The experiments were performed in accordance with Italian national legislation (D. lgs. 26/2014) and EU recommendations (Directive 2010/63/EU), with the authorization of the Italian Ministry of Health number code 665/2016-PR.
Animals
Sea breams were obtained from the commercial hatchery Ittica Caldoli s.r.l. (Lesina, Italy). After 3 weeks of acclimation, fish (mean weight ± SD: 314.6 ± 49.1 g) were implanted with ID100 radio frequency identification (RFID) tags (Trovan, The Netherlands) and separated into three fiberglass tanks of 1.2 m3 (n= 115 fish per tank; 30 kg/m3), forming triplicates. Fish were reared in marine water at a constant temperature of 18 °C, a salinity of 35 PSU and pH of 7.1. Three complete water replacements per day were guaranteed, and the oxygen levels were continuously monitored by an automatic system programmed to keep the dissolved oxygen concentration higher than 5.0 ±1.0 ppm.
The European sea bass were obtained from the commercial hatchery Panittica Pugliese SpA (Torre Canne, Italy). After 3 weeks of acclimation, fish (mean weight ± SD: 335.5 ± 62.4 g) were implanted with RFID tags (ID100), and separated into three fiberglass tanks of 1.2 m3 (n= 35 per tank; 10 kg/m3), forming triplicates. Fish were left without disturbance during 2 months before the start of the experiment, thereafter presented. Water parameters (temperature, salinity and oxygen) were constant and similar to those for sea breams.
During all the experimental duration, both sea bream and sea bass were reared with a constant photoperiod of 12L:12D and fish were fed 1% of the body mass using commercial food (Skretting Marine 3P, Italy) administered by automatic feeders for three hours in the morning.
Experimental procedure
At the beginning of experiment (t0; figure 1), fish were gently caught from the rearing tank and anesthetized with a hydroalcoholic clove oil solution (with a 30 mg/L; [16,17]). Morphometric parameters (body weight and total lengths) were recorded to further evaluate the fish growth performance: specific growth rate (SGR, see “Growth measurement and SGR calculation” section).
Tag implantation
Eighteen days after the beginning of the experiment for sea bream and at the beginning of the experiment for sea bass (d(days)=18 and d=0; see figure 1), n=5 and n=9 fish were randomly selected for sea bream and sea bass respectively and implanted with VEMCO V9AP acoustic accelerometer tag (AMIRIX Systems Inc., Nova Scotia, Canada), as described in Carbonara et al. [7] (at least n=2 fish per tank, except n=1 fish in one tank for the sea bream experiment). Briefly, for both species, fish were fasted 24 h before the implantation and were anaesthetized using 30 mg/L of a hydroalcoholic clove oil solution [16,17]. The transmitter was inserted into the body cavity through a 1.5 cm incision. The body cavity was then carefully closed using sutures and fish were treated with antibiotic injection (sodic-ampicillin–cloxacillin 1 mg/kg 24 h-1; [18]) before sending back to their home tank until further processing at t1. The tag represented 1.63 ± 0.32 % and 0.90 ± 0.21 % (mean ± SD) of the sea bream and sea bass mass respectively. All operated fish recovered in few days and no mortality linked to the surgical operation was observed for both species [7]. For evaluating the possible tag effects, n=12 untagged sea bream and n= 9 untagged sea bass were randomly chosen from the group to serve as control (at least n=3 per tank; Table 1) and followed during the experimental duration.
Table 1. Sample size and mass (g; mean ± SD) of tagged and untagged fish for sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) used in the present study at both t0 and t1.
Species
|
Treatment
|
N
|
Mass at t0 (g)
|
Mass at t1 (g)
|
Sparus aurata
|
Untagged
|
12
|
309.4 ± 65.3
|
389.5 ± 90.8
|
|
Tagged
|
5
|
312.6 ± 48.2
|
407.8 ± 52.4
|
Dicentrarchus labrax
|
Untagged
|
9
|
425 ± 76.4
|
479.22 ± 71.4
|
|
Tagged
|
9
|
423.8 ± 80.7
|
466.9 ± 79.5
|
Growth measurement and SGR calculation
At the end of the experiment, (t1; i.e. d=46 and d=95 days after marking for sea bream and sea bass respectively; see figure 1), tagged and untagged fish were gently caught once again from their rearing tank and anesthetized with clove oil solution as already described above. The body weight (g) was measured for evaluating the Specific Growth Rate (SGR) between the end (t1) and the beginning of the experiment (t0). For both sea bream and sea bass, the SGR was calculated according to the following equation [19]:
where W is the total weight of the fish (g) respectively at the end of the experiment t1 (Wt1) and the beginning of the experiment t0 (Wt0) respectively, and T is the number of feeding days between these two dates of the experiment.
Blood sampling and stress indicators analysis
After the morphometric measurements (~ after 2-3 minutes in anesthetic), a blood sample of 0.5 mL was immediately taken from the first branchial arch of the tagged and untagged fish using a heparinized syringe. Blood was then centrifuged (15,000 g for 3 min), and plasma was collected and stocked at – 20°C until further processing, described below.
For both species, the cortisol, glucose and lactate plasmatic concentrations were measured as described in Carbonara et al. [7]. Briefly, the plasma cortisol concentration was determined using a Cortisol II kit (cobas®) based on a solid-phase, competitive chemiluminescent enzyme immunoassay. Plasma glucose and lactate concentrations were determined using a commercial kit 17630H and 17285 for glucose and lactate respectively (Sentinel, Italy) based on the enzymatic colorimetric Trinder reactions (GOD/PAP for glucose and PAP for lactate).
Statistical analysis
Statistical analyses were carried out using the R software version 3.6.2 [20] at the level of significance of 95%. Homoscedasticity of the data was a priori tested using a Shapiro test and then the appropriate statistical test was performed to compare the SGR and the physiological stress indicators (cortisol, glucose and lactate) between tagged and untagged fish (i.e. either Wilcoxon or t-test) for each species.