This research programme aimed to review the nutritional requirements for the main minerals, (namely calcium, phosphorus, magnesium and zinc) formulated in commercial diets that are essential for the health and growth performance for salmonid fish. This was undertaken with the aim of improving our knowledge of their physiology, metabolism and fate in the rainbow trout, Oncorhynchus mykiss. Phosphorous (P) featured strongly in this work due to its adverse role in pollution and the environmental impact of intensive fish farming. The first chapter surveyed the gross nutritional requirements of fish and focused on the mineral requirements in particular. Typically the P requirements for trout were found to range from 0.5-0.8% of the diet. The problems of P loading as a consequence of dietary loses was addressed and the physiological and metabolic roles of both calcium and phosphorous were especially noted in relation to fish health and for phosphorous its environmental implications were addressed. Experimental approaches were evaluated and it was decided to conduct both standard growth trial studies as well as digestibility trials to provide the basis of most investigations with the rainbow trout. Novel approaches and strategies were used in relation to specific experiments such as examining the mineral levels in blood, and various tissues and the testing of different feed ingredients, dietary supplements and mineral sources in successive investigations. Initial investigations appraised commercial diets of varying nutritional profile with respect to mineral retention and availability for rainbow trout under controlled laboratory conditions. The effects of diets containing different fishmeal sources: i.e. brown versus white fishmeals, elevated ash content and also varying in the levels of oil were tested on juvenile rainbow trout in closed recirculated systems. Diet composition caused a significant effect on mineral retention and distribution profile in fish tissues and organs. Typically, both P and Ca were of highest concentration in vertebrae of trout (60mg/g-dry weight), compared with P concentrations for all other major organs/tissues, which were fairly even between 11-20mg/g. A small increase in dietary P level (1.08% vs. 1.22%) did not affect any growth parameters for trout for the first two commercial feeds tested but there were interesting observations with respect to the amount of P excreted in the bile with a 25% increase from 0.8mM to 1.2mM. The P levels in plasma of these fish did not reflect any dietary changes. However, there was a noticeable reduction in the digestibility of P in the diet containing the white fishmeal source (26%) compared with 49% for the higher grade fishmeal diet. High ash content feeds resulted in a marked reduction in the net mineral retention of this element (16% compared to 27% for the lower ash diet). The same was also true for Ca (12% compared with 26%). The effect of oil levels in diets on mineral utilization was investigated under farmed conditions and was of particular interest given the demand for nutrient dense feeds in the industry. There was a strong tendency for improved P and Ca digestibility coefficients at each incremental increase in oil level for juvenile production sized fish (50-l00g). This ranged from 55% to over 70% when oil levels were over 26%. However this was not observed for larger fish of over 200g in weight. Experimental investigations followed are described in (Chapter 4) where fishmeal based diet was supplemented with varying levels of inorganic phosphorous. Phosphorous, calcium and other mineral absorption characteristics in addition to retention were measured in a series of growth and digestibility trials. Interestingly, there was no apparent change in the distribution of P with increasing dietary levels ranging from 1.39-2.16%. These were above known requirements for these fish with minerals being in excess. Similar results were noted for all other minerals measured in rainbow trout. There was a significant rise in the P concentration of plasma of rainbow trout fed a diet containing over 2% P and this may infer that the homeostatic regulation of P is unable to function at this level. Other haemato-logical parameters were not affected. Although not significant, there appeared to be slight trend in elevated bile P with increasing dietary P supplementation. The faecal concentrations for each of the minerals showed that elevated P in fish meal diets led to increased faecal output from 25mg/g to over 40mg/g for the highest P diet. Overall digestibility coefficients were lower as dietary P increased above that in the fishmeal control diet. These ranged from 50% to 39% for P, Ca and Mg were not greatly affected. The net retention of P was calculated and this fell from 30% to just below 20% for the range of dietary P used in the investigation. A preliminary study, reported in chapter 5A, was useful in providing information about the relative absorption profiles for differential mineral absorption from the various regions of the gastro intestinal tract of rainbow trout. A standard commercial diet was fed to large trout (>200g) and subsequently, digesta was removed from fish and analysed. For all minerals and protein, the pyloric and mid intestinal region was the main site for digestion, release and absorption of the macro elements concerned. The protein and mineral digestibility of suitable feedstuffs commonly employed in the formulation of complete diets for trout resulting from a sequence of experimental trials are presented in chapter 5B. These included a selection of marine, animal and plant by-products which were substituted into a reference basal diet designed for salmonids. This involved the inert marker- yttrium oxide and calculations based on nutrient digestibility from diet and faecal concentrations. Mineral digestibility coefficients were found to vary considerably and a number of anomalies such as negative values were obtained for Ca and Zn in certain feedstuffs. Combined diets (reference and test ingredient) gave values that were more consistent and P digestibility ranged from 47-59% in marine and animal protein concentrates compared with plant sources (24-37%). Negative values for Ca and Zn were thought to be attributable to complex interactions with other feed components. Additionally, a group of inorganic mineral supplements were tested by inclusion into a series of diets. These included mono calcium phosphate, di-calcium phosphate (DCP), mono di-calcium phosphate and magnesium phosphate. DCP produced lower Ca and P digestibility values, 31 and 50% respectively, compared with the other sources (44-62%), indicating the importance of choice of mineral supplement in aquafeeds. A critical appraisal of this work is provided in Chapter 6 and formed a retrospective review of the results generated and integrated these findings into a foundation for further research and development. Nutritional investigations using the rainbow trout as the model salmonid species raised many more questions and possibilities and broadened the scope of the topic. The subject of mineral requirements for fish is very complex and numerous factors are involved at several physiological and biochemical levels in fish. Although the research on rainbow trout involved whole animal studies under both laboratory and commercial farm conditions, the need to explore alternative in vitro methods and to utilize larger scale farm and sea cage trials for salmon were suggested. The advent of more advanced diet formulations and feeding strategies were mentioned and the scope for more scientific investigations to improve the utilization and reduce phosphorous discharge into the environment proposed.

Conventional fish monitoring requires considerable investments of equipment and labour, and often harmful and potentially fatal techniques. Emerging methods allow detection of aquatic animals by collecting water and extracting DNA that has been shed to the environment (eDNA). Present knowledge gaps in the field include minimum densities necessary for consistent detection, and persistence of eDNA after a target species has left a site. I conducted three experiments at a salmon hatchery in British Columbia to address these knowledge gaps. Water samples were taken from flow-through tanks with juvenile Coho Salmon densities ranging from 38.0g/1000L to 0.6g/1000L. To simulate field surveys in recently abandoned habitats, I sampled water from tanks after removing fish, at flow-through volumes ranging from 20,000L to 1,000,000L. Post removal sampling occurred starting at one hour and ending after just over four days of flow-through time. Water samples from tanks containing one or more fish tested positive for Coho DNA at least 70% of the time, increasing at higher densities. Samples taken after removing the fish had detection probability of 75% at flow-through volume of 40,000L. Detection failed at flow-through volumes greater than 80,000L. In stream samples, all sites with Coho or salmonid presence confirmed by conventional trapping also tested positive for target species’ eDNA. Two sites tested positive for Coho eDNA where conventional methods failed, indicating a possible higher sensitivity of eDNA sampling. I also mapped the distribution of juvenile Coho Salmon through multiple tributaries of a productive salmon system with conventional and eDNA detections. This study improves on an emerging method with a new species by addressing existing uncertainties regarding eDNA detection threshold, and signal persistence through dilution in a simulated stream pool habitat. It also demonstrates that eDNA methods can be used to assess coastal streams for presence of juvenile and resident salmonid fishes. / Graduate

<p>Animals show a great variety in physiological and behavioural responses to stressors. These responses are often bimodally distributed within populations and show consistency on an individual level over time and across situations, which in terrestrial vertebrates have been identified as proactive and reactive stress coping strategies. Proactive animals show lower cortisol responses, higher sympathetic activation and brain serotonergic activity compared to reactive animals. Behaviourally, proactive animals are more aggressive, more active in avoiding stressors, they form routines and show fewer cases of conditioned immobility compared to reactive animals. Our aim has been to reveal if such stress coping strategies exist in fish. Our results show that rainbow trout with high (HR) or low (LR) cortisol responses to stressors differs in sympathetic activation and brain serotonin turnover in the same manner as proactive and reactive mammals. HR fish showed less locomotor activity when reared in large groups (30 individuals) compared to LR fish. When reared in isolation there were no differences between HR and LR fish when exposed to stressors within a familiar environment. The adaption of a proactive coping style among reactive coping individuals when they are challenged within a familiar environment has previously been shown to be distinction between proactive and reactive coping mammals. However, when they were transferred to unfamiliar environments a behavioural difference between the two lines was observed indicating different stress coping strategies akin to those described in mammals. Finally, we observed a consistency over time in the cortisol response of an unselected line of rainbow trout. Fish from this line also demonstrated a correlation between behavioural responses to different stressors. However, there was no apparent connection between these behavioural responses and the cortisol response. Overall, the results of this thesis have strengthened the hypothesis that different stress coping strategies exist in teleost fish.</p>

Animals show a great variety in physiological and behavioural responses to stressors. These responses are often bimodally distributed within populations and show consistency on an individual level over time and across situations, which in terrestrial vertebrates have been identified as proactive and reactive stress coping strategies. Proactive animals show lower cortisol responses, higher sympathetic activation and brain serotonergic activity compared to reactive animals. Behaviourally, proactive animals are more aggressive, more active in avoiding stressors, they form routines and show fewer cases of conditioned immobility compared to reactive animals. Our aim has been to reveal if such stress coping strategies exist in fish. Our results show that rainbow trout with high (HR) or low (LR) cortisol responses to stressors differs in sympathetic activation and brain serotonin turnover in the same manner as proactive and reactive mammals. HR fish showed less locomotor activity when reared in large groups (30 individuals) compared to LR fish. When reared in isolation there were no differences between HR and LR fish when exposed to stressors within a familiar environment. The adaption of a proactive coping style among reactive coping individuals when they are challenged within a familiar environment has previously been shown to be distinction between proactive and reactive coping mammals. However, when they were transferred to unfamiliar environments a behavioural difference between the two lines was observed indicating different stress coping strategies akin to those described in mammals. Finally, we observed a consistency over time in the cortisol response of an unselected line of rainbow trout. Fish from this line also demonstrated a correlation between behavioural responses to different stressors. However, there was no apparent connection between these behavioural responses and the cortisol response. Overall, the results of this thesis have strengthened the hypothesis that different stress coping strategies exist in teleost fish.

<p>Diss. (sammanfattning) Umeå : Umeå universitet, 1995, härtill 5 uppsatser.</p> / digitalisering@umu

Rainbow trout (Oncorhynchus mykiss) are introduced in large parts of the world and are commonly farmed for consumption as well as a valued sport fish. Many species of salmonids show large intraspecific variation in pigmentation, which has been shown to correlate with stress response and several other behavioral traits. In this study, the behavior and stress response of rainbow trout linked to pigmentation is investigated, depending on previous sport fishing experience. Through data from a previous study on rainbow trout in semi-natural ponds, locomotor activity was measured as a proxy of stress, under three different sport fishing treatments. From previous photos, the pigment spots of each rainbow trout are counted to test the correlation with locomotor activity. In addition, it is tested whether different previous experience of sport fishing means a higher stress response when re-exposed to fishing. No significant relationship between pigment spots and locomotor activity could be obtained for the three treatment groups. However, significant differences in locomotor activity due to angling experience between treatment groups upon re-exposure to fishing were found, with fish that were inexperienced in angling having higher locomotor activity compared to previously caught fish. Sport fishing and catch-and-release had the effect of reducing locomotor activity in rainbow trout, which can be interpreted as fishing could both stress them and trigger the fish's feeding response, depending on previous experience. Fishing is believed to create a passivation due to the negative association of being caught, while fish not previously caught were activated by sport fishing. The study's missing correlation between pigment spots and stress has been both confirmed and denied in other studies and may depend on the origin and the degree of domestication, which means that the correlation within other species of salmonids or fish with different origins would be relevant to investigate. / Regnbåge (Oncorhynchus mykiss) förekommer introducerade i stora delar av världen och är vanliga att odlas för konsumtion samt en uppskattad sportfisk. Många arter av salmonider visar stor intraspecifik variation i pigmentering, vilket visats korrelera med stressrespons och flera andra beteendemässiga karaktärsdrag. I denna studie undersöks regnbågarnas beteende och stressrespons kopplat till pigmentering, beroende på tidigare erfarenhet av sportfiske. Genom data från en tidigare studie på regnbåge i semi-naturliga dammar mäts rörelseaktivitet som indirektmått på stress, under tre olika sportfiskebehandlingar. Från tidigare foton räknas varje regnbåges pigmentfläckar för att testa korrelationen med rörelseaktivitet. Därtill testas om olika tidigare erfarenhet av sportfiske, innebär högre stressrespons vid återexponering för fiske. Inget signifikant samband mellan pigmentfläckar och rörelseaktivitet kunde erhållas för de tre behandlingsgrupperna. Signifikanta skillnader i rörelseaktivitet på grund av erfarenheten av sportfiske mellan behandlingsgrupperna vid återexponering för fiske fanns dock, där fisk som var oerfaren sportfiske hade högre rörelseaktivitet jämfört med fisk som fångats tidigare. Sportfiske och catch-and-release hade effekten att minska rörelseaktiviteten hos regnbågar, vilket kan tolkas som att fisket både kunde stressa och trigga fiskens födorespons, beroende på tidigare erfarenhet. Fisket tros skapa en passivisering på grund av den negativa associationen att bli fångad, medan fisk som inte fångats tidigare aktiverades av sportfisket. Studiens uteblivna samband mellan pigmentfläckar och stress har både bekräftats och dementerats i andra studier och kan bero på ursprung och graden av domesticering, vilket gör att sambandet inom andra arter av salmonider eller fisk med olika ursprung vore aktuellt att undersöka.