Kokou, F., Hellenic Centre for Research, Institute for Marine Biology, Biotechnology and Aquaculture, Marine, Greece, Institute of Animal Science, Agricultural Research Organization, P.O. Box 170, Bet Dagan, Israel
Adamidou, S., BioMar Hellenic SA, Str no 3-5 2nd Industrial Zone of Volos, Greece
Karacostas, I., BioMar Hellenic SA, Str no 3-5 2nd Industrial Zone of Volos, Greece
Sarropoulou, E., Hellenic Centre for Research, Institute for Marine Biology, Biotechnology and Aquaculture, Marine, Greece

One of the main concerns in gene expression studies is the calculation of statistical significance which in most cases remains low due to limited sample size. Increasing biological replicates translates into more effective gains in power which, especially in nutritional experiments, is of great importance as individual variation of growth performance parameters and feed conversion is high. The present study investigates in the gilthead sea bream Sparus aurata, one of the most important Mediterranean aquaculture species. For 24 gilthead sea bream individuals (biological replicates) the effects of gradual substitution of fish meal by plant ingredients (0% (control), 25%, 50% and 75%) in the diets were studied by looking at expression levels of four immune-and stress-related genes in intestine, head kidney and liver. The present results showed that only the lowest substitution percentage is tolerated and that liver is the most sensitive tissue to detect gene expression variations in relation to fish meal substituted diets. Additionally the usage of three independent biological replicates were evaluated by calculating the averages of all possible triplets in order to assess the suitability of selected genes for stress indication as well as the impact of the experimental set up, thus in the present work the impact of FM substitution. Gene expression was altered depending of the selected biological triplicate. Only for two genes in liver (hsp70 and tgf) significant differential expression was assured independently of the triplicates used. These results underlined the importance of choosing the adequate sample number especially when significant, but minor differences in gene expression levels are observed. © 2015 The Authors. Published by Elsevier B.V.