The nutritional value of dietary fibre for rainbow trout, Salmo gairdneri was investigated using juvenile fish (lO-30g) maintained in freshwater at ambient temperatures under a natural photo period. A preliminary experiment was conducted using five purified dietary fibre sources, namely, a~cellulose, lignin, lignosulphonate, galactomannan and chitin ·which varied in physical, chemical and textural characteristics. A commercially available, powdered polyethylene was also used as an inert control ingredient and all sources of fibre were included at a, realistically low level of 5'% in separate semi-purified diets. Although there were no significant differences in the growth of fish at the end of the 10-week trial, several nutritional parameters were affected for rainbow trout fed the different experimental treatments. Mean daily food intake was re"d uced for trout receiving the lignin, chitin and galactomannan diets compared to the polyethylene control. Similarly the food conversion ratios (FeR) and protein efficiency ratios (PER) were relatively inferior for diets containing chitin and galactomannan compared to the lignosulphonate treatment. Maximum net dietary nitrogen utilization was obtained for the polyethylene control t' ... ra ~on whilst lower values were again observed with chitin and galactomannan. Apparent dry matter (DM) and nitrogen digestibility coefficients however were in close agreement for each of the dietary treatments except for the lignin diet which was poorly digested. Generally the results implied that the properties of chitin and galactomannan were worthy of further study at higher inclusion levels and in different physical states. A specific·investigation in which 0, 5, 10, 15 and 20% additions of a purified a-cellulose replaced dietary starch in separate experimental di e t s f a~'1 e d to produce any significant changes in the growth performance of trout and only slightly influenced nutrient utilization at the higher 15 and 20% inclusion levels. Negative digestibility coefficients for the 'unavailable' carbohydrate fraction of diets calculated from the 'total' and 'available' carbohydrate contents of diet and faecal samples was considered to be evidence of the non-nutritive and bulking qualities of a-cellulose. Growth and digestibility trials were then undertaken to examine the effects of including different levels (10 and 20%) and particle size ranges (45-500, 500-1000 microns) of chitin (poly-Nacetyl- D glucosamine) as a natural source of dietary fibre for trout. In a similar experiment, graded amounts of galactomannan polysaccharide (0, 10 and 20%) were added to moist pelleted diets to examine the long term effects of feeding a gel-type fibre characteristic' of many commercially available binding agents. Negative digestibility coefficients for both chitin and galactomannan based on specific biochemical measurements together with the failure to detect any chitinase activity in stomach and intestinal tissue was confirmation of the inability of rainbow trout to degrade and utilize these materials. Coarse grades of chitin at the 10 and 20% levels impaired food intake, growth performance and nutrient utilization as shown by the Poorer FCR, PER, net nitrogen utilization and digestibility coefficients compared to the diets containing finely ground chitin or the α-cellulose control treatment. Similar findings were obtained with increasing additions of galactomannan and there were associated reductions in the serum glucose and protein concentrations with each increment of dietary galactomannan. The final carcass compositions of fish were also affected by the gel fibre which caused a significant reduction in the tissue lipid content and an inverse trend in moisture content compared to trout receiving an a-cellulose control diet. Further investigations using a sacrificial method to follow and quantify the passage of food through the gastrointestinal tract revealed that the physical properties of fibre such as particle size composition, water retaining capacity and viscosity were among several factors which modified gastric evacuation and digestion rates in rainbow trout. From the predicted gastric emptying times (GET), it was apparent that coarsely graded chitin (20%) and both 10 and 20% inclusions of galactomannan considerably increased the residence time of the gastrointestinal contents compared to finely ground chitin and a control diet without added fibre. Although an exponential relationship was found to best describe the stomach emptying profiles obtained, linearization of the data was achieved by applying surface area and volume dependent mathematical models which emphasized the importance of these physical factors. The combined nutrition and physiological studies supported the contention that fibre is the non-nutritive part of the diet, but it Was concluded that the level and nature of the fibrous material has· important consequences on the processes controlling food intake and the efficiency of digestion, which in turn may affect the assimilation of nutrients and the performance of growing rainbow trout.

No description available.

Determining the physical and biological habitat variables that influence the abundance of juvenile salmonids in British Columbia streams will improve management practices. Habitat models are tools that provide insight into organisms’ habitat needs and provide a more efficient mechanism for estimating population abundance than direct measurement. Models have been developed for salmonids in other jurisdictions, but very few have included invertebrate drift (a primary food source for juvenile salmonids) as a predictive variable. This is because temporal and spatial variation of drift abundance are widely assumed to be so high that drift cannot be reliably estimated without unreasonable effort. This thesis investigates the temporal and spatial variability of invertebrate drift and the impact of its inclusion in habitat models for juvenile salmonid abundance in two chapters. The first objective of the first chapter was to evaluate the temporal variability of invertebrate drift by comparing the seasonal and day-to-day variation in drift abundance to spatial variation within and between sites. The second objective was to develop predictive models for invertebrate drift abundance. Aquatic, terrestrial and total invertebrate drift abundances varied primarily between sites and very little between days or months at the same site, indicating that a single day of sampling is sufficient to assess drift abundance for comparison among sites. The abundance of invertebrate drift was related to productivity- and flow-related habitat variables. The objectives of the second chapter were to develop predictive models for juvenile salmonid abundance in southwestern BC using physical and biological habitat variables, to determine whether habitat variables differ between the Coast and Interior regions of BC, to determine the contribution of invertebrate drift to the relative predictive ability of the models, and to determine cost:benefit ratios for the predictive models and their component variables. The final models for predicting abundance of all young-of-year salmonids combined, and rainbow trout (Oncorhynchus mykiss) and coho salmon (O. kisutch) individually, included variables related to stream structure and productivity, and the models for rainbow and coho showed regional differences. Invertebrate drift did not improve model fit.

In early rainbow trout development, a switch from high-affinity embryonic hemoglobin to lower-affinity adult hemoglobin occurs along with a turnover of round, embryonic erythrocytes to oval, adult erythrocytes. The objective of my thesis was to determine how the ontogeny of blood oxygen transport was affected by chronic hypoxia (30% of saturation) in rainbow trout. Hemoglobin-oxygen affinity, cooperativity, and the Bohr and Root effects were unaffected by hypoxia treatments, whereas hemoglobin content, erythrocyte counts, and hematocrit were significantly reduced. In hypoxia, larvae had higher concentrations of embryonic hemoglobin mRNA and embryonic erythrocytes than stage-matched normoxia-reared larvae. Overall, these results indicate that chronic hypoxia suppresses erythrocyte development prior to complete yolk absorption. Ultimately, this suggests that in early ontogeny rainbow trout conform to low oxygen conditions, rather than mounting the hypoxia response observed in oxygen-regulating adult trout.

The molecular biology of macrophage activating and deactivating cytokines and their receptors was discussed. Comparison of IFN-γ amino acid sequences of several mammalian species reveals a low conservation of amino acids. The interaction of IFN-γ with its receptor system is complicated and coherent with the species specificity of IFN-γ. Identification by PCR of an IFN-γ-like gene in the trout genome was not possible. In contrast with IFN-γ, TGF-β is very conserved in its amino acid sequence. The PCR-amplification of a TGF-β fragment from amphibian, <I>Xenopus</I>, and rainbow trout cDNA libraries was possible. Two oligonucleotide primers were used in PCRs to amplify a 360 bp fragment of trout Mhc class II β chain. Using these two oligos, this 360 bp fragment could be amplified from trout spleen cDNA library and HK leucocytes. cDNA synthesized from RNA extracted from ConA/PMA stimulated HK leucocytes was used as template DNA in PCR, and a class II specific fragment was amplified. This class II fragment could not be amplified from HK macrophages treated with a MAF containing supernatant, although HK macrophages treated with a control supernatant did express class II molecules. This could suggest that priming or activation of trout macrophages results in a decreased expression of Mhc class II antigens. A novel method for analysing 5'nucleotidase activity of head kidney macrophages was optimised for use with cell monolayers, with respect to the effect of cell numbers, temperature and substrate concentration. Both lysed and whole cells could be used for determination of 5'nucleotidase activity. Maximal 5'nucleotidase activity was found in the range of 27°C to 33°C and using a substrate concentration of ≥ 1 μmol AMP ml^-1 for whole cells and ≥ 1.5 μmol AMP ml^-1 for lysed cells. 5'nucleotidase activity was also correlated with respiratory burst activity in cells treated with a variety of supernatants containing MAF activity. A significant inverse relationship between these two activities was found. MAF-treated cells were also found to lose 5'nucleotidase activity faster than control cells in the presence of cycloheximide, suggesting such cells may have a higher membrane turnover of this enzyme.

Carotenoid utilization in rainbow trout (Oncorhynchus mykiss Walbaum) and Atlantic salmon (Salmo salar L.) has been investigated with respect to tissue distribution of carotenoids and the role of the liver on the bioavailability of the lipid soluble carotenoids, astaxanthin and canthaxanthin. Species-specific and tissue-specific accumulations were noted for astaxanthin and canthaxanthin in the rainbow trout and Atlantic salmon, possibly indicating fundamental differences in their utilization in these species. The liver and the kidney were revealed to be the major tissues involved in carotenoid metabolism in both rainbow trout and Atlantic salmon. Apparent digestibilities (-96% and -30% for rainbow trout and Atlantic salmon, respectively) and flesh carotenoid retentions (-12% and -5.4% for rainbow trout and Atlantic salmon, respectively) differed significantly between species, suggesting that rainbow trout are more efficient depositors of carotenoids within the flesh. Isolated rainbow trout liver perfusion experiments revealed small differences in the uptake of astaxanthin and canthaxanthin. Uptake of astaxanthin in both synthetically-derived and serum-derived models showed saturable uptake mechanism that occurred earlier than for canthaxanthin. These results can potentially offer an explanation for the better utilization of astaxanthin in rainbow trout, where the liver reduces the bioavailability of canthaxanthin through continued uptake. Results show a low hepatic extraction ratio (0.03-0.07), in line with published post-prandial elimination rates. Neither astaxanthin nor canthaxanthin significantly induced hepatic or renal xenobiotic-metabolizing enzymes in the rainbow trout, contrary to published reports in rats and mice. This may imply fundamental species-specific differences in the metabolic pathways for these carotenoids. Histochemical investigations revealed that both carotenoids significantly impacted liver structure, resulting in higher levels of total lipids and mucopolysaccharides. This is thought to be due to their antioxidant functions and their provitamin A activity. Carotenoid-treated fish also had higher levels of glycogen phosphorylase in liver sections, providing the first evidence in fish for the possibility of glucuronidation of their metabolites. The present investigations demonstrate the liver to be a major organ in carotenoid metabolism, and consequently affects carotenoid distribution and availability. In addition, carotenoid supplementation significantly affects liver structure and may potentially enhance its function. Furthermore, these investigations have provided new avenues of investigation into the use of isolated organ perfusions for biochemical nutrition research, and expanded the knowledge of liver physiology and biochemistry.

Metabolic power budgeting, the regulation of metabolism with respect ot metabolic scope, was studied in the laboratory in zebra fish using respirometry, and in the field on pike using heart-rate telemetry. Increased food consumption by zebra fish resulted in higher growth, mortality and metabolism. The magnitudes of the components of metabolism: maximum metabolism, standard metabolism, routine metabolism and feeding metabolism were measured. Power budgets for zebra fish fed high and low rations were constructed. Fish fed high rations worked harder than fish fed lower rations, but were apparently not working near the upper limit of the metabolic scope. Possible mechanisms for growth-related mortality are considered. Biological information on the populations of pike in Lochs Kinord and Davan (Grampian Highlands) were gathered. The population of L. Kinord was dominated by young, small fish; apparently due to exploitation. L. Davan is unexploited and had a pike population consisting of a much wider range of ages and sizes. Methods for assessing regurgitation by pike were developed. Effects of long and short-term temperature fluctuations, and feeding on heart rate of captive pike were studied. Resting heart rate increased exponentially with increasing temperature; heart rate appeared to accommodate all changes in resting metabolism. Post-prandial heart-rate records could be used to accurately estimate meal size. Gastric evacuation rates corresponded to digestion times estimated from heart-rate records. Heart-rate telemetry was used to study metabolic power budgeting, feeding and activity of wild pike from Lochs Kinord and Davan in June 1988. Pike worked mainly at low power levels relative to metabolic scope. Tachycardias associated with localized movement were frequent, and such movement was accurately recorded by heart-rate telemetry but frequently undetected by conventional means. Feeding events were identified and the metabolic costs of survival estimated. Some unusually energetically-expensive localized movements were recorded; the possible reasons for this are discussed. Intraperitoneal implantation techniques were developed for transmitter attachment on pike. Experments using dummy transmitters on pike and rainbow trout showed no effect on growth, survival or reproduction, but tissue reactions differed. Male and female pike, location-tracked with implanted transmitters before, during and after spawning time exhibited increased overall activity during the apparent spawning period, as well as changes in diet activity. Males were significantly more active than females in three out of seven weeks. Spawning appears to be a period of high energy expenditure for pike.

Cadmium (Cd) is a nonessential metal present in sublethal concentrations within the aquatic environment. Cd is an endocrine disruptor and high concentrations of this metal suppress stressor-induced cortisol production in fish. However, few studies have examined the effect of Cd at concentrations that are environmentally relevant on the functioning of the hypothalamus-pituitary-interrenal (HPI) axis. The HPI axis activity is essential in the stressor-induced cortisol production, a highly conserved adaptive response to stress in vertebrates. Elevation of plasma glucose in response to a rise in plasma cortisol is mediated through steroid activation of glucocorticoid receptors (GRs), but the mechanism of action of Cd in disrupting target tissue cortisol action is not known in fish. The overall objective of this thesis was to examine the impact of sublethal and environmentally relevant levels of Cd on the stress response and target tissue metabolic capacities, and to investigate the mechanisms of action of this metal in disrupting cortisol production and target tissue cortisol action in rainbow trout (Oncorhynchus mykiss). The impact of subchronic exposure to environmentally relevant levels of Cd on metabolic capacity and stress performance was identified through a 28 day (d) in vivo exposure of juvenile rainbow trout to either of two Cd concentrations (0.75 µg/L or 2.0 µg/L). During the exposure period, juvenile rainbow trout accumulated Cd within the liver, kidney and gills, but were able to adapt to exposure concentrations as no changes were observed in plasma cortisol, glucose and lactate levels. However, changes in abundance of mRNAs encoding proteins involved in corticosteroidogenesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and P450 side chain cleavage enzyme (P450scc), and liver GR protein expression suggesting endocrine disruption over the 28 d period. Also, target tissue metabolic capacities, including lower liver glycogen content and changes in intermediary metabolic enzyme activities in the liver and gill, were compromised by the 28 d exposure to Cd. The response to a secondary handling stressor at either 7 or 28 d exposure was attenuated suggesting that subchronic exposure to low levels of Cd disrupts the highly conserved adaptive stress response in rainbow trout. Upon further investigation using in vitro head kidney slices exposed to 0, 10, 100 or 1000 nM of Cd and stimulated with adrenocorticotropic hormone (ACTH), a similar inhibition of cortisol production was observed, as demonstrated in vivo, suggesting that Cd disrupts interrenal corticosteroidogenesis in fish. The impact of Cd on ACTH-stimulated cortisol production involved the suppression in the abundances of MC2R, StAR and P450scc transcripts. This response was also mimicked when head kidney slices from 7 d Cd exposed fish were incubated ex vivo with ACTH confirming that interrenal tissue is a key target for endocrine disruption by Cd. In both the in vitro and ex vivo incubations of head kidney slices 8-Bromo-cAMP (a cAMP analog) completely abolished the Cd-mediated cortisol inhibition demonstrating for the first time that Cd disruption of corticosteroidogenesis is occurring upstream of cAMP production. Further investigation of Cd-mediated impact on MC2R showed alterations in MC2R mRNA transcripts during in vivo exposure after 7 days and an attenuation of MC2R mRNA levels after Cd-exposed fish were subjected to a handling stressor. Disruptions in the mRNA abundance of MC2R was associated with disruptions of melanocortin receptor accessory protein 1 (MRAP1), but not MRAP2; a phenomenon that was also observed in ex vivo head kidney slices. Cell transfection studies confirmed that rainbow trout MC2R/MRAP1 receptor complex displayed decreased activity in the presence of Cd. Taken together these results suggest that Cd directly targets the MC2R/MRAP1 complex to inhibit ACTH-stimulated cortisol production in juvenile rainbow trout. In addition to Cd inhibiting interrenal steroidogenesis, the results also suggest that Cd may impact the negative feedback regulation of cortisol through the suppression of brain mineralocorticoid receptor (MR), but this requires further investigation. At the target tissue level, Cd by itself did not significantly affect liver metabolism, but inhibited the cortisol-induced glucose production in liver slices. This involved suppression of GR protein expression along with the suppression of GR-responsive genes, including phosphoenolpyruvate carboxykinase (PEPCK) and suppressor of cytokines signaling 1 (SOCS1) and changes in enzyme activities, including hexokinase, glucokinase, pyruvate kinase and PEPCK, pointing to a disruption in liver GR signaling by Cd. Altogether, Cd exposure disrupts the organismal stress responses in juvenile rainbow trout. Furthermore, Cd impairs the ability of juvenile rainbow trout to respond to a secondary stressor, which is a vital adaptive process that is fundamental to successful stress performance. Most importantly, these studies highlight for the first time that disruption of the HPI axis to attenuate cortisol production occurs at the level of the MC2R/MRAP1 complex, suggesting that the mechanism of action for attenuation of cortisol occurs at the level of MC2R activation. Also, GR signaling is a key target for Cd and may be a mechanism leading to altered metabolic capacities in stressed fish from Cd-contaminated sites. Overall environmentally relevant levels of Cd disrupt cortisol production and target tissue action of this steroid in rainbow trout.

Cadmium (Cd) is a nonessential metal present in sublethal concentrations within the aquatic environment. Cd is an endocrine disruptor and high concentrations of this metal suppress stressor-induced cortisol production in fish. However, few studies have examined the effect of Cd at concentrations that are environmentally relevant on the functioning of the hypothalamus-pituitary-interrenal (HPI) axis. The HPI axis activity is essential in the stressor-induced cortisol production, a highly conserved adaptive response to stress in vertebrates. Elevation of plasma glucose in response to a rise in plasma cortisol is mediated through steroid activation of glucocorticoid receptors (GRs), but the mechanism of action of Cd in disrupting target tissue cortisol action is not known in fish. The overall objective of this thesis was to examine the impact of sublethal and environmentally relevant levels of Cd on the stress response and target tissue metabolic capacities, and to investigate the mechanisms of action of this metal in disrupting cortisol production and target tissue cortisol action in rainbow trout (Oncorhynchus mykiss). The impact of subchronic exposure to environmentally relevant levels of Cd on metabolic capacity and stress performance was identified through a 28 day (d) in vivo exposure of juvenile rainbow trout to either of two Cd concentrations (0.75 µg/L or 2.0 µg/L). During the exposure period, juvenile rainbow trout accumulated Cd within the liver, kidney and gills, but were able to adapt to exposure concentrations as no changes were observed in plasma cortisol, glucose and lactate levels. However, changes in abundance of mRNAs encoding proteins involved in corticosteroidogenesis, including melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and P450 side chain cleavage enzyme (P450scc), and liver GR protein expression suggesting endocrine disruption over the 28 d period. Also, target tissue metabolic capacities, including lower liver glycogen content and changes in intermediary metabolic enzyme activities in the liver and gill, were compromised by the 28 d exposure to Cd. The response to a secondary handling stressor at either 7 or 28 d exposure was attenuated suggesting that subchronic exposure to low levels of Cd disrupts the highly conserved adaptive stress response in rainbow trout. Upon further investigation using in vitro head kidney slices exposed to 0, 10, 100 or 1000 nM of Cd and stimulated with adrenocorticotropic hormone (ACTH), a similar inhibition of cortisol production was observed, as demonstrated in vivo, suggesting that Cd disrupts interrenal corticosteroidogenesis in fish. The impact of Cd on ACTH-stimulated cortisol production involved the suppression in the abundances of MC2R, StAR and P450scc transcripts. This response was also mimicked when head kidney slices from 7 d Cd exposed fish were incubated ex vivo with ACTH confirming that interrenal tissue is a key target for endocrine disruption by Cd. In both the in vitro and ex vivo incubations of head kidney slices 8-Bromo-cAMP (a cAMP analog) completely abolished the Cd-mediated cortisol inhibition demonstrating for the first time that Cd disruption of corticosteroidogenesis is occurring upstream of cAMP production. Further investigation of Cd-mediated impact on MC2R showed alterations in MC2R mRNA transcripts during in vivo exposure after 7 days and an attenuation of MC2R mRNA levels after Cd-exposed fish were subjected to a handling stressor. Disruptions in the mRNA abundance of MC2R was associated with disruptions of melanocortin receptor accessory protein 1 (MRAP1), but not MRAP2; a phenomenon that was also observed in ex vivo head kidney slices. Cell transfection studies confirmed that rainbow trout MC2R/MRAP1 receptor complex displayed decreased activity in the presence of Cd. Taken together these results suggest that Cd directly targets the MC2R/MRAP1 complex to inhibit ACTH-stimulated cortisol production in juvenile rainbow trout. In addition to Cd inhibiting interrenal steroidogenesis, the results also suggest that Cd may impact the negative feedback regulation of cortisol through the suppression of brain mineralocorticoid receptor (MR), but this requires further investigation. At the target tissue level, Cd by itself did not significantly affect liver metabolism, but inhibited the cortisol-induced glucose production in liver slices. This involved suppression of GR protein expression along with the suppression of GR-responsive genes, including phosphoenolpyruvate carboxykinase (PEPCK) and suppressor of cytokines signaling 1 (SOCS1) and changes in enzyme activities, including hexokinase, glucokinase, pyruvate kinase and PEPCK, pointing to a disruption in liver GR signaling by Cd. Altogether, Cd exposure disrupts the organismal stress responses in juvenile rainbow trout. Furthermore, Cd impairs the ability of juvenile rainbow trout to respond to a secondary stressor, which is a vital adaptive process that is fundamental to successful stress performance. Most importantly, these studies highlight for the first time that disruption of the HPI axis to attenuate cortisol production occurs at the level of the MC2R/MRAP1 complex, suggesting that the mechanism of action for attenuation of cortisol occurs at the level of MC2R activation. Also, GR signaling is a key target for Cd and may be a mechanism leading to altered metabolic capacities in stressed fish from Cd-contaminated sites. Overall environmentally relevant levels of Cd disrupt cortisol production and target tissue action of this steroid in rainbow trout.

Characterization of the 5' flanking region of rainbow trout ki-ras gene was begun with the cloning and sequencing of this region by the inverse PCR technique and dideoxynucleotide chain termination method. In total, a nucleotide sequence of 1080 bp upstream from the first coding ATG was sequenced. Although this region showed certain promoter elements, it does not share common features with other mammalian ras promoters, which lack the TATA and contain multiple GC boxes with Spl binding activities. In contrast, this region in trout ras contains typical TATA and CCAAT boxes. This structural difference of the trout ki-ras promoter from that of other mammalian ras genes may suggest that different transcriptional regulation mechanisms of the ras ger.e are used at various levels in evolution. The chromatin structure of the trout ki-ras gene was studied by probing invivo for DNase I hypersensitive sites. To overcome the difficulties of using the traditional indirect end labeling method for a single-copy gene, the technique of ligation-mediated PCR was applied. No hypersensitive sites were observed at or near the codon 12 region of the gene, either in normal (protooncogene) or tumor (oncogene) tissue from the liver. This result suggests that the local chromatin structure of trout ki-ras gene may not be an important factor for codon 12 mutations induced by genotoxins, and that changes of chromatin structure are unlikely to be promoted after tumor formation. Studies by micrococcal nuclease demonstrate that this ras gene, in the region around 12, lacks ordered nucleosome positioning or may be even free of nucleosomes. Such an irregular organization of ras oncogenic chromatin would resemble that of many other "normal", highly active eukaryotic genes. The intrinsic affinity of trout ki-ras gene for aflatoxin B₁ was determined by in vitro alkylation experiments. Exon 1 of the gene was synthesized and labeled at the 5'end of the coding strand by the PCR technique. Taking advantage of the selective cleavage of AFB1-DNA adducts by piperidine under alkali conditions, the frequency of AFB 1 attack to each guanyl site was determined by densitometric scans after the cleaved fragments were electrophoresed on sequencing gels. The results demonstrated that two guanyl sites of codon 12 had differential affinity to AFBl, the more 5' G was relatively inaccessible but the more 3' G was accessible, indicating that the sequence selectivity of AFB I may contribute to the preference of the initial adduction in vivo. / Graduation date: 1993