This study examined the metabolic response of juvenile turbot (Scophthalmus maximus) to diets with graded fishmeal (FM) replacement with plant, animal, and emerging protein sources (PLANT, PAP, and MIX) in comparison to a commercial-like diet (CTRL). The feeding experiment was carried out from April to July 2019 in the Centre for Aquaculture Research (ZAF) at the Alfred Wegener Institute for Polar and Marine research in Bremerhaven, Germany. The juvenile turbot (Scophthalmus maximus) were purchased from France Turbot (L'Épine, France) and acclimated to the recirculating aquaculture system (RAS) for 2 weeks prior to starting the 16 weeks experimental trial. To elucidate the effects of the protein sources and the level of FM replacement on the metabolic response of the fish, a 1H‐nuclear magnetic resonance (NMR) spectroscopy was used to assess the metabolic profiles of muscle and liver tissue after feeding the fish the experimental diets for 16 weeks. Feed, muscle, and liver samples were ground under liquid nitrogen and approx. 200–250 mg tissue was homogenized in 5x volume of ice‐cold 0.6 M perchloric acid (PCA) (w:v). After one cycle of 20 s at 6000 rpm and 3 °C, using Precellys 24 (Bertin Technologies, Montigny‐le‐Bretonneux, France), samples were sonicated for 2 min at 0 °C and 360 W (Branson Sonifier 450, FisherScientific, Schwerte, Germany). Homogenates of the experimental diets, muscle and liver tissues were centrifuged for 2 min at 0 °C and 16,000 g, and supernatants were neutralized with ice cold potassium hydroxide (KOH) and PCA to pH 7.0–7.5. To remove precipitated potassium, perchlorate samples were centrifuged again for 2 min at 0 °C and 16,000 g. The entire supernatant was transferred, shock‐ frozen in liquid nitrogen, and stored an −80 °C for later analysis. One‐dimensional 1H‐NMR spectra for feed and tissues extracts were acquired using a vertical 9.4 T wide bore magnet with Avance III HD (Bruker‐GmbH, Ettlingen, Germany) at 400.13 MHz with a 1.7 mm diameter triple tuned (1H‐13C‐15N) probe. Each spectrum was processed and analyzed with Chenomx NMR Suite 8.4 software (Chenomx Inc., Edmonton, Canada). Before analyzing, the spectra were corrected for phase, shim and baseline and calibrated to trymethylsilyl proprionate (TSP) signal (at 0.0 ppm).
