Thesis (M. Sc. (Agriculture)) -- University of Limpopo, 2016 / Fish assemblages in relation to environmental variables within the Broederstroom and Debengeni Rivers were investigated. Both rivers were characterized by coarse substrates (pebble and gravel), temperatures below 20°C and moderate depth. Trout dominated fish assemblages in terms of numbers caught and was only distributed at higher altitude sites >1400 m (a.s.l). Coarse substrates, temperatures below 15°C, flow rate, depth and riparian cover were the variables shown important for the distribution of trout by multivariate analysis. The confinement of the trout to higher altitude and lack of optimal habitat variables at lower altitude sites contributed to the assertion that the area is marginal for trout distribution. It was therefore inferred that the marginality of the area is the possible explanation for trout failure to self-sustain its population, therefore dependent on the continuous restocks by the local hatchery. The continuous restocks of small size trout in the Broederstroom River prompted a subsequent study where trout’s impact on macroinvertebrate communities was evaluated through surveys and field experiments. The ability of small trout to utilize macroinvertebrates made them suitable candidates to evaluating their impact in the area. Aquatic invertebrates were found to be the main food source for the trout in the area. Taxa such as Gomphidae and Potamonautidae were the most frequent food items from the analyzed stomachs of trout. However, observations from both field surveys and experiments showed that trout is a weak regulator of macroinvertebrate diversity in the area, since there were no significant differences (ANOVA, P<0.05) in the diversity of invertebrates from trout invaded and uninvaded sites. Trout being a weak regulator of macroinvertebrate diversity in the area, it prompted surveys to the Ebenezer Dam to determine its competitive interactions with native predatory species. If the introduced species is a more efficient predator than the native predator species, it may affect changes in the structure of the habitat and food resource. Trout in the Ebenezer Dam was found to be selective to habitat variables whilst C. gariepinus was cosmopolitan to all habitat categories. The catfish also had a broader food preference than trout and the diversity of the food items was significantly different (ANOVA, P<0.05) between the two species. Unfortunately, the interspecific food overlap between trout and the catfish could not be determined in Ebenezer Dam, because of the small sample size of trout but food selection between vii them was evident. It was then concluded that the native catfish has a wider niche and it is a more efficient predator than the introduced trout. This observation contributed further to the assertion that the area is marginal for trout to thrive. Due to trout selection of habitat variables, it became prudent to carry out another study where the past climate and land use changes were analyzed to determine their effect on the habitat that could have affected the distribution of trout in the area. Future projections were also made to determine possible future impacts of climate change on the distribution of trout in the area. The effects of climate and land use change resulted in warmer water temperature, altered riparian cover and altered stream flow patterns. The changes could have influenced the confinement of trout to higher altitude catchments. The projected maximum temperatures by 2050 shows an increase from 2014 with a decline in precipitation. If these projections are to be the same for water temperature and flow regimes, coupled with current land uses in the area, they will continue to affect the distribution of trout negatively.

Kaliumkanäle ermöglichen es, den geladenen Kaliumionen selektiv die hydrophobe Lipidphase von Zellmembranen zu passieren. Sie operieren in ihren Funktionen als Antagonisten von Natrium- und Kalziumkanälen, um die elektrische Erregbarkeit von Zellen zu kontrollieren. Im ZNS der Vertebraten existiert eine Vielzahl verschiedener Kaliumkanäle, welche die zeitliche Struktur von Aktionspotentialen festlegen. Im Rahmen der vorliegenden Dissertation wurden bisher unbekannte Kanäle aus den Familien der spannungsgesteuerten Kv1-/Kv3- und der kalziumsensitiven SK-/BK-Kaliumkanäle aus dem ZNS der Regenbogenforelle kloniert. Diese wurden durch heterologe Expression in Insekten- und Säugerzellen einer biophysikalischen und pharmakologischen Charakterisierung zugeführt. Gegen fünf der bekannten Kanäle wurden spezifische Antikörper hergestellt. Mit Hilfe immunhistochemischen und PCR-Techniken konnte die gewebetypische und entwicklungsspezifische Expression der Kaliumkanäle untersucht werden. Mit den in dieser Arbeit präsentierten Daten konnte die Grundlage für eine differentielle Betrachtung der funktionellen Bedeutung und des Expressionsmusters der entsprechenden Kaliumkanäle im nativen Kontext gelegt werden.

Snack food development studies were conducted to iii utilize trout (Oncorhynchus mykiss) frames, a by-product of the filleting operation, using extrusion and conventional technology. Twin screw extrusion studies were conducted to study the effect of fish mince, non-fat dry milk, process temperature, and moisture content on the physicochemical properties of the extruded snack food. Response surfaces were plotted to understand the effects of the independent variables on dependent variables such as bulk density, expansion ratio, shear strength, and water absorption index. Quadratic models expressed the relationship between the dependent and independent variables. Based on the extrusion studies, conditions suitable for further development of a ready-to-eat snack food were obtained. Conventional technology was also studied in the development of a fish cracker called keropok. A well expanded, tasty snack food was obtained using the minced fish and tapioca starch. Physicochemical characteristics of the developed snack were determined. Taste panel ratings for texture and taste of the cracker indicated a good potential for acceptance of this product for production and sale by local fish processors. Further studies may be undertaken to develop a continuous process to prepare the crackers on a larger scale.

<p>Teleosts take up metals by two major pathways: gills and/or gut. Past research is heavily focused on branchial uptake despite evidence that the gastro-intestinal tract (GIT) is the dominant route in some natural environments. To address this information gap, my thesis characterizes uptake mechanisms of Cd and Ca along the GIT of <em>Oncorhynchus mykiss</em>. Toxic effects of Cd, protective effects of Ca against Cd uptake, and Cd distribution within fish after ingestion are also explored.</p> <p>Four-week dietary Cd exposure affected growth and Ca regulation, while causing toxicity at the subcellular level. Elevated Ca in diets protected against accumulation and altered subcellular handling of Cd. Pre-exposure to different diets changed unidirectional uptake and binding rates of Cd and Ca, although they remained highly correlated. Binding rates of Cd to mucus can predict absorption rates of Cd<strong>.</strong></p> <p>A variety of <em>in vitro </em>and <em>in vivo </em>experiments on four distinct GIT segments (stomach, anterior-, mid-, and posterior- intestine) were undertaken to determine specific mechanisms of Cd and Ca uptake in freshwater trout. Cd transport was unaffected by solvent drag, but was stretch- and temperature- sensitive. Strong evidence for a common pathway for Ca and Cd transport was obtained. Cd also appeared to be taken up in part by zinc (ZIP-like) transporters and the divalent metal transporter DMT1. Ca uptake along the GIT appeared to be carrier-mediated, time- and concentration- dependent, but was not affected by solvent drag, or by Na concentration. Mucosal Cd did not inhibit Ca uptake. Ca, but not Cd, uptake rates were much lower in sea water-acclimated trout. Seawater and freshwater fish accumulated similar whole body Cd concentrations when fed contaminated diets for three weeks, but the majority of Cd in seawater trout remained in the posterior intestine tissue, while freshwater trout had higher internal burdens.</p> <p>This thesis advances the knowledge of metal uptake along the GIT of trout.</p> / Doctor of Science (PhD)

<p> The aim of this research was to determine how freshwater fish respond to dietary lead (Pb) in their environment with a respect to both physiological and toxicological endpoints. Previous acute exposure studies have shown that waterborne Pb accumulates in the internal tissues, resulting in hematological alterations and disruptions in Na+, cr and Ca2+ ion-regulation, with waterborne Ca2+ being protective against Pb tissue burden. In comparison with other metals such as silver, zinc, cadmium and copper, very little is known about Pb toxicity via the trophic food chain. </p> <p> Using rainbow trout as the model freshwater fish species, it was discovered that dietary Pb accumulated mainly in the gastrointestinal tract, with the intestine and the stomach being suggested as the primary tissues involved in Pb toxicity. Mild physiological disruptions in terms of plasma Ca2+, Mg2+ and Na+ levels were evident. On a per fish weight basis, the bone accumulated the greatest Pb burden (38%) followed by the anterior intestine (19%) and white muscle (12%). The order of Pb accumulation reflected the exposure pathway of dietary Pb in the internal tissues after 42 days; anterior intestine> stomach> mid intestine> posterior intestine> bone> posterior kidney> anterior kidney> liver> spleen> gill> carcass> brain> white muscle (JJ.g Pb/g tissue wet weight). Despite this accumulation of Pb into the internal tissues, there were no effects on growth and survival rates, indicating that growth is not a sensitive indicator of dietary Pb toxicity in fish. Elevated dietary Ca2+ levels had an overall effect in reducing dietary Pb burdens in the whole body and most of the internal tissues analysed. The red blood cells (RBC) accumulated 99% of the Pb in the blood, with less than 1% in the plasma. The enzyme 8-aminolevulinic acid dehydratase (ALAD), important in hemoglobin synthesis was significantly inhibited by the end of the experiment in fish exposed to high concentrations of dietary Pb. In contrast to waterborne Pb studies, ALAD was not a sensitive biomarker of dietary Pb toxicity. </p> <p> Overall, this study has shown the mild physiological and toxicological disturbances of dietary Ph exposure to juvenile rainbow trout. It encourages studies on transport mechanisms at the intestine and in the blood of fish exposed to dietary Pb. It is the hope that the work from this thesis will be used in risk assessments and environmental policies that mitigate the toxic effects of dietary Pb in the aquatic environment. </p> / Thesis / Master of Science (MSc)

In aquatic environments Cu is both a vital nutrient and an important toxicant. Consequently fish require Cu as a micronutrient and can obtain this metal from either water or their diet. Inadequate intake of Cu is associated with reduced growth and development, while decreased growth rates, mortality and reduced swimming capacity have been reported in fish when Cu accumulates in excess of cellular needs. Characterization of Cu uptake is therefore critical in understanding the dynamics that govern toxicity and the risks associated with exposure to an aquatic contaminant. While mechanisms of waterborne uptake and toxicity are well understood, far less is known about gastrointestinal Cu uptake in fish. In vivo and in vitro techniques were therefore used in this study to investigate dietary Cu uptake in freshwater rainbow trout. The mid and posterior regions of the intestine emerged as important sites for Cu absorption in trout, while the role of the stomach and anterior intestine in Cu absorption requires further investigation. The intestinal uptake route was kinetically characterized as a low affinity absorption pathway as compared to the branchial route. Cu uptake appeared to occur via a hypoxia-resistant, carrier-mediated, saturable process which could be fueled by Cu(II)^2+ at concentrations typically found in the fluid phase of chyme in the trout intestine. Experimental manipulation of mucosal NaCl levels stimulated Cu uptake, Na2SO4 had an identical effect, implicating Na rather than the anion. These responses were unrelated to solvent drag, osmotic pressure or changes in TEP. The presence of excess luminal Ag and L-histidine stimulated Cu and Na uptake indicating that a portion of Cu transport was mediated by a Na-Cu co-transport system. Partial inhibition of Cu and Na uptake by phenamil and hypercapnia stimulated Na and Cu transport suggest Cu entry could also occur via the apical Na channel. The Na-dependent mechanism thus either involves more than one component or a unique Na-Cu co-transport mechanism with these combined characteristics mediates part of Cu uptake. Cu uptake was sensitive to pH and competed by Fe and Zn implicating DMT1 in the transport of Cu in the trout intestine. These factors had no effect on Na uptake, leading to the identification of a Na-independent mechanism for Cu uptake in the trout intestine. While the Na dependent nature of Cu uptake and Ag stimulated Cu transport argue against a role for Ctrl in this process, Cu transport characteristics identified in this study compare well with a recently identified Cu transporter in Ctr1 deficient mouse embryonic cells, indicating the existence of a similar transport mechanism in the trout intestine. / Thesis / Master of Science (MSc)

Das Vorkommen von über den Umgebungsdruck erhöhten Sauerstoffpartialdrucken in Geweben ist im Tierreich nur von der Schwimmblase und dem Auge von Knochenfischen bekannt. Die physiologischen Mechanismen dieser Sauerstoffkonzentrierung wurden an der Schwimmblase insbesondere des Aals intensiv untersucht, entsprechende Untersuchungen zu den Vorgängen im Auge der Knochenfische fehlen jedoch. In dieser Arbeit wurde daher eine Methode etabliert, mit der die Sauerstoffkonzentrierung im Auge der Knochenfische an isolierten Augen untersucht werden kann. 1. Das Gefäßsystem der Regenbogenforelle (Oncorhynchus mykiss, Walbaum 1792) wurde durch Gefäßausgüsse dargestellt. Das im Auge gelegene choroidale Rete mirabile wird über die Arteria ophthalmica, die gleichzeitig die efferente Pseudobranchienarterie darstellt, versorgt. Die Pseudobranchie selber empfängt das Blut aus der efferenten Arterie des ersten Kiemenbogens. Diese Gefäßtopologie, Versorgung der Pseudobranchie mit bereits im ersten Kiemenbogen oxygeniertem Blut und auschließliche Versorgung des choroidalen Rete mirabile mit Blut aus der Pseudobranchie, weist auf die Bedeutung der Pseudobranchie für die Prozesse im Auge hin. 2. Die Retina der Regenbogenforelle wurde mikroskopisch untersucht. Die einzelnen Schichten der Retina entsprechen denen anderer Wirbeltiere. Die Forellenretina ist mit 407 µm aber wesentlich dicker als z.B. die Retina von Mammaliern. Zusätzlich fehlt der Retina der Forelle die bei vielen Mammaliern vorhandene retinale Blutgefäßversorgung. Mit den der Retina am nächsten gelegenen Gefäßen in der Choroidea bestehen bei der Forelle wesentlich größere Diffusionsstrecken von den Blutgefäßen zum Ort des Sauerstoffverbrauchs in den Zellen der Retina als bei anderen Wirbeltieren. Dies wir durch den erhöhten Sauerstoffpartialdruck in den Choriokapillaren kompensiert. 3. Für die Messung des intraretinalen Sauerstoffpartialdrucks wurden im Rahmen dieser Arbeit Sauerstoffmikroelektroden inklusive der zur Betreibung der Elektroden notwendigen Elektronik entwickelt. Die Elektroden waren vom Recess-Typ: die reaktive Oberfläche schloss nicht mit dem Ende der Glaskapillare ab, sondern war unter Belassung eines Hohlraumes in der Elektrodenspitze (Recess) in das innere der Glaskapillare verlagert. Dadurch waren die Elektroden unempfindlich gegenüber Rühreffekten. Die Mikroelektroden hatten eine Spitzendurchmesser von < 10 µm und eine mittlere Empfindlichkeit von 172 fA Torr-1 O2. Die Linearität der Elektroden über eine Partialdruckbereich von 0 bis 760 Torr O2 war besser als r = 0,9998. 4. Zur Überprüfung des normale Sauerstoffpartialdrucks in der Retina wurden Regenbogenforellen betäubt und künstlich ventiliert. Die systemischen Parameter wie arterieller PO2-Wert, Blutdruck und Herzschlag entsprachen dabei im Mittel mit 99 Torr (arterieller PO2), 28 Torr (Blutdruck) und 1,23 Hz (Herzschlag) den Werten unbetäubter, freischwimmender Forellen. Lediglich der arterielle pH-Wert war mit 8,02 während der künstlichen Ventilation durch eine ungenügende Kompensation einer respiratorischen Alkalose leicht gegenüber dem normalen Wert von 7,9 erhöht. 5. Die Auswirkungen der Präparation des Auges zur Messung des intraretinalen Sauerstoffpartialdrucks auf den Augeninnendruck wurde überprüft. Der Augeninnendruck betrug 4,9 Torr. Die Einbringung der Sauerstoffmikroelektrode hatte keine Auswirkung auf den Augeninnendruck. Erst das Entfernen der Elektrode nach der Messung führte zu einer deutlichen Verringerung des Augeninnendrucks. 6. Zum ersten Mal wurde in der vorliegenden Arbeit die Durchblutungsrate des Auges eines Knochenfisches bestimmt. Aus präparatorischen Gründen wurde die Messung an der afferenten Pseudobranchienarterie durchgeführt. Der mittlere Blutfluss in dem Gefäß betrug 745 µl min-1 kg-1. Durch Verschluss des kontralateralen Gefäßes stieg der Fluss auf 135% des ursprünglichen Wertes, da jetzt durch das ipsilaterale Gefäß und über die zwischen den beiden Gefäßen bestehende Anastomose beide Augen durch das ipsilaterale Gefäß mit Blut versorgt wurden. 7. Die Funktion der Retina während der intraretinalen Sauerstoffmessung wurde durch Ableitung des Elektroretinogramms kontrolliert. Trotz der während der Versuche fortbestehenden Betäubung mit dem Betäubungsmittel MS222, welches sich negativ auf das Elektroretinogramm auswirken soll, konnten normale Elektroretinogramme aufgezeichnet werde. Die invasive Messung des intraretinalen PO2 wirkte sich nicht negativ auf die Funktionalität der Retina aus. 8. Der intraretinale Sauerstoffpartialdruck wurde an betäubten, künstlich ventilierten Forellen überprüft. Der mittlere maximale PO2 im Bereich der Bruch´schen Membran betrug 382 Torr. An der Innenseite der Retina wurde ein PO2 von 10 Torr gemessen. Der Bereich des O2-Partialdruckgradienten in der Retina, gemessen über den Vorschub der Sauerstoffmikroelektrode vom niedrigsten bis zum höchsten PO2, betrug 433 µm, ein Wert der gut mit der mikroskopisch bestimmten Dicke der Retina (407 µm) übereinstimmt. 9. Zur Untersuchung der Einflüsse von Blutparametern auf die Sauerstoffkonzentrierung im Auge der Forelle wurden isolierte Augen künstlich mit verschiedenen Erythrozytensuspensionen perfundiert. Dazu wurde zum einen eine Suspension von Forellenerythrozyten eingesetzt, bei der erst der Root-Effekt in Abhängigkeit vom pH-Wert der Suspension bestimmt wurde. Bei basischem pH-Werten war das Hämoglobin der Erythrozyten vollständig gesättigt. Bei einem pH-Wert von 7,68 war noch eine Sättigung von 90% vorhanden, die bis zu einem pH-Wert von 7,31 auf 50% abnahm. 10% Sättigung wurden bei einem pH-Wert von 6,94 erreicht. Für die Perfusion wurde die Suspension der Forellenerythrozyten mit einem pH-Wert von 7,48 eingesetzt. Bei diesem pH-Wert liegt die Sättigung bei ca. 82% und eine Änderung des pH hat in diesem Bereich eine deutliche Änderung der Sättigung zur Folge, was die Freisetzung von O2 von Hämoglobin erleichtert. Als alternative Perfusionslösung wurde eine Suspension von Humanerythrozyten eingesetzt. Humanhämoglobin weist keinen Root-Effekt auf, es kann also durch die im Auge der Knochenfische vorhandenen Mechanismen kein Sauerstoff freigesetzt werden. 10. Der mittlere intraretinale Sauerstoffpartialdruck isolierter Augen betrug bei Perfusion mit der Suspension von Forellenerythrozyten 99 Torr. Nach dem Wechsel auf die Suspension mit Humanerythrozyten fiel der intraretinale Sauerstoffpartialdruck sofort auf 30 Torr ab. Der Perfusionsdruck änderte sich bei dieser Umstellung nicht. Bei Perfusion mit Ringerlösung fiel der intraretinale PO2 auf 20 Torr, gleichzeitig fiel jetzt auch der Perfusionsdruck ab. 11. Eine Konzentrierung des Sauerstoffs konnte bei der künstlichen Perfusion isolierter Augen nicht nachgewiesen werden. Die Notwendigkeit des Root-Effekts für einen hohen Sauerstoffpartialdruck im Auge wird trotzdem deutlich, da bei Perfusion mit einer Suspension von Humanerythrozyten, die einen höheren Sauerstoffgehalt aufwies als die Suspension der Forellenerythrozyten, der intraretinale Sauerstoffpartialdruck deutlich geringer war. 12. Das Modell des isoliert perfundierten Auges ist zur physiologischen Untersuchung des choroidalen Rete mirabile und Sauerstoffkonzentrierung besser geeignet als in vivo - Experimente, da systemische Einflüsse vermieden und die Auswirkungen der Pseudobranchie auf die Blutchemie ausgeschaltet werden können. / Oxygen partial pressures (Po2) above ambient levels are found only in the swimbladder and the eye of teleost fish species. The mechanisms related to the involved oxygen concentration processes have been investigated to some detail in eel swimbladder, but little attention has been paid to analogous mechanisms of the teleost eye. This thesis has accordingly concentrated on developing a method promoting investigation of oxygen concentrating mechanisms in teleost eyes by application of an isolated perfused eye preparation. 1. The vascular supply of eye and pseudobranch in rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) was depicted by corrosion casts of the vascular system. The choroid rete mirabile is supplied with blood by the ophthalmic artery as the efferent blood vessel of the pseudobranch, with a small anastomosis to the contralateral vessel. The pseudobranch in turn is fed by the efferent artery of the first gill arch. This pattern of serial vascular connection suggests a role for the pseudobranch in the process of providing high oxygen tensions required for the high metabolic demand of the visual perception in the retina. 2. Eyes of various fish species were sectioned after paraffin embedding and examined microscopically. The organisation of teleost fish retinae is characterised by the same features and organisation well known from other vertebrates. However, the total thickness of 407 µm found for the retina of the rainbow is much larger than that observed in other animals such as mammals. The conditions of diffusive oxygen supply of retinal nerve cells is further hampered by the complete lack of intraretinal blood vessels commonly found in mammals. In trout, blood is supplied to only the choroid layer, leaving large diffusion distances to the retinal nerve cells to be spanned by oxygen partial pressures, elevated as compared to the bulk arterial system in the choroid capillaries. 3. Intraretinal oxygen partial pressure was determined in long-term anaesthetised specimens of Oncorhynchus mykiss, using specially developed oxygen microelectrodes and required electronic circuitry. Characteristic features of the electrodes include insensitivity of the signal to stirring, achieved by a recess of the reactive metal surface in a glass capillary, an average tip diameter of less than 10 µm and a characteristic sensitivity to oxygen of 172 fA Torr-1. The response of the electrodes to oxygen was linear over a range of partial pressures from 0 to 760 Torr O2 (r= 0,9998). 4. Normal oxygen partial pressures in trout retina were measured in long-term anaesthetised and artificially ventilated specimens of Oncorhynchus mykiss. In order to conduct the experiments under conditions as close as possible to normal homoiostasis, several systemic valuables were monitored continuously. During experimentation arterial Po2 averaged 99 Torr, mean blood pressure was 28 Torr and heart frequency was on the average 74 bpm, respectively, data in the range of values determined in unanesthetized, freely swimming trout. As the one exception, arterial pH (8,02) was slightly elevated during artificial ventilation as compared to control values (7,89), a shift induced by an inadequately high rate of artificial ventilation. 5. The impact of insertion of electrodes required for intraretinal Po2 measurement into the eye was found to have little effect. The intraocular pressure remained unaffected by opening the eye by puncture with leading cannula and inserted micro-Po2-electrode. The intraocular pressure fell only after removing the electrode after completion of the experiment. 6. Normal function of the retina during experimentation in vivo was checked by repeatedly measuring electroretinograms provoked by light flashes under dark ambient conditions. Although extended anaesthesia with MS222 is expected to affect the electroretinogram in fish, there was no indication of abnormal or reduced electroretinograms during experimentation. Also puncture of the eye with insertion of electrodes did not affect the electroretinogram. 7. Intraretinal Po2 was measured in anaesthetised and artificially ventilated trout. Oxygen profiles were recorded during advancement of the electrode tip though the retina, from the lowest Po2 at the inner surface with about 10 Torr to the highest Po2 in the region of Bruch´s membrane with about 382 Torr, travelling a total distance of 433 µm. This distance closely corresponds to the thickness of the retina measured by microscopic sectioning. 8. Blood flow to the eye as an important parameter for the establishment of an isolated eye preparation was measured in the afferent artery of the pseudobranch. Due to the anatomical situation this represents the closest possible approximation to measurement of flow in the ophthalmic artery. The blood flow was estimated by ultrasonic Doppler techniques with in situ calibration to be 216 µl min-1. Occlusion of the contralateral vessel resulted in an increase of the blood flow in the ipsilateral vessel to 135% of the original as a result of flow through the small anastomosis between the two ophthalmic arteries. This estimate is the first performed for blood flow to the eye of a fish. 9. An isolated eye preparation was used to investigate the influence of specific blood parameters on oxygen concentrating processes in the eye of Oncorhynchus mykiss. Eyes were perfused with suspensions of trout erythrocytes, human erythrocytes and Ringer´s solution. The Root Effect of trout haemoglobin was confirmed in suspension of erythrocytes. The haemoglobin was fully saturated at pH 8.45, still 90% saturated at 7.68, 50% at 7.31 and 10% at pH 6.94. In order to provide optimal starting conditions for the release of oxygen by relatively small changes in pH, pH 7.48 (resulting in 82 % saturation) was chosen for the perfusion of isolated eyes. As a control perfusion, suspensions of human erythrocytes provided a perfusate lacking any Root effect, thus not supporting the oxygen concentrating processes in fish eyes. 10. Perfusion of isolated trout eyes with pH 7.48 trout erythrocyte suspension resulted in an average intraretinal Po2 of 99 Torr, whereas perfusion with human erythrocytes resulted in an immediate decrease of Po2 to (of) 30 Torr, with a further drop to 20 Torr during perfusion with Ringer´s solution. Changes in perfusion between erythrocyte suspensions were performed without any changes in perfusion pressure, but the switch to control perfusion with Ringer´s solution exhibited a drop in perfusion pressure due to the reduced viscosity of the medium. 11. Although perfusion of isolated eyes did not exhibit Po2 above ambient values, the effect of oxygen concentrating processes were clearly indicated by comparison with Root effect-lacking human erythrocyte perfusate (Po2´s of 99 vs. 30 Torr, respectively). 12. It is concluded that studies of oxygen concentrating mechanisms are to be performed preferably on isolated eye preparations. For closer analysis of the involved mechanisms this preparation allows for isolated control of individual parameter of the perfusate. In particular, uncontrolled effects of the pseudobranch on the perfusate can be eliminated.

Cette thèse précise les conditions pour introduire une sélection sur apparentés de caractères de qualité assistée par empreintes génétiques dans un programme de sélection massale sur la croissance chez la truite arc-enciel. Ce travail confirme l’intérêt à maîtriser l’effet maternel avec une doublement de l’héritabilité du poids à 70 g (0,36 vs 0,16), des estimations d’héritabilités des caractères mesurés de valeurs intermédiaires (0,37-0,54), l’intérêt d’utiliser la résiduelle des parties mesurées régressées linéairement au poids vif pour une sélection indépendante du poids pour les rendements, l’intérêt à remplacer la mesure du rendement au filetage par celle du rendement en carcasse éviscérée-étêtée plus héritable et très corrélé au rendement au filetage (0,97),la corrélation génétique élevée entre les rendements avec le ratio échographique e8/e23 (0,72-0,85) permettant une sélection sur candidats plus efficace qu’une sélection sur apparenté sur le rendement lui-même, des précisions élevées des valeurs génétiques (0.63–0.82) malgré très peu d’individus par famille (3,5-4) et une efficacité surestimée d’une sélection sur apparentés pré-sélectionnés (de 14 % à 62 %). Les conclusions sont qu’il est possible d’introduire une sélection sur apparentés dans un programme de sélection massale avec des gains génétiques au moins équivalents à ceux attendus en sélection familiale classique avec familles élevées initialement de façon séparées / The thesis aims at describing conditions to improve mass selection on growth by sib-selection on quality traits assisted by DNA parentage assignment in rainbow trout. Main results are that the control and management of differences in egg size between dams doubles heritability of body weight at 70 g (0.36 vs 0.16), heritabilities of quality traits are intermediates (0.37-0.54), the opportunity to use the residual of the body part (e.g. fillet weight) regressed to the whole body weight to select independently of body weight for ratios, the higher efficiency to replace fillet yield by deheaded and gutted carcass weight more heritable and highly genetically correlated to fillet yield (0.97), the possibility to use e8/e23ultrasound measures to predict genetically yields (0.72-0.85) allowing a direct selection on breeding candidates more efficient than in using sib information, a surprising intermediate to high accuracy of EBV even with a very low mean number of sib per full-sib family (3.5-4) and the medium to high overestimation of EBV when using pre- selected sibs (14 % to 62 %). The general conclusion are that sib-selection on quality traits can be introduced in mass selection with at least similar expected genetic gains than in traditional breeding design based on families reared initially separated

La pression sur les quotas de pêche et l’augmentation de la production aquacole ont contribué à une substitution importante des farines et des huiles de poisson incorporées dans les aliments pour poissons carnivores, par des farines et des huiles végétales. La truite arc-en-ciel, qui est un poisson carnivore, est affectée par ce changement de régime. Ainsi un retard de croissance apparaît dès le plus jeune stade si certaines transformations et supplémentations ne sont pas apportées aux végétaux. L’objectif de ce travail a été d’évaluer, aux stades alevins et juvéniles, l’impact d’une substitution totale des huiles et farines de poisson sur le tractus digestif de la truite arc-en-ciel, et plus particulièrement sur ses capacités digestives et sur la composition de son microbiote intestinal. Le but in fine étant de déterminer si certaines enzymes de la digestion, transporteurs intestinaux, ou sous-communautés bactériennes sont impactés par le changement de régime et peuvent expliquer le retard de croissance observé. Chacun de ces facteurs ont été étudiés via une approche de métagénomique par séquençage de nouvelle génération NGS pour la caractérisation du microbiote, et via de la PCR quantitative et des mesures d’activités enzymatiques pour la comparaison des capacités digestives. Des lignées isogéniques de truites, identifiées comme divergentes dans leur réponse à l’alimentation végétale (capables d’adaptation ou réfractaires) ont permis de disposer d’un matériel biologique pertinent pour répondre à cette question. Une modification du microbiote intestinal associé à la muqueuse digestive pourrait également contribuer à la baisse de l’homéostasie intestinale. Le changement de régime conduit en effet à une équitabilité plus faible chez les truites ayant reçue un aliment végétal, ce qui reflète un changement dans la représentativité de certains OTUs. Ce changement de régime s’est également traduit par des communautés dissimilaires en moyenne à 70 %, d’après l’estimation de la β-diversité entre les communautés de truites nourries avec l’aliment marin et celles nourries avec l’aliment végétal. La sélection opérée par l’aliment a conduit à un remplacement des OTUs rencontrés au sein des Firmicutes, c’est-à-dire que différentes espèces bactériennes de Firmicutes sont rencontrées suivant le régime considéré. La comparaison de communautés bactériennes entre les différentes lignées isogéniques a montré que la sélection opérée par le génotype de l’hôte a davantage eu lieu sur le remplacement des β-Protéobactéries. Enfin, les comparaisons d’abondances en certaines espèces bactériennes particulières suggèrent que les bactéries Cetobacterium somerae, capables de synthétiser de la vitamine B12, et Shewanella, dont l’implication dans la stimulation des cellules β du pancréas endocrine a déjà été observée chez d’autres espèces, pourraient être impliquées dans la réponse métabolique des truites aux végétaux. Les modifications identifiées dans ce travail constituent des indicateurs biologiques qui pourront être mis à profit pour évaluer la réponse du tractus digestif des truites à de nouvelles formules alimentaires. / Over-fishing pressure and increasing aquaculture production led to an important substitution of fish oil and fish meal with oil and meal from plant origin in feed meant for farming fish. However this replacement has some deleterious incidence on fish. For rainbow trout, which are carnivorous fish, some growth delay often appears from the early life stages when they are fed with plant based diet. The aim of this work was to assess, at alevin and juvenile stages, the impact of a total replacement of fish meal and oil on rainbow trout gastrointestinal tract, and more particularly on the digestive capacity and the associate microbiota. The objective, in fine, being to determine if some digestive enzymes, intestinal transporters, or bacterial communities are impacted by the dietary replacement and if these biological factors can be related to the observed growth delay. Metagenomic approach using next generation sequencing was used to characterize the gut bacterial communities, while digestive capacity was assessed through quantitative PCR and enzymatic measurements in order to compare rainbow trout responses to a plant-based diet. In our investigations, rainbow trout isogenic lines that diverge in their response to this alternative diet (tolerant or rather reluctant) were adopted because they constitute a pertinent biological material for answering this question.In alevin rainbow trout, a plant-based diet led to an increase of pepsinogen, trypsinogen, and chymotrypsinogen genes which codes for proteolitic enzymes. Two main assumptions can explain this response, and their effectivness remains to investigate: wether this is a physiological response due to a lower weight of trout fed with the plant-based diet, or so it is due to an increase transcritpion of pancreatic enzymes to compensate for a reduction of protein digestibility. In the intestine, it appears that an increase transcription of IAP, SGLT1, CCK-t, and PEPT1 genes, and a decrease transcription of GLUT2 gene under a plant-based diet could reflect a disability to grow under a vegetable diet.In juvenile rainbow trout, a plant-based diet led to a decrease of lipid digestibility, and of triglycerides and total amino acid plasmatic levels. These perturbations could be explained in part by a decrease of the phosphatase alkaline activity, which suggest perturbancesof intestinal homeostasis, and by a decrease of phospholipase A2 activity. Transcriptional decrease of the triglycerides transporter MTP and of the prolidase, which is a peptidase from intestinal cell cytosol, has also been observed. Some modification of the microbiota associated to the intestinal mucosa could also contribute to the decrease of the intestinal homeostasis. The dietary replacement effectively led to reduce evenness of the bacterial communities in trout fed with a plant-based diet, which reflected a shift in the representativeness of some OTUs. Bacterial community from trout fed with a marine diet and trout fed with a plant-based diet were on average 70 % dissimilar. Dietary substitution led to the replacement of OTUs from the Firmicutes class, different bacterial species being observed according to the considered diet. The comparison of bacterial community between the isogenic lines showed that the genotype led to the replacement of β-Proteobacteria. Finally, abundance comparison suggested that Cetobacterium somerae, which is able to synthesise vitamin B12, and Shewanella, which has already been reported to stimulate pancreatic β cells, could be implicated in the trout response to vegetable.Modifications observed in this work constitute biological indicator that could be used to assess the response of the digestive tract to future feed formulations.

Juvenile rainbow trout (Oncorhynchus mykiss) fed L-tryptophan (TRP) supplemented feed for 7 days show a suppression of aggressive behaviour along with a reduction in post-stress plasma cortisol levels. However, 3 days of elevated dietary TRP does not have any effects on either aggressive behaviour or post-stress plasma cortisol levels. The effects of TRP on aggression and stress responsiveness could be related to elevated brain serotonergic activity, TRP being the precursor of serotonin (5-HT). However, 5-HT is, in turn, the precursor of melatonin (ME) and we found that elevated dietary intake of TRP results in an increase in daytime plasma levels of ME. Elevated plasma ME concentrations in rainbow trout fed TRP supplemented feed appear to be of gastrointestinal origin. ME has been suggested to have inhibitory effects on aggressive behaviour and to act as a glucocorticoid antisecretagogue. However, we found that exogenuos ME did not affect aggressive behaviour or post-stress plasma cortisol in rainbow trout. Instead, the effects of TRP on aggressive behaviour and plasma cortisol were mimicked by treatment with the selective serotonin re-uptake inhibitor, citalopram, suggesting that the effects of TRP are mediated by a stimulation of brain 5-HT activity and not by increased ME synthesis and secretion. However, the slow time-course of the effects of TRP on aggressive behaviour and plasma cortisol suggests that effects on 5-HT receptors mechanism are likely to be involved.