The survival and physiology of rainbow trout in alkaline waters was the focus of this thesis. It is known that salmonids have problems with ammonia excretion in alkaline water (Cameron and Heisler, 1983; Wright and Wood, 1985). The first set of studies attempted to increase the survival rates of rainbow trout planted into alkaline lakes by acclimating them to the alkaline conditions before their release. The first field study acclimated rainbow trout to alkaline waters by acidifying the lake water with C0₂ in order to reduce the magnitude of the pH change experienced by the fish. The second field study acclimated rainbow trout to alkaline waters by increasing the alkalinity of the hatchery water in which the fish were held, over a six day period. In both studies the acclimated fish experienced higher survival rates relative to non-acclimated fish. Plasma sodium concentrations ([Na⁺]p1) of the fish were shown to increase, while plasma chloride concentrations decreased. These changes were attributed to an increase in the exchange of external Na⁺ with endogenous H⁺, and the decrease in the exchange of endogenous HC0₃⁻ with external Cl⁻, respectively. The increased [Na⁺]pl may have also been the result of the exchange of plasma ammonium (NH₄⁺) with external Na⁺. The second set of studies investigated the physiological response of rainbow trout to alkaline waters. The first study, the chronic exposure of rainbow trout to alkaline water, showed that trout in hard alkaline water experienced higher survival rates and regulated plasma ammonia and ion concentrations more competently than trout in soft alkaline water. This increased ability to regulate plasma ammonia and ion concentrations was attributed to the possible "reactivation" of the Na⁺/NH₄⁺ exchange. The purpose of the second study, the acute exposure of rainbow trout to alkaline water, was to further investigate the mechanisms that enable fish in hard alkaline water to survive better than fish in soft alkaline water. The possible activity of the Na⁺/NH₄⁺ exchange was again observed in the hard alkaline water. The addition of amiloride to the alkaline hard treatment water increased plasma total ammonia and stabilized [Na⁺]pl′, which supported the "reactivation" of the Na⁺/NH₄⁺ exchange in hard alkaline water. As a result of these studies, the acclimation of rainbow trout to hard alkaline water before being planted into any alkaline body of water was recommended. / Land and Food Systems, Faculty of / Graduate

This study examines three possible methods for improving the digestibility and bioavailability of zinc to rainbow trout (Oncorhynchus mikiss). The first method was to examine the availability of the zinc utilisation from commonly used protein sources; the second was to assess the efficacy of the upstream use of an enzyme treatment of the raw materials; the third was to assess the use of organically complexed mineral supplements as opposed to the inorganic salts widely used at present. The first section indicated that the zinc from the soyabean meal was the most available (49.4%). The zinc digestibilities of the animal based protein used in this current study were 15.1% for LT94 fish meal, 26.6% for the Provimi 66 white fishmeal and 15.8% for poultry meat meal. The zinc in the maize/corn gluten meal was 31.9% digestible and from the NuPro 26.1%. Gram for gram maize gluten meal supplied the least amount of zinc to the fish (3.66 mg per kg). Based on these results the diets for the subsequent supplementation trials were formulated. The liver, eye and caudal fin were identified biomarkers of a severe zinc deficiency. The second part of the study revealed a soybean product, treated by exogenous enzymes, had a higher phosphorus digestibility, (49.0%, vs. 36.6%) and zinc digestibility (30.7% vs. 7.9%) The treatment did not improve the protein digestibility (85%). The third part of the study showed the organic source proved more digestible than the inorganic, 37.4% and 26.9% respectively. The fish fed the organic source maintained a higher level of zinc in both the eye and caudal fin. The liver zinc levels were unaffected by both dietary level and zinc source. Analysis of the liver for a zinc dependant protein showed that under stress conditions only the organic supplemented fish were able to synthesis this protein. The analysis of the mRNA levels coding for this protein indicate the fish on both zinc forms up regulated the production of the mRNA to the same extent when stressed. Finally this study also examined the viability of using a stable isotope to identify different ‘preferences’ for one form of supplementation over the other in different tissues. This method illustrated a tissue dependant difference to how the fish attempted physiologically to compensate for zinc deficiency. The rate of turnover was fastest in the liver, then the caudal fin and then the eye, and also showed that when the diet was more deficient there was an increased ability for the tissues to take up the organic form.

Cyclopropenoid fatty acids (CPFA), which are a group of fatty acids produced by plants of the order Malvales, are known to induce adverse physiological effects when administered to a variety of animal species. A structurally strained cyclopropene ring is present in all CPFA and is believed responsible for the toxic action of these fatty acids. Dietary consumption of CPFA by mammals, poultry and fish has resulted in toxic responses including hepatic damage, impaired reproductive capabilities and sizeable alterations in lipid metabolism. Furthermore, CPFA have been identified as mildly carcinogenic and strongly cocarcinogenic towards rainbow trout (Salmo gairdneri). The mechanism by which CPFA enhance carcinogenesis is currently not understood. The research in this thesis has therefore been directed toward obtaining a better understanding as to how CPFA induce toxic responses in rainbow trout. Hepatic plasma membranes were isolated from both control trout and trout which had consumed dietary CPFA. The plasma membranes were then compared via the use of electron microscopy, chromatographic analysis of phospholipid and fatty acid content, two dimensional polyacrylamide gel electrophoresis of proteins, and Western blot analysis of concanavalin A sensitive glycoproteins. Electron micrographs revealed that control plasma membranes appeared more homogeneous than CPFA membranes and were characterized by more membrane sheets and less vesicularization. The analysis of enzyme activities revealed that CPFA caused a decrease in whole liver glucose-6-phosphatase activity and that control plasma membranes expressed slightly higher glucose-6-phosphatase and 5'-nucleotidase activities as compared to CPFA membranes. Although dietary CPFA appeared to have no effect on the phospholipid content of the plasma membranes, significant alterations in the fatty acid profiles of ethanolamine and choline phospholipids were observed. CPFA caused a decrease in palmitic, palmitoleic and oleic acids while the level of stearic and docosahexaenoic acids subsequently increased. Differences between the protein content of control and CPFA plasma membranes were made clear through the analysis of electrophoretic and Western blotting data. Membranes isolated from fish fed CPFA contained several proteins of high molecular weight (above 66,000 daltons) and other proteins of high isoelectric point that were not present in control plasma membranes. Additionally, two families of glycoproteins which had previously been identified as microsomal in origin were detected only in CPFA plasma membranes. A discussion concerning the possible causes and biological ramifications of the observed subcellular alterations caused by CPFA insult is also presented in this thesis. / Graduation date: 1989

A partial cystatin cDNA from rainbow trout was generated by reverse transcription polymerase chain reaction with two degenerate primers. The partial cystatin PCR product was 168 bp and used to screen trout liver λgt 11 cDNA library. Four positive clones were isolated and designated as cstl, cst2, cst3 and cst4. Only cst2 contained the full-length cystatin cDNA which was 674 bp and included 5' untranslated region and the polyadenylation signal sequence AATAAA in the 3' region. Translation of the cDNA contains 132 amino acid residues. Comparison of the amino acid sequence with those of family II cystatin indicated that the 21 amino acids at N-terminal end is a signal peptide that leads to cystatin secretion, and the 111 amino acids are mature cystatin. Four cysteine residues in the cystatin may form two disulfide bonds for the secondary structure. Cst2 was subcloned into pGEM-3z for Northern and Southern blot experiments. Northern blot indicated that trout cystatin mRNA is about 750 bp. Cystatin is expressed in all tissues examined but at various levels. This difference may reflect the regulation of cysteine proteinase activities. Southern blot of trout genomic DNA showed that the copy number of the trout cystatin gene is probably one per haploid genome. / Graduation date: 1997

Dimethylbenz (a) anthracene (EMBA), a carcinogen that requires metabolic activation to produce active metabolites capable of binding to DNA, has been studied in the trout and other fish. Polycyclic aromatic hydrocarbons (PAH) are of importance as they are ubiquitous in the environment and their carcinogenic effects in fish from contaminated waters are an important indication of the pollution risks to man. Since such pollution risk assessment presents the involvement of multiple agents, the study of the modulation of PAH-DNA binding produced by other agents is important. In this study the effect, of dietary pretreatment at 500 ppm, 100 ppm and 2000 ppn, using BNF, Aroclor 1254, or indole-3-carbinol (I3C) respectively on DMBA-DNA binding was examined. To study the effect of age on sensitivity to DMBA-DNA binding, adult trout and fry were used in two separate studies. The fish were fed treatment diet for at least two weeks. Fry were then injected with [³H] DMBA, at 22.4 μCi/3.9 x 10⁻² μmole/fish and adult trout at 284 μCi/1.58 μmole/fish. Liver DNA was isolated, purified and binding of radioactivity to DNA was examined and computed as the covalent binding index (CBI). Mean CBI for control dietary group vising adult trout was 1000 fold lower than for fry. Statistical analysis of covalent binding index for the treatment groups revealed that a statistically significant (p < 0.05) inhibition in DNA-DMBA binding response in adult trout and fry was produced fcy the DNF dietary treatment only. Diethylnitrosamine (DENA), a potent hepatocarcinogen in several animal species belongs also to the class of compounds that require metabolic activation. Dietary treatment and continuous exposure of trout to the carcinogen in water, have produced hepatocellular carcinctnas. The water exposure also produced a dose related DNA ethylation of the O⁶ position of guanine, believed to be the promutagenic adduct produced after DENA exposure. This study examines two other routes of exposure to DENA, in vitro hepatocyte incubations and i.p. injection. Adult trout and fry were injected with [³H] DENA. Adult fish received 3.3, 16.5, and 33 mg/kg DENA, and fry received 10, 50 and 100 mg/kg. Hepatocyte incubation was performed with doses up to 220 μM [³H] DENA, or 1 mM unlabelled DENA. DNA from fish livers and from hepatocyte pellets was isolated, purified and examined for radioactive binding of the DENA metabolites or in the case of the unlabelled DENA, was analyzed for O⁶ and N7 adduct using an HPIC technique with fluorescence detection. O⁶-EtG adduct after DENA exposure, in DNA of hepatocytes obtained from trout pretreated with beta-naphthoflavone (BNF, a known inducer of cytodhrcme P-450 dependent enzyme activities involved in the activation of xenobiotics) was below the limits of detection of the HPDC-fluorescenoe detection procedure used. To examine further if the lack of DNA binding and absence of the O⁶-EtG adduct was due to rapid repair, the persistence of O⁶-EtG after exposure to 40 nM ethylnitrosourea (ENU, a direct ethylating agent) was studied in hepatocytes at 2, 4, and 5 hours after treatment. The activity of the alkyltransferase protein involved in the repair of alkylguanines also was determined using liver extracts from adult rainbow trout. The studies did not reveal a significantly high rate of repair. It is concluded that i.p. injection and in vitro hepatocyte incubations are not a good method for studying the kinetics of activation and DNA binding of DENA in the rainbow trout. The i.p. route may lead to substantial loss of the dose of the carcinogen administered and hepatocyte incubations are limited by the toxic effects of increasing carcinogen concentration. The reasons mentioned above, coupled with low levels of metabolism of nitrosamines in trout results in the inability to detect and study the appearance, persistence and repair of the O⁶-EtG adduct. / Graduation date: 1989

The DNA repair capabilities of rainbow trout (Salmo gairdneri) were studied vising the method of autoradiography. Trout were fed a semi-purified control diet containing 0 ppm, 50 ppm, or 300 ppm cyclopropenoid fatty acids (CPFA) for 6-9 weeks. Liver slices were prepared and exposed in vitro to a control treatment, ultraviolet irradiation (UV), ethidium bromide (EB), UV/EB in succession, or aflatoxin B₁. The degree of DNA repair was analyzed in terms of net grains per cell. Except following the EB treatment, fish on the control diet revealed an absence of ongoing DNA repair. Trout fed 50 ppm CPFA exhibited a consistently low level of repair over time following the in vitro control treatment. Fish fed 300 ppm CPFA revealed a relatively higher degree of ³H-Me-thymidine incorporation indicative of induced DNA repair following the in vitro control treatment, and the degree of repair increased with time on the diet. UV-irradiation caused a marked increase in the degree of induced DNA repair in 300 ppm CPFA fish at 6 and 7.5 weeks, and in 50 ppm CPFA fish at 7.5 weeks. Follcwing UV-irradiation, liver slices were exposed to EB, a DNA intercalating agent used to inhibit normal DNA replication. However, in contrast to the desired effect, EB caused a marked decrease in the degree of repair synthesis observed in 300 ppm CPFA fish at 6 and 7.5 weeks. Indicative of intercalation, the in vitro EB treatment caused a moderate degree of ³H-Me-thymidine incorporation in fish fed the control diet. Repair was also induced in 300 ppm CPFA fish following exposure to EB at 6 and 7.5 weeks. Aflatoxin B₁ induced DNA repair to various degrees in fish on all diets at 7.5 and 9 weeks. In comparison to the in vitro control treatment, it was observed that the degree of induced DNA repair was decreased significantly - "completely" following the UV, UV/EB, and EB treatments - in fish fed the 300 ppm CPFA diet for 9 weeks. In view of the low level of DNA repair observed in rainbow trout using autoradiography, the repair capabilities were studied using a more sensitive assay, bromodeoxyuridine (BrdU) photolysis. Isolated hepatocytes were prepared from fish fed the various diets and exposed in vitro to a control treatment, UV-irradiation, or 4-nitroquinoline-N-oxide. The obtained results were nonconclusive indicating technical improvements on the assay need to be made. / Graduation date: 1988

Before 1980, Ashurst Lake had an exceptionally productive rainbow trout fishery. More recently, Ashurst has become turbid and trout growth rates have declined substantially. The growth, condition, and food habits of a single cohort of rainbow trout was followed from May 1985 until October of 1986. The first year, trout in Ashurst grew from 66 mm to 164 mm in 5 months. Growth reached an average of 24 mm/month from June to September, a time when trout fed primarily on Daphnia. By July of the second year, growth had stopped and condition declined substantially in conjunction with a change in their diet from Daphnia to crayfish and minnows. The decrease in growth rate was attributed to a decrease in the food base coupled with hampered foraging abilities of rainbow trout in water with high turbidity. High tubidity may have resulted from erosion of the surrounding watersheds.

Spilled heavy fuel oil (HFO) sinks within the water column and accumulates in sediments, affecting aquatic organisms that are not typically exposed to oils that float. Previously, the 3-4 ring alkyl substituted polycyclic aromatic hydrocarbons (PAHs) have been identified as the major toxic components in crude oil. Since HFO is comprised of higher concentrations of 3-4 ringed alkyl PAH and an abundance of 5-6 ringed PAH relative to crude oil, it is predicted to be more toxic to the early life stages of fish. An effects-driven chemical fractionation (EDCF) of HFO 7102 was undertaken to establish the toxicity relative to crude oil, and to identify the compounds that are bioavailable and chronically toxic to the early life stages of fish. In this EDCF, the complex HFO 7102 mixture was separated by low temperature vacuum distillation into three distinct fractions, 2, 3 and 4. Each fraction was assessed using a chronic bioassay to determine whether it contained components that caused toxicity to rainbow trout embryos similar to that of the whole oil. Acute bioassays with juvenile trout demonstrated the presence of compounds that induce cytochrome P450 enzymes, an indicator of exposure to PAH. Fraction 3, the fraction more toxic than the parent mixture, was further separated by cold acetone extraction into fraction 3-1 (PAH-rich extract) and fraction 3-2 (wax residue), and assessed with the same bioassays. Simultaneous chemical analysis with bioassays guided the fractionation, and identified compounds abundant and consistently present in toxic fractions. Due to resistance to dispersion of HFO, a chemical dispersant was used with vigorous mixing to drive the maximum amount of oil into solution to minimize the potential for false negatives and the volume of test material used. The potency of HFO 7102 and its fractions were also measured using water accommodated fractions (WAFs) produced by a continuous flow system of water flowing through oil coated gravel. Both exposure methods traced the toxicity from whole oil into fractions containing higher concentrations of 3-4 ring alkyl PAH, similar to crude oil. This research is the first toxicological assessment of HFO 7102, which is essential for determining the risk of spills of HFO to fish, and whether the risk of oils can be predicted from their alkyl PAH composition. / Thesis (Master, Biology) -- Queen's University, 2013-01-24 14:14:16.278

Three new NILT genes were successfully cloned and characterized from rainbow trout, with one NILT alternatively spliced into a long isoform containing two Ig domains and a short isoform containing one Ig domain.  The expression of NILTs was studied in six different tissues and two different cell lines, with expression apparent in immunologically important tissues.  Furthermore, phylogenetic analysis showed that NILTs are more closely related to triggering receptor expressed on myeloid (TREM) cells and Nkp44 from humans than to NITRs from rainbow trout. The genomic organisation and structure of the multigene family of NILTs in Atlantic salmon was investigated using a BAC sequencing approach.  This revealed the presence of six novel NILT genes, which either contained one or two Ig domains and several immunoreceptors tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic region. By homology search two NILT-like genes in zebrafish (<i>Danio rerio</i>) located on chromosome 1 have been obtained. Chromosome 1 in zebrafish also contains the <i>Dare</i>-ZE genes, which are equivalent to the human MHC class I genes located on chromosome 6.  The distance between the later and the TREM genes on chromosome 6 is similar to the distance between the NILT-like genes and <i>Dare</i>-ZE genes on zebrafish chromosome 1.  In addition, two NILT-like Ig domains were obtained from the green spotted pufferfish (<i>Tetraodon nigroviridis</i>), putatively part of the same receptor. The results will contribute to our knowledge of the immune system in fish and provide useful information for the control of inflammatory processes in salmonids.

Through the implementation of in vivo feeding trials, the efficacy of three wheat gluten (WG) products, vital (Amytex®), hydrolysed (Merripro®) and soluble hydrolysed (Solpro®) wheat gluten as replacement of soy protein concentrate, and scFOS prebiotic (Profeed®) supplementation were analysed to assess their impacts on intestinal health and production of juvenile rainbow trout. Microbial community analysis in experiment one revealed a degree of diet based modulation with 7.5% and 15% inclusions of wheat gluten (WG) products. Bacterial species diversity was significantly reduced with 15% hydrolysed wheat gluten (HWG) inclusion compared to the plant protein control and 15% vital wheat gluten (VWG) treatments, with sequenced OTUs dominated by the phylum Firmicutes and possible promotion of probiotic species. No detrimental effects were observed on intestinal morphology. These findings led onto a longer duration feed trial with a more holistic, higher resolution approach. Experiment two revealed modulation of the allochthonous intestinal microbiota, with increased proportions of Enterococcus and Weissella in the 10% and 20% VWG treatments. Bacillus and Leuconostoc relative abundances were significantly increased with 10% HWG and soluble hydrolysed (Sol) wheat gluten inclusions. HSP 70 transcripts were significantly down-regulated in all WG treatments compared to the basal soy protein concentrate treatment (SPC) and increased intraepithelial leukocyte counts were observed with 10% VWG inclusion. Growth performance was unaffected by 10% dietary inclusions of WG, however, FCR’s were significantly improved in the 20% VWG treatment compared to the 10% HWG and Soluble treatments. This led to the investigation of increased inclusion levels of WG products in experiment three. All WG treatments in experiment three yielded significantly improved growth performance. Somatic indices were significantly increased with 30% blended WG inclusion compared to the SPC treatment. Modulation of allochthonous intestinal microbiota was observed to a lower degree than the previous experiments, with a dose response observed with increasing blended WG inclusion. In the final experiment two basal diets (SPC and 20% Blended) and two scFOS supplemented diets (SPC + FOS and 20% Blended + FOS) were investigated for the effect on growth performance, gut health and allochthonous microbial population. Growth performance was unaffected, however, modulation of the allochthonous microbial population was observed with an apparent synergistic effect of scFOS supplementation in WG diets. This synergistic trend was also observed in the transcription level expression of immune relevant genes. 20% WG inclusion with additional scFOS supplementation observed significant down regulation of the pro-inflammatory cytokine TNF-α, as well as HSP 70, CASP 3 and Glute ST compared to the 20% Blend treatment. The present research demonstrates dietary inclusions of WG products, solely or blended, at the expense of soy protein concentrate to modulate the allochthonous microbial population, potentially promoting probiotic species, whilst reducing the levels of intestinal stress in juvenile rainbow trout. Supplementation of the prebiotic scFOS modulated the microbial populations, enhancing the proportion of potential probiotic species, and combined with WG inclusions, reduce intestinal and oxidative stress and inflammation biomarkers, with no observed deleterious effects.