title: Impact of plant-based oils and other feed ingredients on growth, tissue composition, gene expression, and health of Atlantic salmon (Salmo salar)
Creators: name: Katan, Tomer 
Publisher: name: Memorial University of Newfoundland
Year: 2022

description: The ω6 to ω3 (ω6:ω3) fatty acid (FA) ratio is known to affect many biological processes (e.g. inflammation, FA metabolism) and human diseases. However, its impacts on salmon physiology, immune response, and the underlying molecular mechanisms are less well understood. The current thesis applied a nutrigenomics and lipidomics approach to study the impacts of plant-based feeds with varying dietary ω6:ω3 ratios and ω3 long chain polyunsaturated fatty acid (LC-PUFA) levels on farmed salmon growth, tissue composition and lipid metabolism, gene expression, and antibacterial immune response. In Chapter 2 Atlantic salmon were fed with diets containing the same sources and equal levels of marine and plant proteins, and differed in plant oil mixes to generate a range of ω6:ω3 (i.e. 0.3-2.7). A targeted qPCR study was used to measure the mRNA expression of lipid metabolism and eicosanoid synthesis-related genes in the liver. This study revealed that while growth performance and organ indices were not affected by dietary ω6:ω3, liver and muscle FA composition was highly reflective of the diet and suggested elongation and desaturation of 18:3ω3 and 18:2ω6. Compound-specific stable isotope analysis further demonstrated that liver 20:5ω3 and 20:4ω6 synthesis was largely driven by dietary 18:3ω3 and 18:2ω6, respectively. Moreover, significant correlations between LC-PUFA synthesis-related transcripts and liver LC-PUFA further supported FA biosynthesis. In Chapter 3, I used the same fish from the previous feeding trial in order to investigate how the two extreme ω6:ω3 diets (i.e. high ω6 and high ω3) affected the hepatic transcriptome (using 44K microarrays), and to identify novel biomarker genes that respond to variation in ω6:ω3. The microarray study identified transcripts with important roles in lipid metabolism (helz2a), cell proliferation (htra1b), immune and inflammatory response (lect2a, itgb5, helz2a, p43), control of muscle and neuronal cell development (mef2d), and translation (eif2a, eif4b1, p43). Further, the PPARα activation-related transcript helz2a was down-regulated by high ω6 diet compared with high ω3 diet, and two of its paralogues showed significant correlations with ω6 and ω3 FA in two tissues (liver, muscle). These data indicated their potential as biomarkers of tissue response to dietary ω6:ω3 variation. The focus of Chapter 4 was to apply a two-factorial design to evaluate if diets with different combinations of ω6:ω3 (high ω6, balanced, high ω3) and EPA+DHA levels (0.3, 1.0, or 1.4%, as formulated) impact head kidney lipid composition, and the transcript expression of genes involved in FA and eicosanoid metabolism (using qPCR). Head kidney FA composition was reflective of the diet and responded to ω6:ω3 variation. Proportions of 20:5ω3 were similar among fish fed 0.3% EPA+DHA with high ω3, 1% EPA+DHA (both high ω3 and high ω6 treatments) and 1.4% EPA+DHA/balanced fed fish, although dietary 20:5ω3 varied by 2.5- to 3-fold. Further, positive correlations were identified between head kidney ω3 LC-PUFA and elovl5a transcript levels. This suggested that high dietary 18:3ω3 promoted the synthesis of ω3 LC-PUFA in salmon fed lower dietary EPA+DHA levels (0.3%). This Chapter also showed significant correlations between head kidney FA composition and the expression of eicosanoid synthesis-related transcripts (i.e. 5loxa, 5loxb, cox1, cox2, ptges2, ptges3, and pgds), which illustrated the constitutive relationships among FA and eicosanoid metabolism in salmon. In Chapter 5 salmon were fed high ω6 and high ω3 diets combined with two EPA+DHA levels (0.3 and 1.0% of diet) to investigate dietary impacts on antibacterial and eicosanoid responses. Eicosanoid-metabolism-related transcripts (e.g. cox1, 5loxa, 5loxb, pgds, lkha4) in the head kidney were down-regulated in formalin-killed Aeromonas salmonicida (ASAL)-injected compared with the phosphate-buffered saline (PBS)-injected fish (within diet), suggesting an attempt to mitigate the proinflammatory response in bacterin-challenged fish. Fish fed 0.3% EPA+DHA with high ω6 diet showed the strongest fold-change induction (ASAL vs. PBS) of antibacterial genes (ccl19b, il8, il10, il8, saa5, hamp). Further, this Chapter showed positive correlations between head kidney ω6 PUFA and the transcript expression of immune-related genes (e.g. il8, il10, hamp, camp, stlr5, lect2). These findings suggested that 0.3%EPA+DHA with high ω6 diet may have enhanced the innate antibacterial immune response of Atlantic salmon. Finally, diet and ASAL and/or PBS injection significantly impacted the plasma prostaglandin (PGE2, PGF3α) levels [measured with electrospray ionization mass spectrometry (ESI-MS/MS)] of salmon. The 0.3%EPA+DHA with high ω6 diet also resulted in lower levels of PGE2 in ASAL- when compared with PBS-injected fish, and this was concurrent with lower head kidney expression of PGE2 receptor-encoding transcript (i.e. ptger4a).