Animals
Sea breams (mean weight ± SD: 314.6 ± 49.1 g) were obtained from the commercial hatchery Ittica Caldoli (Lesina, Italy). After three weeks of acclimation, ID100 radio frequency identification (RFID) tags (Trovan, Netherlands) were implanted in the fish, which were then separated into three fiberglass tanks of 1.2 m3 (n= 115 fish per tank; ~30 kg/m3), forming triplicates. The implantation of pit-tag was performed under anaesthesia conditions (hydroalcoholic clove oil solution; 30 mg/L) under the skin in the region near the first dorsal fin. The fish were reared in marine water at a constant temperature of 18 °C, salinity of 35 PSU and a pH of 7.1. The water was completely replaced three times a day, and the oxygen levels were continuously monitored by an automatic system programmed to maintain the dissolved oxygen concentration above 5 ± 1 ppm.
European sea bass fish (mean weight ± SD: 335.5 ± 62.4 g) were obtained from the commercial hatchery Panittica Pugliese SpA (Torre Canne, Italy). After three weeks of acclimation, RFID tags (ID100) were implanted in the fish, which were then separated into three fiberglass tanks of 1.2 m3 (n= 35 fish per tank; ~10 kg/m3), forming triplicates. The implantation of pit-tag in sea bass was performed under similar conditions (anaesthesia and area of implantation) as for sea bream. The fish were left undisturbed for two months before the start of the experiment. The water parameters (temperature, salinity and oxygen) were constant and similar to those for the sea breams.
Throughout the experimental period, all fish were exposed to a 12L:12D photoperiod and were fed 1% of their body mass using commercial feed (Skretting Marine 3P, Italy) dispensed by automatic feeders for 3 h every morning.
Experimental procedure
At the beginning of the experiment (t0; Fig. 1), the fish were gently removed from their rearing tanks and anaesthetized with a hydroalcoholic clove oil solution (30 mg/L) [16, 17]. Morphometric parameters (body weight and total length) were recorded to calculate the SGR (see the “Growth measurements and SGR calculations” section).
Tag implantation
At the beginning of the experiment (Day 0) for sea bass and 18 days later for sea breams (Day 18) (Fig. 1), V9AP acoustic accelerometer tags (Vemco Systems Inc., Nova Scotia, Canada) were implanted in nine randomly selected sea bass and five randomly selected sea breams (at least two fish from each tank, except one fish from one tank for the sea bream experiment), as described in Carbonara et al. [7]. Briefly, the fish were subjected to fasting for 24 h before implantation and were anaesthetized using a hydroalcoholic clove oil solution in doses of 30 mg/L [20,21]. The transmitter was inserted into the body cavity through a 1.5-cm incision. The incision was then carefully sutured, and the fish were injected with antibiotic (sodic ampicillin–cloxacillin; 1 mg/kg 24 h-1) [22] before being returned to their home tanks until the end of the experiment (t1; Fig. 1). The mean tag weight in air accounted for 1.63% ± 0.32 and 0.90% ± 0.21 of the sea bream and sea bass body mass, respectively. All tagged fish recovered within a few days, and no mortality linked to the surgical procedure was observed [7]. To evaluate possible tag effects, 12 untagged sea breams and 9 untagged sea bass were randomly selected as controls (at least three fish per tank; Table 1) and were monitored during the experimental period.
Table 1. Sample sizes and mean masses of tagged and untagged sea breams and European sea bass
|
Species
|
Status
|
N
|
Mass at t0 (g)
|
Mass at t1 (g)
|
Sea bream (Sparus aurata)
|
Tagged
|
5
|
312.6 ± 48.2
|
407.8 ± 52.4
|
Untagged
|
12
|
309.4 ± 65.3
|
389.5 ± 90.8
|
European sea bass (Dicentrarchus labrax)
|
Tagged
|
9
|
423.8 ± 80.7
|
466.9 ± 79.5
|
Untagged
|
9
|
425 ± 76.4
|
479.2 ± 71.4
|
t0: beginning of experiment; t1: end of experiment
|
Growth measurements and SGR calculations
At t1 (Days 46 and 95 after tagging the sea breams and sea bass, respectively; Fig. 1), the tagged and untagged fish were once again gently removed from their rearing tanks and anaesthetized with clove oil solution as described above. Their body weight was measured (in grams) to calculate the differences in SGR between t0 and t1. The SGR was calculated according to the following equation [23]: (see Equation 1 in the Supplementary Files)
where W is the total weight at the end (t1) and the beginning of the experiment (t0), and T is the number of feeding days between t0 and t1.
Blood sampling and stress indicator analysis
After the morphometric measurements (2–3 minutes after anaesthesia inducement), blood samples of 0.5 mL were immediately taken from the first branchial arch of the tagged and untagged fish using a heparinized syringe. The samples were then centrifuged at 15,000 g for 3 min, and plasma was collected and stored at –20 °C until further processing, described below.
The plasmatic cortisol, glucose and lactate concentrations were measured as described in Carbonara et al. [7]. Briefly, the cortisol concentration was determined using solid-phase competitive chemiluminescent enzyme immunoassays with a cobas Cortisol II kit (Roche, Switzerland). The glucose and lactate concentrations were determined using kits 17630H and 17285 (Sentinel Diagnostics, Italy), respectively, based on the enzymatic colorimetric Trinder reaction (GOD/PAP for glucose and PAP for lactate).
Statistical analysis
Statistical analyses were performed using the R software version 3.6.2 [24] at a 95% level of significance. Homoscedasticity of the data was a priori tested using the Shapiro-Wilk test. The appropriate statistical test (either the Wilcoxon test or the t-test) was then performed to compare the SGRs and physiological stress indicators (cortisol, glucose and lactate) between the tagged and untagged fish of each species.