evidence of fungal toxins was evident, fungal spore size appeared to exceed macrophage phagocytic capabilities. Taken together, such a loss of MH II expression at low temperature may allow for establishment of fungal and bacterial diseases and that upon the return to warmer temperatures, saprolegniales have the ability to maintain MH II down-regulation and evade immune recognition. Concurrent to the study of MH expression, this report includes the first cloning and characterization of calreticulin (CRT) in fish. Like its mammalian homologue and primary chaperone to MHC receptors and other immune proteins, trout CRT appears to be a single copy gene with ubiquitous tissue distribution, displaying anomalous migration as a doublet with relative molecular mass of 60kD. Despite its promoter containing endoplasmic reticulum stress elements (ERSE), trout CRT expression did not increase upon treatment with several calcium homeostasis antagonists. Treatment of peripheral blood leukocytes with phytohemaglutinin did reveal a qualitative increase in cell surface expression, as seen in mammals; however, cellular protein levels did not change, suggesting that, in trout, CRT function may be regulated through cellular sub-localization, rather than through changes in gene expression, as it is in mammals.

An experiment was conducted to determine the effect of addition of antioxidants or encapsulation of linseed oil on the oxidative stability of linseed oil and the effect on growth and fatty acid composition of rainbow trout fed these products. Four diets differing only in their lipid sources were prepared by cold extrusion: 1) fish oil (FO), 2) linseed oil (LO), 3) linseed oil (980 g/kg) stabilized with vitamin E (7.5 g/kg) and butylated hydroxytoluene (BHT) (12.5 g/kg) (stabilized linseed oil; SLO) and 4) linseed oil (350 g/kg) containing vitamin E (7.5 g/kg), BHT (12.5 g/kg) and encapsulated in a coating material primarily consisting of hydrogenated palm oil (630 g/kg) (encapsulated linseed oil; ELO). Diets were fed twice daily to rainbow trout to apparent satiation (n=22 / replicate; 7 replicates per treatment) during a 168 day growth trial. Following the growth trial, the fish were humanely euthanized by a sharp blow to the cranium and analyzed for fatty acid composition, thiobarbituraric reactive substances (TBARS), fillet colour and sensory attributes (trained and consumer panels). There were no significant differences between treatments on any of the growth parameters investigated or TBARS levels of fish fillets. Omega-3 polyunsaturated fatty acids of trout fed LO were significantly higher than those fed FO (35.5% of total fatty acids vs. 27.6%) and ELO (28.9%) (P < 0.05). EPA and DHA levels were not significantly different between treatments. Diet samples were stored for 168 days at room temperature in sealed plastic containers. Following storage, the oxidative stability index (OSI) of the FO and LO diets were reduced to 0.00 hours while that of the SLO diet 9.20 hours and the ELO diet was 11.40 hours. Trained panelists determined fish fed FO had a significantly higher aroma intensity and significantly lower aroma desirability and overall acceptability than those fed SLO. The rancid aroma and flavour of the FO-fed fish was significantly higher than fish fed the other treatments (P < 0.05). Consumer panelists found no significant differences between the sensory attributes of fish fed the four experimental diets and exhibited no preference between treatments (P > 0.05). Fillets from fish fed FO had significantly higher values than the other three treatments for redness (3.59 vs values between 1.86 and 2.07) and yellowness (25.35 vs values between 20.51 and 21.22) (P < 0.05). Addition of antioxidants to linseed oil improves its oxidative stability during storage and processing and results in fish fillets with fatty acid composition and consumer acceptance equal or superior to fish fed fish oil.

Major Histocompatibility Complex (MHC) receptors serve a critical role in self/non-self recognition through the presentation of peptide antigen to circulating T lymphocytes and are also believed to play a role in mate selection. Through the development of antibodies to MHC homologues in trout, this report demonstrates the presence of MHC expression in germ cells, as well as a soluble form in seminal fluid. What role these immune molecules may perform in reproduction and mate selection is discussed. In addition, as ectotherms, fish are often subjected to low temperatures. Previous data indicates that the expression of these genes is abolished by low temperatures. Employing these same antibodies, this report further demonstrates that trout maintain the expression of MH I and its critical light chain component, beta-2-microglobulin when subjected to 2oC for 10 days. Expression of the MH II receptor sub-units however, was sensitive to both confinement stress and low-temperature in vivo, as well as to factors secreted from a known fungal pathogen in cultured macrophage. As the cause of "winter kill", Saprolegniales cultures induced homotypic aggregation and pro-inflammatory gene expression in the macrophage cell line, RTS11 as well as down-regulation of MH II. Though no evidence of fungal toxins was evident, fungal spore size appeared to exceed macrophage phagocytic capabilities. Taken together, such a loss of MH II expression at low temperature may allow for establishment of fungal and bacterial diseases and that upon the return to warmer temperatures, saprolegniales have the ability to maintain MH II down-regulation and evade immune recognition. Concurrent to the study of MH expression, this report includes the first cloning and characterization of calreticulin (CRT) in fish. Like its mammalian homologue and primary chaperone to MHC receptors and other immune proteins, trout CRT appears to be a single copy gene with ubiquitous tissue distribution, displaying anomalous migration as a doublet with relative molecular mass of 60kD. Despite its promoter containing endoplasmic reticulum stress elements (ERSE), trout CRT expression did not increase upon treatment with several calcium homeostasis antagonists. Treatment of peripheral blood leukocytes with phytohemaglutinin did reveal a qualitative increase in cell surface expression, as seen in mammals; however, cellular protein levels did not change, suggesting that, in trout, CRT function may be regulated through cellular sub-localization, rather than through changes in gene expression, as it is in mammals.

Municipal wastewater effluent (MWWE) is an important source of pollution in the aquatic environment impacting fish. MWWE is a complex mixture of chemicals including pharmaceuticals, personal care products, industrial chemicals and pesticides. A link between reproductive endocrine disruption and MWWE exposure has been established in fish, but less is known about the effects of MWWE on non-reproductive endocrine disruption. The overall objective of this thesis was to examine the impacts of MWWE exposure on the stress response and intermediary metabolism in rainbow trout (Oncorhynchus mykiss). In fish, the primary adaptive organismal stress response involves the activation of hypothalamic-sympathetic-chromaffin axis to produce catecholamines, predominantly epinephrine, and the hypothalamic-pituitary-interrenal (HPI) axis to produce cortisol. Both of these hormones play a key role in elevating plasma glucose levels that is essential to fuel the increased energy demand associated with stress. Along with the organismal stress response, the cellular stress response, involving the synthesis of a suite of heat shock proteins (hsps), also plays an important role in protecting cellular protein homeostasis in response to stressors, including toxicants. The impact of MWWE on stress-related pathways were identified using a low-density trout cDNA microarray enriched with genes encoding for proteins involved in endocrine-, stress- and metabolism-related processes. This was further confirmed by assessing plasma hormone and metabolite levels and stress-related targeted genes and proteins expression and enzyme activities in select tissues in rainbow trout. Studies were carried out in controlled field (caging) and laboratory experiments to examine the impacts of MWWE on stress and tissue-specific metabolic responses in rainbow trout. Further in vitro studies using rainbow trout hepatocytes in primary cultures were carried out to investigate the mechanism of action of two pharmaceuticals, atenolol and venlafaxine, found in relatively high concentrations in MWWE in impacting the stress-mediated glucose response. In caged fish, MWWE exposure significantly elevated plasma cortisol and glucose concentrations, and altered the mRNA abundance of a number of stress-related genes, hormone receptors, glucose transporter 2 and genes related to immune function. When fish were exposed to an acute handling stress following a 14 d exposure to MWWE, the cortisol response was abolished and the glucose response was attenuated. The effects on cortisol did not correlate with changes in the expression of genes involved in cortisol biosynthesis, but were associated with an increase in hepatic glucocorticoid receptor (GR) protein expression. Upon further investigation in controlled laboratory studies, MWWE exposure elevated constitutive hsp 70 and hsp90 expression after 8 d exposure, which correlated with a decrease in glycogen levels in the liver in fish exposed to a high concentration of MWWE compared to control fish, pointing to a MWWE-induced increase in liver energy demand. By 14 d, glycogen stores were replenished, and this was commensurate with increases in liver gluconeogenic capacity, including increases in the activities of phosphoenolpyruvate carboxykinase (PEPCK) and alanine aminotransferase (AlaAT), along with a decrease in liver GR expression. In the heart, GR protein expression increased in treated fish, and the activity of pyruvate kinase increased, indicating an increase in glycolytic capacity. Subjecting the MWWE exposed fish to a secondary handling disturbance (acute stress) led to an attenuated plasma cortisol and glucose response compared to the control group. This corresponded with a reduced liver gluconeogenic capacity and a lower liver and heart glycolytic capacities, reflecting a disturbance in the energy substrate repartitioning that is essential to cope with stress. While it is difficult to establish causative agents from a complex mixture such as MWWE, the two pharmaceutical that were tested impacted glucose production. Specifically, atenolol and venlafaxine disrupted the epinephrine-induced glucose production, but did not modify cortisol-mediated glucose production in trout hepatocytes. The suppression of epinephrine-mediated glucose production by atenolol and venlafaxine was abolished by cAMP analogue (8-bromo cAMP) or glucagon (a metabolic hormone that increases glucose production). This suggests that both drugs disrupt β-adrenoceptor signaling, while it remains to be determined if the response is receptor isoform-specific. Altogether MWWE exposure disrupts the organismal and cellular stress responses in trout. Key targets for MWWE impact leading to the impaired cortisol and metabolic responses to stress include liver and heart GR expression, liver gluconeogenic capacity, and liver, heart and gill glycolytic capacities. Most significantly, MWWE impairs the ability to metabolically adjust to a secondary acute stressor, which is an important adaptive process that is integral to successful stress performance. From an environmental stand-point, long-term exposure to MWWE will lead to reduced fitness and will compromise the capacity of fish to cope with additional stressor, including escape from predators.

An experiment was conducted to determine the effect of addition of antioxidants or encapsulation of linseed oil on the oxidative stability of linseed oil and the effect on growth and fatty acid composition of rainbow trout fed these products. Four diets differing only in their lipid sources were prepared by cold extrusion: 1) fish oil (FO), 2) linseed oil (LO), 3) linseed oil (980 g/kg) stabilized with vitamin E (7.5 g/kg) and butylated hydroxytoluene (BHT) (12.5 g/kg) (stabilized linseed oil; SLO) and 4) linseed oil (350 g/kg) containing vitamin E (7.5 g/kg), BHT (12.5 g/kg) and encapsulated in a coating material primarily consisting of hydrogenated palm oil (630 g/kg) (encapsulated linseed oil; ELO). Diets were fed twice daily to rainbow trout to apparent satiation (n=22 / replicate; 7 replicates per treatment) during a 168 day growth trial. Following the growth trial, the fish were humanely euthanized by a sharp blow to the cranium and analyzed for fatty acid composition, thiobarbituraric reactive substances (TBARS), fillet colour and sensory attributes (trained and consumer panels). There were no significant differences between treatments on any of the growth parameters investigated or TBARS levels of fish fillets. Omega-3 polyunsaturated fatty acids of trout fed LO were significantly higher than those fed FO (35.5% of total fatty acids vs. 27.6%) and ELO (28.9%) (P < 0.05). EPA and DHA levels were not significantly different between treatments. Diet samples were stored for 168 days at room temperature in sealed plastic containers. Following storage, the oxidative stability index (OSI) of the FO and LO diets were reduced to 0.00 hours while that of the SLO diet 9.20 hours and the ELO diet was 11.40 hours. Trained panelists determined fish fed FO had a significantly higher aroma intensity and significantly lower aroma desirability and overall acceptability than those fed SLO. The rancid aroma and flavour of the FO-fed fish was significantly higher than fish fed the other treatments (P < 0.05). Consumer panelists found no significant differences between the sensory attributes of fish fed the four experimental diets and exhibited no preference between treatments (P > 0.05). Fillets from fish fed FO had significantly higher values than the other three treatments for redness (3.59 vs values between 1.86 and 2.07) and yellowness (25.35 vs values between 20.51 and 21.22) (P < 0.05). Addition of antioxidants to linseed oil improves its oxidative stability during storage and processing and results in fish fillets with fatty acid composition and consumer acceptance equal or superior to fish fed fish oil.