Genetic variation of flesh colour in canthaxanthin fed rainbow trout

Genetic experiments were conducted using either random independent full-sib families (9 and 11 respectively) or sire-half-sib families (18) of rainbow trout who were fed an experimental diet supplemented with canthaxanthin. The resulting orange-red colour of the flesh from each fish was analyzed through spectrophotometry and expressed in standard terms of luminosity (Y), dominant wavelength (λd) and excitation purity (Pe). The following results were obtained : - There is a substantial genetic variability among families in each colorimetric parameter. Estimated values of heritability from full-sib and from half-sib families do not differ significantly. - Positive correlation between λd . and Pe, and negative correlations between Y and λd and between Y and Pe, are consistent with the pattern of canthaxanthin deposition in the flesh. Genetic correlations do not differ significantly from phenotypic ones. - Pigmentation intensity is correlated to fish weight. This relationship, however, accounts for but a minor part of colour variation among market-size fishes.Des expériences génétiques ont été réalisées chez la Truite arc-en-ciel sur des familles aléatoires et indépendantes de pleins-frères (au nombre de 9 et 11) ou demi-frères de pères (18), alimentées par un régime expérimental supplémenté en canthaxanthine. La couleur de chair orange-rouge obtenue chez chaque poisson a été analysée par spectrophotométrie et exprimée en termes standards de luminosité (Y), longueur d’onde dominante (λd) et pureté d’excitation (Pe). Les résultats obtenus sont les suivants : - Il y a une variabilité génétique notable entre familles pour chaque paramètre colorimétrique. Les valeurs d’héritabilité estimées à partir des familles de plein-frères et de demi-frères ne diffèrent pas significativement. - Les corrélations, positives entre λd et Pe et négatives entre Y et λd et entre Y et Pe, sont conformes au mode d’action de la canthaxanthine se déposant dans la chair. Les corrélations génétiques ne diffèrent pas significativement de leurs homologues phénotypiques. - L’intensité de la pigmentation est corrélée avec le poids des poissons. Cette relation toutefois n’explique qu’une part minime de la variation de couleur chez des animaux de taille marchande

Kirjolohen tie fileeksi: geneettiset tunnusluvut

vo

PHYSIOLOGICAL AND BIOCHEMICAL FACTORS AFFECTING CAROTENOID UTILIZATION IN SALMONID FISH

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.EWOS Innovation AS, Dirdal, Norwa

Carotenoid profiles in relation to maturation, Moulting, food and habitat in the Indian spiny Lobster Panulirus homarus (Linnaeus, 1758) (TH 113)

Carotenoids are responsible for natural pigmentation in crustaceans. The changes in the amount and redistribution of carotenoid pigments in crustaceans depend on several factors including sexual cycle, moulting, food and habitat. Carotenoid changes in Panulirus homarus were studied during thc premoult, postmoult and intermoult stages. Highest carotenoid concentration was recorded in the premoult stage (774I.1g/g in hepatopancreas, 725I.1g/g in the exoskeleton and 133 I.Ig/g in the muscle). During postmoult stage the carotenoid concentration was lowest. Moulting accounted for 66% loss in carotenoid pigments. Astaxanthin levels were high in premoult stage in the exoskeleton and the hepatopancreas. During postmoult stage there was a sharp decrease in astaxanthin concentration in these tissues. Significant differences in carotenoid concentration were recorded in different tissues during ovarian maturation. The carotenoid levels were low in the immature ovary (128I.1g/g) and in the fully mature ovary the concentration increased to 512I.1g/g. The increase in carotenoid concentration of the ovary coincided with a decrease in carotenoid concentration of hepatopancreas and increase in carotenoid concentration in the exoskeleton. Spent stage was characterized by decrease in carotenoid content in the ovary, hepatopancreas and exoskeleton. Astaxanthin was found to accumulate in the hepatopancreas during early maturation and during vitellogenesis they were mobilized to the ovaries. Different naturally occurring carotenoids were studied for their effect on pigmentation of P. homarus. Among the four feeds (Donax cuneatus, Perna viridis, Metapenaeus dobsoni, Paphia malabarica) used P. homarus fed on M. dobsoni and green mussel P. viridis had the best pigmentation pattern. The least pigmented lobsters were those fed on D. cuneatus. D. cuneatus was enriched with Spirulina and Haematococcus pluvialis and these were offered as food to P. homarus. The results revealed that pigmentation of P. homarus can be increased by enriched feed. Enrichment studies revealed that Spirulina enriched clams were superior to clams enriched with H. pluvialis when used as feed for P. homarus. The study on the influence of habitat on pigmentation pattern revealed that in captive conditions the colour of rearing tanks had a direct influence on pigmentation pattern. Among the four different colored tanks used in the study (blue, black, translucent and white) lobsters reared in black coloured tanks had the best pigmentation pattern

Effects of outbreeding on farmed Chinook salmon product quality

Approximately 20% of British Columbia's salmon farming industry is represented by native Pacific Chinook salmon (Oncorhynchus tshawytscha). Few commercial facilities rear Chinook salmon, limiting their breeding stocks which may allow for inbreeding and can potentially lead to downstream effects on product quality. As consumers refuse to pay for low quality products even when prices are reduced, product quality metrics have become increasingly important to the aquaculture industry. As such, there is a need to determine whether product quality of farmed Chinook salmon can be improved through hybridization between wild and farmed populations. Product quality metrics were assessed in adult Chinook salmon generated from hybrids between six wild populations and one inbred commercial population to determine the impact of hybridization on product quality. Assessed quality metrics included slaughter yield, fillet yield, condition factor, colour, and lipid content. Overall, I found that fillet quality metrics differed across populations, and that hybrid populations did not outperform the farmed control in most metrics except for colour. I further aimed to examine the relationship between growth rate and product quality. I found that growth had significant relationships with traits of commercial interest such as, colour, fat content, jacking rate, and condition factor. Although hybrid populations did not outperform the farmed population, this thesis provides evidence that hybrid chinook salmon may be competitive commercially as they exceed or perform at desired market values for traits

Skin health and product quality of Atlantic Salmon (Salmo salar L.) fed increased LC-PUFAs from micro algae (Schizochytrium sp.)

The Atlantic salmon is a valuable protein source that can increase food security for the growing world population. However, there is an urgency of novel sustainable ingredients to support production growth in the Aquaculture industry. Micro algae have the potential to replace fish oil in Atlantic salmon feed. It is a viable source, rich in n-3 LC PUFA. The heterotrophic micro algae studied in this thesis, Schizochytrium sp. is sustainable and efficient to cultivate. The micro algae has high EPA (C20:5n-3) and DHA (C22:6n-3) content and studies show diets with Schizochytrium sp. result in improved EPA+DHA retention efficiency. This study investigated the effects of dietary modifications on skin health, product quality (fillet pigmentation) and nutritional quality of Atlantic Salmon. The three test diets consisted of control diet, resembling commercial (7.5% EPA+DHA, FO) feed, test diet 1 containing increased levels of n-3 LC-PUFA from marine sources (10% EPA+DHA, FO) and test diet 2 with increased levels of n-3 LC-PUFA supplemented with Schizochytrium sp. (algae oil, AO) (5% FO+5% AO). The test feeds were given to the Atlantic salmon in artic farming environment in Hammerfest, Finnmark in triplicate commercial sea-cages from October 22nd. The sampling period for this thesis were from November 2022 and April 2023. The result showed an overall positive development on bodyweight for all diet groups, indicating no negative effects of micro algae inclusion. And fish fed with control diet, diet 2 and diet 2 had similar trends in biometric traits. The test feed had no significant effect on skin health or fillet pigmentation. The chemical composition of the fillets showed increased level of oleic acid, linoleic acid and DHA in salmon fed diet supplemented with micro algae. Diet with increased n-3 LC PUFA had the highest level of EPA in the salmon fillet. Astaxanthin was not affected by the modified diets, but had a positive development

Genetics of the yeast, Phaffia rhodozyma

The aim of this thesis is studying the genetics of Phaffia and to develop a genetic transformation system for this yeast. The genetic properties of Phaffia were studied on the gene and genome level.As a first step the molecular structure of the Phaffia actin gene was analyzed. Actin genes are highly conserved throughout nature, and as such they have been used for the classification of significantly diverging eukaryotic groups, like (in-)vertebrates, plants and fungi.We anticipated that the analysis of the primary structure of Phaffia actin gene and comparison with the actin genes from fungi, including 2 ascomycetous filamentous fungi, 2 basidiomycetous yeasts and 5 ascomycetous yeasts would provide further phylogenetic information on this yeast.It was found that the Phaffia actin gene encoded a protein consisting of 375 amino acids. In addition 4 (non-coding) intervening sequences were present. Comparison of both the coding DNA sequence and its predicted protein product with their fungal counterparts, revealed that least homology was found with the ascomycetous yeasts, like Saccharomyces cerevisiae and Kluyveromyces lactis . It was also shown, that based on these comparisons Phaffia is closer related to the filamentous ascomycetous fungi Thermomyces lanuginosus and Aspergillus nidulans , whereas most homology was found with the basidiomycetous yeast Filobasidiella neoformans (perfect stage of Cryptococcus neoformans ).In addition to the phylogenetic analysis of the actin exons, the architecture of the introns (splice site consensus sequences, size, position in the gene) was compared. it was shown that the Phaffia introns most resembled that of Filobasidiella neoformans where as least resemblance occurred with the ascomycetous yeasts. This result was in agreement with the actin exon homology studies. Furthermore, the presence of multiple introns in the Phaffia actin gene resembled the situation in the actin genes from F. neoformans and the filamentous fungi, whereas the ascomycetous yeasts only carry one intron in their actin genes.Similar results were obtained by (phylo-)genetic analysis of the five introns containing Phaffia glyceraldehyde-3-phosphate dehydrogenase gene.The genomic organization of the multiple rDNA genes in Phaffia was elucidated. It was found that Phaffia carries the rDNA genes in three clusters, of 12, 14 and 35 copies, on three different chromosomes. In the ascomycetous yeasts and fungi the rDNA is mainly present on one chromosome.The significant differences on the gene and genome level with the ascomycetous yeasts affected the strategy for the development of a transformation system for Phaffia . Whereas several marker gene sequences or sequences for plasmid replication and maintenance can be readily interchanged between most ascomycetous species as a result of high homologies, it was shown that this was not the case for Phaffia . Therefore an almost entirely homologous transformation sytem was developed using plasmids carrying the dominant G418 resistance gene (Km R), driven by either the Phaffia actin or the gpd promoter and a Phaffia ribosomal DNA (rDNA) fragment for homologous integration.It was found that the rDNA clusters could serve as a target for high copy number integration. This integrative transformation system was used to determine the ploidy of Phaffia , strain CBS 6938, by monitoiring chromosomal shifts as a result of multiple integrations. It was found that this strain was haploid.Plasmids carrying the gpd promoter driven Km Rgene transformed Phaffia with significant higher efficiencies than constructs with the actin promoter. Furthermore, the plas-mid copy number and transformation efficiencies of the first were found to be influenced by the presence of the gpd terminator downstream the Km Rgene.It was shown that plasmid amplification occurred independent from selection pressure to an extend that appeared to be negatively related to the effectiveness of expression of the Km Rgene. This observation indicated that the rising metabolic burden, as a consequence of amplification, imposes limits to the number of plasmid copies.The effectiveness, stability, and plasmid amplifying properties of the Phaffia transformation system offer possibilities for the use of recombinant DNA technology in developing industrially attractive Phaffia strains