Lysozyme activity (serum and gut mucus)
Five serum samples/replicate were collected, and then the fish were euthanised, and the entire intestine was removed. The guts were opened and scraped carefully with a rubber spatula. The intestinal mucus samples were collected and centrifuged at 1500g. The supernatants were filtered with 0.22 μm Millipore filters before testing. The serum and the mucus lysozyme activities were measured using the turbidometric method, as previously described by Esteban et al. [29]. A twenty-five microlitres sample of serum and mucus was added to 175 μl (0.75 mg/ml Micrococcus lysodeikticus) in flat-bottomed, 96-well plates. The reduction in the absorbance at 450 nm was measured from 0 to 15 min at 25 °C in the ELISA reader. One unit of lysozyme activity was defined as a reduction in absorbance of 0.001 min−1, and the units of lysozyme activity were calculated using the hen egg white lysozyme standard curve.