*2.2. Plasma Humoral Parameters*

In experiment one, plasma humoral parameters (NO production, antiprotease and peroxidase activities) remained unaffected by the different dietary treatments at both sampling points (Figure 1A–C). However, antiprotease activity increased from 1 to 2 weeks of feeding, while peroxidase followed an opposite trend.

**Figure 1.** Plasma immune parameters of gilthead seabream juveniles. Experiment one: (**A**) Antiprotease activity; (**B**) Peroxidase activity; (**C**) Nitric oxide. Data are the mean ± SEM (*n* = 12). Experiment two: (**D**) Antiprotease activity; (**E**) Peroxidase activity; (**F**) Nitric oxide. Data are the mean ± SEM (*n* = 9). Different capital letters represent significant differences in time regardless diet (*p* < 0.05).

Following heat-inactivated bacteria inoculation, peroxidase activity increased after 48 h (Figure 1E), while both NO concentration and antiprotease activity decreased at 24 and 48 h (Figure 1D,F).


**Table 3.**

Haemoglobin,

 mean corpuscular

haemoglobin

 (MCH), red blood cells (RBC) and white blood cells (WBC) in gilthead seabream


**Table 4.**

Absolute values of peripheral blood leucocytes

(thrombocytes,

Lymphocytes,

 monocytes

 and neutrophils)

 in gilthead seabream

Different capital letters represent significant differences in time regardless of diet (*<sup>p</sup>* < 0.05), ns (not significant).

#### *2.3. Gut Innate Immune and Oxidative Stress Biomarkers*

Peroxidase, NO production and SOD activity remained unchanged during the health status experiment in gut samples (Figure 2A–C). Nonetheless, D2 fed fish showed increased gut lipid peroxidation compared to D3 and CTR (Figure 3A), and catalase activity increased from 1 to 2 weeks of feeding.

**Figure 2.** Gut immune parameters of gilthead seabream juveniles. Experiment one: (**A**) Peroxidase activity; (**B**) Nitric oxide (NO). Data are the mean ± SEM (*n* = 12). Experiment two: (**C**) Peroxidase activity; (**D**) Nitric oxide (NO). Data are the mean ± SEM (*n* = 9) Different capital letters represent significant differences in time regardless of diet (*p* < 0.05).

In experiment two, all measured parameters changed over time. Peroxidase activity increased from 24 to 48 h and NO production decreased after 24 and 48 h (Figure 2C,D). Antioxidant defences, such as catalase activity decreased 48 h after inoculation (Figure 3E), while lipid peroxidation increased at 24 and 48 h (Figure 3D). Superoxide dismutase activity increased at 24 h post-stimulus and D1 fed fish had higher activity than D3, irrespective of the sampling point (Figure 3F).

**Figure 3.** Gut oxidative stress parameters of gilthead seabream juveniles. Experiment one: (**A**) Lipid peroxidation (LPO); (**B**) Catalase activity; (**C**) Superoxide dismutase activity (SOD). Data are the mean ± SEM (*n* = 12). Experiment two: (**D**) Lipid peroxidation (LPO); (**E**) Catalase activity; (**F**) Superoxide dismutase activity (SOD). Data are the mean ± SEM (*n* = 9). Different symbols represent significant differences between diets regardless of time (*p* < 0.05). Different capital letters represent significant differences in time regardless of diet (*p* < 0.05).
