*4.2. Diet Composition*

The trial comprised 4 isoproteic (50% protein in dry matter (DM)), isolipidic (17% fat in DM) and isoenergetic (23 kJ/g) dietary treatments. A fishmeal-based (FM), practical diet was used as a control (CTR), whereas three experimental diets based on CTR were further supplemented with a 2% inclusion of *C. vulgaris* powdered biomass (Diet D1); 0.1% inclusion of *C. vulgaris* peptide-enriched extract (Diet D2) and finally 0.1% inclusion of *C. vulgaris* insoluble residue (Diet D3) (Table 9). Diets were manufactured by SPAROS (Olhão, Portugal). All powder ingredients were initially mixed and ground (<200 micron) in a micropulverizer hammer mill (SH1, Hosokawa-Alpine, Germany). Subsequently, the oils were added to the powder mixtures, which were humidified with 25% water and agglomerated by a low-shear and a low-temperature extrusion process (ITALPLAST, Parma, Italy). The resulting pellets of 2.0 mm were dried in a convection oven for 4 h at 55 ◦C (OP 750-UF, LTE Scientifics, Oldham, UK). Diets were packed in sealed plastic buckets and shipped to the research site (CIIMAR, Matosinhos, Portugal), where they were stored in a temperature-controlled room.

**Table 9.** Ingredients and proximate composition of experimental diets.


<sup>1</sup> 66.3% CP, 11.5% CF, Pesquera Diamante, Peru; <sup>2</sup> 94% WEISHARDT, Slovakia; <sup>3</sup> 62.2% CP, 0.7% CF, Soycomil P, ADM, Netherlands; <sup>4</sup> 80.4% CP, 5.8% CF, VITAL, Roquette, France; <sup>5</sup> 61.2% CP, 5.2% CF, COPAM, Portugal; <sup>6</sup> Dehulled solvent extracted: 47.4% CP, 2.6% CF, Cargill, Spain; <sup>7</sup> Solvent extracted: 34.3% CP, 2.1% CF, Ribeiro e Sousa Lda, Portugal; <sup>8</sup> Solvent extracted: 29.1% CP, 1.8% CF, Ribeiro e Sousa Lda, Portugal; <sup>9</sup> 11.7% CP, 1.6% CF, Molisur, Spain; <sup>10</sup> 98.1% CF (16% EPA; 12% DHA), Sopropêche, France; <sup>11</sup> 98.6%, JC Coimbra, Portugal; <sup>12</sup> Vitamins (IU or mg/Kg diet): DL-alphatocopherol acetate, 100 mg; sodium menadione bisulphate, 25 mg; retinyl acetate, 20,000 IU; DL-cholecalciferol, 2000 IU; thiamine, 30 mg; riboflavin, 30 mg; pyridoxine, 20 mg; cyanocobalamin, 0.1 mg; nicotidin acid, 200 mg; folic acid, 15 mg; ascorbic acid, 1000 mg; inositol, 500 mg; biotin, 3 mg; calcium panthotenate, 100 mg; choline chloride, 1000 mg, betaine, 500 mg. Minerals (g or mg/kg diet): cobalt carbonate, 0.65 mg; copper sulphate, 9 mg; ferric sulphate, 6 mg; potassium iodide, 0.5 mg; manganese oxide, 9.6 mg; sodium selenite, 0.01 mg; zinc sulphate. 7.5 mg; sodium chloride, 400 mg; calcium carbonate, 1.86 g; excipient wheat middling's, Premix Lda, Portugal; <sup>13</sup> CELATOM FP1SL (diatomite), Angelo Coimbra S.A., Portugal; <sup>14</sup> Windmill AQUAPHOS (26% P), ALIPHOS, Netherlands; <sup>15</sup> 99% Lys, Ajinomoto EUROLYSINE S.A.S, France; <sup>16</sup> 98.5% Thr, Ajinomoto EUROLYSINE S.A.S, France; <sup>17</sup> 99% Met, Rodhimet NP99, ADISSEO, France; <sup>18</sup> *Chlorella vulgaris* lyophilized biomass, Allmicroalgae, Portugal; 19,20 *Chlorella vulgaris* aqueous and insoluble extracts, CBQF—Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Portugal.

#### *4.3. Bacterial Growth and Inoculum Preparation*

*Photobacterium damselae* subsp. piscicida (Phdp), strain PP3, was used for the inflammatory insult. Bacteria were routinely cultured at 22 ◦C in tryptic soy broth (TSB) or tryptic

soy agar (TSA) (both from BD Difco™, Franklin Lakes, NJ, USA) supplemented with NaCl to a final concentration of 1% (*w*/*v*) (TSB-1 and TSA-1, respectively) and stored at −80 ◦C in TSB-1 supplemented with 15% (*v*/*v*) glycerol. To prepare the inoculum for injection into the fish peritoneal cavities, stocked bacteria were cultured for 48 h at 22 ◦C on TSA-1. Afterwards, exponentially growing bacteria were collected and resuspended in sterile HBSS and adjusted against its growth curve to 1 × 107 colony forming units (cfu) mL−1. Plating serial dilutions of the suspensions onto TSA-1 plates and counting the number of cfu following incubation at 22 ◦C confirmed bacterial concentration of the inocula. Bacteria were then killed by heat at 70 ◦C for 10 min. Loss of bacterial viability following heat exposure was confirmed by plating resulting cultures on TSA-1 plates and failing to see any bacterial growth.
