Submitted to: Aquaculture
Publication Type: Proceedings
Publication Acceptance Date: 10/1/2002
Publication Date: 2/1/2003
Citation: Overturf, K.E., Bullock, D.G., Casten, M., Hardy, R. 2003. Real-time pcr analysis of fish growth and health in animals reared on alternative feeds and genetically selected for growth on alternative feeds.. Aquaculture. Aquaculture America, Louisville, KY .p. 215. Interpretive Summary: The continued improvement of fish stocks requires novel techniques that allow a better understanding of the cellular and physiological aspects that underlie traits of interest. While sequence mapping and cellular studies have improved our understanding of cellular mechanisms and transcriptional changes involved with certain physical characteristics in humans and some domesticated animals, this type of knowledge in aquaculture is lacking. To gain a better understanding of the specific genes that are involved with quantitative traits, studies have begun to analyze gene expression levels. Studies have shown gene expression to correlate well with some proteins or with changes in certain physiological aspects. To gain a better understanding of genes involved in selectable traits of interest, specific genes have been selected for monitoring expression level changes under varied experimental conditions. The genetic traits of interest that are currently under study at the Hagerman Fish Culture Experiment Station are growth, on diets using alternative protein sources to fish meal, disease resistance, and health issues related to different diets and environmental conditions. Probes, such as myosin and ILGF-II, pyruvate kinase, and others related to health and growth conditions have been designed and tested for effectiveness in detecting precise changes occurring at the molecular level that are noticeable but difficult to measure at the macroscopic level. We have found that several of our molecular probe and primer sets correlate well with growth, nutritional intake, and other factors that are crucial for in our analysis of fish utilization of alternative feeds and in our studies involving genetic enhancement of rainbow trout reared on cereal grain diets.
Technical Abstract: In order to gain a better understanding of specific genes that could be involved in selectable traits of interest, specific genes were selected to monitor their expression level changes under varied experimental conditions. The genetic traits of interest that are currently under study at the Hagerman Fish Culture Experiment Station are growth, on formulated fish diets using alternative protein sources to fish meal, disease resistance, and health issues related to different diets and environmental conditions. Utilizing knowledge gained from previous work in fish and other animals, and the availability of sequence, certain transcribed genes were chosen for analysis using real-time PCR. Probes and primer pairs, such as myosin and ILGF-II, were designed and tested for growth analysis, pyruvate kinase and lactate dehydrogenase, for analysis of metabolic enzymes, and tumor necrosis factor, CD-8, interferon (MX-1), lysozyme, complement factor C3, interleukin-8, natural resistance associated macrophage protein-alpha and natural killer cell enhancement factor (NKEF), as immunological and health related indicators. Findings from these studies revealed that myosin expression levels in fish correlated with protein intake. Pyruvate kinase, but not ILGF-II, levels correlated with vitamin level in fish reared on formulated diets containing reduced levels of vitamins. Expression of immunological factors was monitored after exposure of fish to pathogenic microorganisms. It was found that pathogens with strong antigens were able to stimulate gene expression of all factors that appeared correlative with pathogen dose. Thus gene expression can be used to monitor physiological changes in fish.