Metabolic fuel use in rainbow trout (𝘖𝘯𝘤𝘰𝘳𝘩𝘺𝘯𝘤𝘩𝘶𝘴 𝘮𝘺𝘬𝘪𝘴𝘴 W.) was investigated using closed system respirometry and proximate body analysis. During short term starvation (15 days, routine activity) the utilization of protein as a substrate, as determined by respirometry, increased from 14 to 24% of total fuel supply. However, even by the end of the experiment, the contribution of protein (24%) did not approach the classically reported values for fish of between 40 and 90%. Indeed, from respirometry data, during the first quarter of the experiment lipid contributed the majority of the fuel ( >60%) while carbohydrate contributed about 20%. Thereafter, lipid and carbohydrate became essentially equivalent in importance (about 37% each). However, from proximate body analysis, a more traditional fuel mixture was found (protein, 58%; lipid, 40%; carbohydrate, 2%) suggesting the possibility that the two procedures were measuring fundamentally different parameters. Instantaneous fuel use during sustainable swimming at different speeds was investigated by respirometry using three day test periods. While protein catabolism remained constant over time, and uniform between groups, its relative contribution tended to increase with time as total M₀₂ declined with sustained swimming. Protein catabolism was highest in nonswimming fish (30-45%) and lowest in the high speed swimmers (20%); lipid was the most abundant (41-55%) fuel used in all groups at all times. In the nonswimmers and lowspeed swimmers, lipid use tended to increase slightly over time whereas in the high speed swimmers, lipid use dropped from 54 to 44%. Carbohydrate use (up to 38%) was higher than predicted by earlier literature, but decreased greatly in both the nonswimmers and low speed swimmers over the three days, whereas in the high speed swimmers the contribution increased with time. The low speed swimmers from the last set of experiments were used as controls for the final set of experiments in which another group of fish were trained for two weeks at 1.0 L·s⁻¹ prior to testing using an otherwise similar regime. Even though there was no difference in gas exchange, the make-up of the fuel mixture was different for the two groups. Protein use was significantly lower, while lipid use was higher in the trained fish. In addition, relative protein use in the trained fish was constant over the three day period, a feature found only in the the high speed swimmers of the previous experiment. A critical evaluation of the respiratory quotient is given since its use by fish physiologists has been without complete conversion from that used by the mammal physiologists. In addition, the often quoted term "fuel use" is differentiated into 𝘪𝘯𝘴𝘵𝘢𝘯𝘵𝘢𝘯𝘦𝘰𝘶𝘴 𝘧𝘶𝘦𝘭 𝘶𝘴𝘦 and 𝘤𝘰𝘮𝘱𝘰𝘴𝘪𝘵𝘪𝘰𝘯𝘢𝘭 𝘧𝘶𝘦𝘭 𝘶𝘴𝘦 since the two describe fundamentally different principles, though this has not always been recognized in the literature. / Thesis / Master of Science (MS)

Thesis / Master of Science (MS)

Dissertation (PhD (Agric))--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: A breeding programme aimed at the genetic improvement of growth rate of rainbow trout was initiated in 1988 by the Department of Genetics, University of Stellenbosch, in collaboration with the local trout producer's organisations. The first phase of the breeding programme included the collection, evaluation and selection of the best available genetic material from 13 different genetic groups (nine local and four overseas) to make up two separate base populations as odd and even year-groups. This was done to establishment a base population with high genetic merit and variation at the onset of the breeding programme. Statistically significant and commercially valuable genetic differences in terms of weight and length gain were detected between the various hatchery groups. The next two generations of the breeding program included a series of single and double crosses in order to increase the levels of genetic variation in the base populations, and to investigate possible heterosis and specific and general combining ability among the crosses. Significant levels of heterosis (6.7% to 9.6%) and general combining ability was found for weight and length gain during consecutive growth stages. No evidence was found for specific combining ability among the crosses. The crossing of selected offspring from the original genetic groups followed by the application of intensive multi-stage selection for growth rate within progeny groups has led to the establishment of second and third generation parental populations with higher levels of genetic variation and improved individual genetic merit with regard to growth rate. The exploitation of non-additive genetic variation within the base populations through crossbreeding and heterosis during the early stages of the selection programme was delayed in favour of the utilization of additive genetic variance through a procedure of multi-stage selection that incorporated high intensities of selection within and between family groups. The estimation of genetic parameters during the fourth generation on the basis of a hierarchical half-sib family structure confirmed the presence of high levels of additive genetic variation within the respective populations/year-groups. High heritability values in the range of 0.40 to 0.53 were recorded for body weight and length at 150 days. Genetic correlations between the traits were also high, in the range of 0.74 to 0.82. The cumulative realized response of 50% in body length for the EVEN year-group after six generations of selection (8.3% per generation), and the 33% for the ODD year-group after five generations of selection (6.6% per generation) confirms the efficiency of the multi-stage selection procedure to exploit the available additive genetic variation for growth rate within the respective populations. The programme is still ongoing, entering its 7th generation in 2004 and is supplying about 50-60% of commercial material through direct supplies of broodstock, ova and fingerlings and indirect supplies via multiplier stations (commercial hatcheries). The programme was the first of its kind in relation to aquaculture species in the Southern African region, and has since initiated the introduction of programmes of genetic improvement in three other indigenous species, namely tilapia (Oreochromis mossambicus), African catfish (Clarias gariepinus) and abalone (Haliotis midae). / AFRIKAANSE OPSOMMING: ‘n Teelprogram gerig op die verbetering van groeitempo in reënboogforel is in 1988 ingestel onder toesig van die Departement Genetika aan die Universiteit van Stellenbosch, in sameweking met die plaaslike forelprodusenteverenigings. Die eerste fase van die teelprogram behels die versameling, evalasie en seleksie van die beste beskikbare genetiese materiaal vanuit, 13 verskillende genetiese groepe (nege plaaslike en vier van oorsee) om twee basispopulasies te ontwikkel in elk van die gelyke en ongelyke jaargange. Die doel daarvan was om ’n basispopulasie met hoë genetiese meriete en variasie te ontwikkel met die aanvang van die teelprogram gerig op genetiese verbetering, deur middel van seleksie. Statisties betekenisvolle en ekonomies belangrike genetiese verskille in massa- en lengtetoename is aangetref, tussen die onderskeie genetiese groepe. Die daaropvolgende twee generasies binne die teelprogram behels die uitvoering van ’n reeks enkel- en dubbelkruisings ten einde ’n verdere toename in genetiese variasie in die basispopulasies te bewerkstellig, sowel as om die voorkoms van heterose en algemene, sowel as spesifieke kombinerings-vermoë tussen die kruisings te bepaal. Betekenisvolle vlakke van heterose (6.7% tot 9.6%) sowel as algemene kombineringsvermoë, is aangetref ten opsigte van massa- en lengtetoename in opeenvolgende groeifases. Daar kon geen aanduiding van betekenisvolle, spesifieke kombineringsvermoë gevind word nie. Die kruising van geselekteerde nageslag vanuit die oorspronklike genetiese groepe, gevolg deur ‘n multi-fase seleksiemetode vir groeitempo binne nageslaggroepe, het bygedra tot die ontwikkeling van ‘n tweede en derde generasie broeipopulasie wat beskik oor hoër vlakke van genetiese variasie en verbeterde individuele meriete ten opsigte van groeitempo. Die benutting van nie-additatiewe genetiese variasie binne die basispopulasies deur middel van kruisteling en heterose tydens die vroee stadium van die teelprogram is uitgestel ten gunste van die benutting van additatiewe genetiese variasie deur middel van ‘n multi-fase seleksiemetode, wat berus het op die toepassing van hoë vlakke van seleksie-intensteit binne en tussen familiegroepe. Die beraming van genetiese parameters tydens die vierde generasie het die voorkoms van hoe vlakke van additatiewe variasie binne die onderskeie jaargroepe bevestig. Hoë oorerflikhede van 0.40 tot 0.53 is beraam vir ligaamsmassa en -lengte op die ouderdom van 150 dae. Genetiese korrelasies tussen die kenmerke was ook hoog met waardes van 0.74 tot 0.82. Die saamgestelde gerealiseerde seleksierespons van 50% vir liggaamslengte vir die “EVEN”-jaargroep na afloop van ses generasies van seleksie (8.3% per generasie) en die 33% van die “ODD”-jaargroep na afloop van vyf generasies van seleksie (6.6% per generasie) het die doeltreffendheid van die multi-fase seleksiemetode bevestig ten opsigte van die benutting van die additatiewe variasie vir groeitempo binne die onderskeie basispopulasies/jaargroepe. Die teelprogram duur steeds voort en sal die 7de generasie in 2004 bereik. Die program voorsien nagenoeg 50-60% van die kommersiele materiaal vanuit direkte voorsiening van teelmaterial, eiers en vingerlinge asook die indirekte voorsiening via kommersiële teelstasies. Die teelprogram was die eerste van sy soort met betrekking tot akwakultuurspesies in Suider Afrika en het bygedra tot die implimentering van programme van genetiese verbetering in drie inheemse spesies, naamlik die tilapia (Oreochromis mossambicus), die baber (Clarias gariepinus) en die perlemoen (Haliotis midae).

Selenium is an important nutritional element that is required in a minute amount for maintenance of proper health in both humans and animals. Many biochemical processes in human and animal depends on Se and selenoprotein function, and various studies have suggested that Se supplementation can improve fundamental immune function in both humans and animals. Se status in the UK is very low; therefore, there is a need to enhance selenium intake through diet. One way of doing that is through the introduction of selenium to farmed animals. In this Ph.D. study, we fed rainbow trout with diet containing different spiked concentrations (0.8 - 8.9 μg g-1) of Se for 14 weeks. Fish were sampled every two weeks and liver, kidney, muscle, gills and whole blood were collected and processed. The first phase of this Ph.D. study focused on selenium distribution and biotransformation in tissues of rainbow trout. Three methods were developed and used to determine total selenium concentration; species' distributions in tissues and Se peptide sequence to determine the possible incorporation of Se into proteins respectively. In the first method total selenium concentration was determined using ICP-MS and the second and third methods using HPLC-ICP-MS/ESI-MS. Total selenium concentrations in trout tissues were determined, and the highest selenium concentrations were found in liver followed by kidney, gills, and muscle. SeMet and SeCys were found to be the major species in all tissues followed by inorganic and other unknown species. To determine the possibility of Se incorporation in to protein, peptide de novo sequencing was carried out. Few selenoprotein were identified using automated de novo sequence and database search. Se species distributions in tissues of beached Pilot whales were studied. Several low molecular weights Se species were identified with selenite pre-dominating other species in most of the adult whales tissues analysed.

The nutritional and chemical characteristics of five earthworm species: Lumbricus terrestris; Allolobophora longa; Eisenia foetida; Dendrobaena veneta and Dendrodrilus subrubicundus were assessed on the basis of crude protein and amino acid composition, lipid and fatty acid composition, and ash and mineral composition. Analyses indicated that all the earthworm species evaluated possessed a high quality protein and lipid fraction suitable for use in fish feeds which was somewhat similar in composition to that of fish meal. During experimental feeding trials (50-84 days in duration) each earthworm species was nutritionally evaluated, on the basis of fish growth performance, feed utilization efficiency and gross carcass composition, as a complete feed (frozen slices of whole worm) for rainbow trout· (Salmo gairdneri) A dried 'earthw9rm meal' derived from each of the species E.foetida, D.veneta and D.subrubicundus was similarly evaluated as a potential replacement for fish meal in trout diets. Fish fed frozen slices of earthworm, with the exception of fish fed E.foetida, achieved growth rates and feed utilization efficiency comparable to. fish fed a control, fish meal based ration. Fish fed solely on frozen slices of E.foetida achieved little or no growth over the experimental period. The possible reasons for the reduced palatability of frozen E.foetida to the fish are discussed, pre-treatment processes applied and a significant improvement in the palatability of frozen E.foetida was achieved by blanching. High dietary inclusion levels of earthworm meal (replacing ~ 50% of the fish meal protein) resulted in depressed feed intake and growth of the fish. At reduced levels of inclusion, dried E.foetida meal (constituting 5-30% of a production salmonid diet) and dried D.subrubicundus meal (constituting 7-36% o~ a semi-synthetic trout diet) adequately replaced the dietary fish meal component without loss in fish growth and feed utilization efficiency. The possible uptake of potentially toxic trace elemeots (Fe, Zn, Mn, Pb, Cu, Cr, Ni, Co and Cd) into the fish carcass through the ingestion of contaminated earthworm diets was also investigated and the levels of certain elements, in particular Pb, were observed to increase in the carcass of fish fed high levels of earthworm in the diet. However, in no instance did the results indicate any harmful or toxic effect of including earthworms in the diets of rainbow trout.

The effects of different dietary lipids on the growth, nutrition and tissue lipid profiles of rainbow trout raised to market size on a commercially available ration were examined. Rainbow trout of 80 g mean initial weight were fed pellets prepared according to Oregon Moist Pellet specifications for 20 weeks. Salmon oil (0MP-1), soybean oil (OMP-2), linseed oil (OMP-3), chicken fat (OMP-4), pork lard (OMP-5) and beef tallow (OMP-6) were used for the 6% lipid component of the diets. No differences in feed conversion or growth rate were observed across diet groups. Trout nutrition was monitored by three blood parameters -- red blood cell (RBC) fragility (hemolysis), packed cell volume (PCV) (hematocrit), and percent hemoglobin. The OMP-1 diet appeared to have caused oxidative stress in trout as measured by hemolysis, while hematocrit and percent hemoglobin values were uniform across diet groups. Tissue levels of total n-3 fatty acids were highest in the OMP-3 group and decreased in the order OMP-1 > OMP-2 = OMP-4 = OMP-5 = OMP-6. However, unaltered linolenic acid (18:3 n-3) comprised almost 50% of the n-3 fatty acid content of OMP-3 trout tissue lipid. The level of total 20:5 n-3 plus 22:6 n-3 (18%), was 33% higher in tissue from OMP-1 trout than tissue from all other diet groups which held fairly constant at 12% across OMP groups 3-6. The lowest tissue level of total 20:5 n-3 plus 22:6 n-3 was found in the OMP-2 trout, 10%. Trout raised on the OMP-1 diet also retained higher tissue stores of 20 and 22 carbon monoenes than trout fed OMP diets 2-6, but less than dietary levels. In contrast, trout fed OMP diets 2-6 retained higher tissue levels of these same fatty acids than dietary levels. The diet ratio of polyunsaturated fatty acids (PUFA)/22:1 appeared to influence whether 22:1 was conserved or oxidized. The most favorable balance of trout tissue monoenes, n-6 fatty acids and total 20:5 n-3 plus 22:6 n-6 for human health was found in trout fed the OMP-6 diet. / Graduation date: 1988

Chemokines are cytokines with chemoattractant ability, and comprise one of the major groups of molecules in immune system. These are small, secreted proteins cause the migration of leukocytes to the sites of injury. Over 40 mammalian chemokines have been identified to date, and they have been implicated in a number of immune mediated processes, including regulation of inflammation, antigen presentation, blood cell development, metastasis, viral infection and wound healing. In rainbow trout, there have been fewer chemokines reported and only one functional study has been published. Rainbow trout chemokine 2 (CK-2) is the only known CC chemokine with a mucin stalk, which has the potential for extensive <em>O</em>-glycosylation. However, no functional characterization has been performed on this molecule yet. CK-2 shares the presence of a mucin stalk with the mammalian chemokines, fractalkine (CX₃CL1), lymphotactin (XCL1), and CXCL16. Another related trout CC chemokine sequence, CK-2. 1, has been discovered recently, which has 98% nucleotide sequence identity with CK-2. CK-2. 1 was believed to be a separate gene due to its apparent differential regulation in challenged rainbow trout. The question remained, however, whether or not CK-2. 1 was a separate gene or an allele of CK-2. The goal of this project was to further characterize both CK-2 and CK-2. 1. <br /><br /> Through genomic PCR on several outbred individuals it was shown that CK-2. 1 is an allele of CK-2 but not a separate gene. Reverse transcriptase (RT) PCR analysis revealed an increased level of transcript both CK-2 and CK-2. 1 in response to phytohaemagglutinin (PHA) stimulation of head kidney leukocytes (HKL) and peripheral blood leukocytes (PBL) collected from fish with different allelic distributions. Similar results were also observed in the rainbow trout macrophage/monocyte cell line, RTS11. Moreover, an anti-CK-2 antiserum was developed in rabbits, which cross-reacted with CK-2. 1. This newly produced antibody was used to determine the protein expression levels in PHA stimulated rainbow trout tissues. RT-PCR was also performed on the same tissues in order to examine the transcript expression. Rainbow trout with both CK-2 and CK-2. 1 were used for this experiment. An overall decreasing pattern of transcript (both CK-2 and CK-2. 1) was observed in brain and HK over 24 hours, while protein was still detected at 24 hours post stimulation. However, in spleen the CK-2 transcript showed a slight upregulation at 4 hours post stimulation along with a very little or no CK-2. 1 expression, although no protein was detected in spleen. Liver showed a very low level of CK-2 and CK-2. 1 transcript at 8 hours post stimulation; while protein was again detected at 24 hours post stimulation. In addition, the sizes of the proteins found in different tissues were larger than expected (&le;30 kDa for CK-2 or &le;35 for CK-2. 1), perhaps due to the presence of extensive <em>O</em>-glycosylation at the mucin stalk of the protein. <br /><br /> A chemotaxis assay was carried out, which is the definitive assay for chemokine activity. This assay showed migration of peripheral blood leukocytes across a membrane with 5??m pores toward CK-2 at an optimal concentration of 500ng/ml (17nm). Moreover, by pre-treating the recombinant chemokine with the polyclonal antisera, it was shown that the chemokine was actually causing the chemotactic activity. Pre-treatment of the cells with pertussis toxin, an inhibitor of G-protein signalling inhibited the migration of PBLs, established the fact that CK-2 caused chemotaxis by binding to a 7 transmembrane, G-coupled receptor just like all other known chemokines. Interestingly, CK-2 was also shown to attract RTS-11 cells. <br /><br /> Overall, the above findings indicate that CK-2 is functionally a chemokine with two very different alleles in rainbow trout. It is probably heavily <em>O</em>-glycosylated and different tissues express different sizes of the protein. This is only the second functional study of a fish chemokine.

The aquaculture sector is currently thriving, and has expanded to meet the demand for fish and shellfish as an alternative protein source to meat. This is especially true for high value products such as Atlantic salmon, where in Scotland salmon farming is reported to be worth> £1 billion to the national economy. Currently around 40% of farmed fish and shellfish destined for human consumption are derived from aquaculture. Therefore, a great deal of attention is paid to problems that the industry faces, with fish diseases of paramount importance. A variety of species of bacteria, viruses and parasites are common in the aquatic environment, which can result in serious diseases amongst fish stocks. As a result, ways to improve disease resistance have been the focus of much attention, with the use of vaccines considered a desirable way forward. However, other approaches are also followed, such as the use of immunostimulants to improve fish health in a more limited, non-specific way, or the use of genetic markers to allow selective breeding of important disease resistance traits. For all of these approaches more information is needed on the pathways that give rise to disease resistance in fish in different situations, to allow their manipulation or monitoring, and the studies in this thesis are directed towards this goal. Fish has been used as a model to study the evolution of vertebrate immunolity for some deacades, especially work on humoral immune responses where knowledge on antibody production has dominated much of the literature on fish immunology. In contrast, little known about specific cell-mediated immunity in fish, even though it also likely plays an important role in the immune system and disease resistance. Therefore, this thesis has been focused on analysing such responses, taking advantage of the recently discovered cytokines of adaptive immune responses in fish, which allow transcriptomic studies in particular to look at the molecules turned on during infection and after vaccination. Thus the goal of this thesis was to take advantage of some successful vaccines that exist for rainbow trout, and examine the gene expression changes that occur in vaccinated trout post-challenge with the homologus pathogen, and to try to dissect pathways that may correlate with disease resistance in this species.

No description available.

Rainbow trout macrophages were stimulated with PMA to produce 02- and H2O2 as detected by the reduction of nitroblue tetrazolium (NBT) and the oxidation of phenol red respectively. Addition of DDC or nitroprusside, inhibitors of superoxide dismutase (SOD) increased O2-levels and decreased H2O2 levels, whereas addition of exogenous SOD had the reverse effect. Such data are indicative of a respiratory burst pathway in teleost macrophages comparable with that of mammals. Respiratory burst activity, acid phosphatase activity and RNA synthesis in rainbow trout macrophages which have been stimulated in vitro with the mitogen Concanavalin A (Con A) or in vivo by injection of formalin-fixed Aeromonas salmonicida in Freund's incomplete adjuvant (FIA) was analysed. With Con A, in vitro stimulated head kidney (HK) or elicited macrophages had increased O2-production and RNA synthesis but no significant increases in H2O2 or acid phosphatase activity after 72h post-stimulation with Con A. In contrast, all functions were increased in in vivo stimulated macrophages compared with FIA-elicited peritoneal macrophages. In a bactericidal assay, Con A stimulated macrophages did not show an increase in killing of an avirulent strain of A. salmonicida (004) above control levels whereas in vivo stimulated macrophages not only displayed increased killing of the avirulent strain of bacteria but also acquired the ability to kill a virulent strain (048). Thus, Con A stimulated macrophages only possessed some of the features of activation whereas in vivo stimulated macrophages were activated as defined by the increased bactericidal activity. Peritoneal washes obtained in the collection of activated macrophages were able to increase NBT reduction in normal HK macrophages suggesting the presence of a soluble activating factor. Lymphokine (LK)-containing supernatants produced using either HK or blood derived leucocytes, by pulsing with 10ug/ml Con and 5ng/ml PMA, were able to increase O2- and H2O2 production, to enhance the killing of an avirulent strain of A. Salmonicida and conferred the ability to kill a virulent strain of A. salmonicida. The LK present in these supernatants was therefore designated a macrophage activating factor (MAF). The use of potential second signals to enhance the killing of bacteria by LK-treated macrophages, met with limited success. Only A. salmonicida (strain 004) LPS was able to produce a small increase in killing above LK-treatment. The MAF produced in this study was tested for antiviral/interferon (IFN) activity. The results showed that the supernatants did contain IFN activity. Attempts to semi-purify the MAF from antiviral activity showed the two activities to co-purify, indicating that both activities may be due to the same molecular species. The retention time of the MAF/IFN, coupled with the results of SDS-PAGE analysis showed the molecular weight of the moiety to be approximately 19K daltons. Both activities were sensitive to low pH (pH 2), high temperature (60oC) and trypsin, providing further evidence that the MAF and IFN activity produced in these studies may be due to the same molecular species, possibly akin to IFN- of higher vertebrates.