2.1. Diet Formulation and Preparation
FM was produced at the Core Facility Food & Bio Processing of the BOKU University Vienna. To produce FM, frozen Atlantic mackerel (Scomber scombrus) was obtained at a local commercial market (METRO, Vienna, Austria) and subsequently freeze dried (Sublimator 30 EKS—R, Zirbus Technology, Harz, Germany, set with condenser temperature of −45 °C and radiation temperature of 30 °C) over three days. The dried product (94% DM) was divided into three groups. The first group was not exposed to heat after freeze drying, described hereafter as FM Control. The second and third groups were placed in a pre-heated laboratory oven (Heraterm, Thermo Scientific, Waltham, MA, USA) for 1 h at 70 and 140 °C, respectively.
In total, four diets were prepared (
Table 1). A conventional FM (64%CP and 20.5 MJ kg
−1 GE; produced at 110 °C) was used to formulate a basal diet (49% CP and 17.5 MJ kg
−1 GE). Plus, three test diets with three different thermal treated FM (supplemented with TiO
2 as an inert marker) were produced by replacing 30% of the basal diet with the tested FM. The apparent total tract digestibility coefficients (ATTD, %) were determined according to methodologies already established in the literature [
15].
All diet ingredients were conditioned and mixed (100 L mixer, 6.0 min conditioning time, 95 °C mixing temperature, 4.6 kg of water addition, and 2.8 kg/h of steam addition). Experimental diets were extruded (OEE 8, Amandus Kahl, Reinbek, Germany) using a 4 mm diameter matrix, ~60 kWh/t, 1:2.5 press ratio, 51 Hz extruder frequency, 83 bar hydraulic pressure, 5.0 kW of performance, throughput of 150.0 kg/h, ~105 °C temperature on extruder head, and ~80 °C after extruder.
2.2. Experimental Setup
The present trial was approved by the ethics committee of the local authority of Schleswig-Holstein (V 244-63725/2022). The trial was conducted at the Fraunhofer IMTE aquaculture facility in Büsum, Germany. Rainbow trout (Oncorhynchus mykiss) were obtained from a commercial fish farm (Forellenzucht Trostadt, Germany) and transferred to the research facilities in Büsum. Afterward, fish were stocked randomly into a recirculating aquaculture system (RAS) consisting of 12 rectangular tanks with 150 L water capacity each. Water treatment consisted of a mechanical and biological filter and a disinfection unit (UV filter). The photoperiod was set at a 16 L: 8 D cycle. Trout were adapted to the environmental conditions for 2 weeks and were fed with commercial dry feed (Aller Aqua Gold, 3 mm, Aller Aqua, Golssen, Germany). After acclimatization, the 216 fish were weighed individually, and 18 trout with an initial weight of 230.1 ± 5.8 g (mean ± standard deviation) were placed into each of the tanks. In total, 12 tanks were assigned to the four experimental groups (basal plus three diets with heat-treated FM) with three replicates (tanks) each. The number of fish per tank is adapted to an optimal stocking density of at least 15 kg/m3 in the individual tanks. This stocking density is necessary for trout to prevent aggression that occurs at lower stocking densities.
Trout were fed manually 1.5% of their body weight once a day at 8:30 am for four weeks. The tanks were cleaned, and water parameters were checked daily (15.6 ± 0.7 °C temperature; 7.1 ± 0.2 pH; 4.2 ± 1.0 ppt salinity, HI 96822 Seawater Refractometer, Hanna Instruments Inc., Woonsocket-RI-USA; 9.1 ± 0.6 mg L−1 O2, Handy Polaris; OxyGuard International A/S, Birkerod, Denmark; 0.7 ± 0.4 mg L−1 NH4-N, 1.1 ± 0.4 mg L−1 NO2-N, Microquant test kit for NH4 and NO2; Merck KGaA, Darmstadt, Germany).
Fecal material was collected once a day (at 1:30 pm) by manual stripping of each fish for 28 days. Therefore, fish were transferred to a separate tank and anesthetized using clove oil (1 mL per 40 L of water). Subsequently, gentle pressure on the abdomen was applied to strip feces out of the posterior intestinal area. Daily feces samples from each tank were pooled and stored at −20 °C pending analyses.
Feed and feces samples were analyzed in duplicate for the proximate composition of dry matter (DM), gross energy (GE), ether extract (EE), crude protein (CP), amino acids (AA), and phosphorus, applying standard methods [
16]. TiO
2 was measured with a standard method [
17], in which 0.5 g of dried samples were digested with 25 mL of H
2SO
4 (98%) and one Kjeldahl tablet for 60 min at 400 °C. After digestion, samples were let overnight to allow crystallization and then filtered in 100 mL PE bottles. A total of 5 mL of the filtrate was mixed with 1 mL of 1M H
2SO
4 and 1 mL of H
2O
2. Samples were placed in disposable cuvettes and measured against distilled water on the spectrophotometer at a wavelength of 405 nm. The concentration of TiO
2 was then calculated with the following formula:
In which y is the absorbance, factor 0.5994 represents the Ti concentration in TiO2, and the volume of the test sample corresponds to the respective volume of the volumetric flask used after digestion.
In fish meal samples, the content of amino acids was conducted after acid hydrolysis and subsequent pre-column derivatization of the extracts with Waters AccQ-Fluor Reagent (6-aminoquinolyl-N-hydroxysuccinimidylcarbamate, ACQ). The individual amino acids were separated and determined using reversed-phase HPLC gradient elution and subsequent fluorescence detection at λ
ex = 250 nm and λ
em = 395 nm. FA profile was additionally analyzed using the one-step methylation method [
18]. Briefly, 0.5 g of samples were extracted and converted to methyl esters for 2 h at 70 °C with toluene and 5% fresh methanolic HCl using nonadecanoic acid (C19:0, Sigma Aldrich, Munich, Germany) as internal standard. Subsequently, 5 mL of 6% K
2CO
3 was added, followed by another 2 mL of toluene. After centrifugation, 1 mL of the organic supernatant was transferred to a GC- Vial and was analyzed on a gas chromatograph (Agilent Technologies 7890A, Santa Clara, CA, USA) equipped with an Agilent 7693 autosampler and an Agilent G4514A injector turret. For separation of the FA Agilent HP-88 capillary column (100 m × 0.25 mm of internal diameter and 0.2 μm film thickness) was used. Hydrogen served as a carrier gas at a constant pressure of 11 psi. The flow rates for the FI-Detector were set at 35 mL/min (hydrogen) and 350 mL/min (synthetic air) at a temperature of 260, respectively. Conditions: Inlet temperature was set to 250 °C, Split ratio to 50:1, and the injection volume was 1 µL. The start temperature of the oven was set to 100 °C, held for 5 min, and after that, ramped at a rate of 4 °C/min to 240 °C and held constant for 30 min (total runtime 70 min). Commercial standard fatty acid methyl esters (FAME) mixtures (Supelco 37 Component FAME Mix, Supelco, PA, USA) were used for the identification of individual FA. Data processing was performed using the Agilent Open Lab V3.6 software.
Apparent total tract digestibility (ATTD) of different nutrients in diets was calculated according to methods established in the literature [
19] after feces and diet nutrient and marker analysis:
The formula for calculation of ATTD of test substance follows the established calculation for fish in the literature [
20]: