2.1.2. Analysis of caprylic acid in fish tissues
Medicated feed and tissue samples were analyzed for caprylic acid content by direst esterification according to Ichihara and Fukubayashi (2010). Briefly, a certain aliquot of sample was placed in a screw-capped glass test tube and depending on the nature of the sample, 1 ml acetylchlorid/methanol (1/20), 1ml methanol and 0.5 ml hexane were added. The mixture was heated at 100 °C for 1 h in a boiling water bath. After cooling, 1 ml of water was added, and caprylic acid was extracted with 1 ml hexane. Gas–liquid chromatography (Varian Model 3300) was used for identification and determination of caprylic acid using an internal standard. Helium was used as the carrier gas. Injector and detector temperatures were 250 and 300 °C, respectively. The column temperature was programmed to be initially 100 °C for 5 min and then to increase at a rate of 6 °C to a final temperature 240 °C.
2.2. Field trials—evaluation of caprylic acid against S. chrysophrii natural infections
2.2.1. Experimental design
The trial was carried out in two cage units experiencing S. chrysophrii infections during the last production cycles. The first location was located in Saronikos gulf where water temperatures are usually recorded to be 2–3 °C higher compared to the second selected unit in Evoikos gulf. Two cages were employed in each site, one served for the treated and the second for the control group. The number of gilthead sea bream and the experimental conditions are given in Table 2. Fish weight was measured by sampling 250 fish on days 0, 30 and 60. Specific growth rate (SGR) was calculated for the whole rearing period using the following equation: SGR = 100 x [ln (W1) − ln (W0)]/days. The experimental diet formulated for the field trials is described in Table 3. A dosing for 200 mg caprylic acid/kg fish was chosen for the field experiments since it was the level achieving the higher tissue concentration for a longer period of time at the kinetic experiment performed at laboratory environment. The experimental diets were given once a day for 60 consecutive days and were readily consumed by the fish.