Experimental fish
Fish used in the experiment were hatchery-reared offspring of wild Atlantic salmon caught by electrofishing in River Storå, Denmark. Fertilized eggs were incubated in egg trays and hatched in late March 2011 at the Danish Centre for Wild Salmon (DCV) in Randers, Denmark. After hatching, the alevins were maintained in the hatching trays until the yolk sac was completely absorbed. Fish were then transferred to flow-through tanks for exogenous feeding and kept under ambient photoperiod and temperature (4°C to 17°C) conditions. The Atlantic salmon were fed daily with commercial trout pellets equivalent to 1.5% to 4% of body mass. All study fish were handled in accordance to the guidelines described in permission (2012-DY-2934-00007) from the Danish Experimental Animal Committee.
Experimental protocol
The laboratory experiment was conducted at the hatchery facilities at DCV from 21 December 2011 to 25 January 2012. A total of 360 Atlantic salmon were divided into three different size classes (n = 120 per size class): I: 80 to 99 mm FL, II: 100 to 119 mm FL, III: 120 to 135 mm FL. Within each size class, fish were randomly assigned to one of five treatment groups: control, sham-operated, 23 mm PIT tag, 23 mm PIT tag and suture, and 32 mm PIT tag. This resulted in 24 fish from each size class per treatment. These fish were evenly and haphazardly distributed among six experimental tanks. Hence, each tank contained fish from all five treatments and each treatment group consisted of four fish from each size class per tank. The net result was 60 fish in each tank (that is, 12 fish per treatment group). Within size classes, there were no significant differences in length and mass among the treatment groups at the beginning of the experiment (one-way ANOVA, F
4,115 ≤2.236, all P ≥0.069).
Treatment fish were placed in an anesthetic bath (benzocaine 20 mg L-1) until the opercular rate became slow and irregular (4 to 5 min). Once unresponsive, the fork length and body mass were measured to the nearest 1 mm and 0.1 g, respectively. Sham-operated fish received a 3 to 4 mm ventrolateral incision, 5 to 7 mm anterior to the muscle bed of the pelvic fins on the left side of the body. Atlantic salmon in the 23 mm PIT-tagged group were treated similarly except a uniquely coded 23 mm PIT tag (RI-TRP-RRHP, half duplex, 134 kHz, diameter 3.85 mm and weight 0.6 g in air; Texas Instruments, Plano, Texas, USA) was inserted into the peritoneal cavity through the incision. For the 23 mm PIT-tagged fish with suture closure treatment, incisions were closed with one stitch of absorbable suture (Vicryl 5–0 FS-2; Ethicon, Piscataway, NJ, USA) tied with a single surgeons knot. Fish in the 32 mm PIT-tagged treatment group were subjected to a 3 to 4 mm ventrolateral incision posterior to the pelvic fins. A 32 mm PIT tag (RI-TRP-WR2B, half duplex, 134 kHz, diameter 3.85 mm and weight 0.8 g in air; Texas Instruments) was gently pushed anteriorly into the body cavity and the incision was left to heal without suture closure. Control fish were handled in the same manner as fish in the other treatments but no surgery was performed and no tag was implanted. Sham-operated fish were included in the experiment to isolate the effects of the surgery procedures from the effects of the PIT tags. The initial mean tag-to-body mass ratio in air was 5.1% (range: 2.2% to 13.6%) for Atlantic salmon tagged with 23 mm PIT tags and 6.7% (range: 2.7% to 14.8%) for those tagged with 32 mm PIT tags.
All control and sham-operated fish received a unique dye-mark combination on the left and/or right side of the caudal peduncle using a Panjet inoculator to allow for individual recognition during the experiment. Fish were marked with Alcian Blue, Irgafin Red P or a combination of the colors and the maximum number of dye-marks per fish was three. When used properly, jet injection of dye has no measurable effect on survival and growth of juvenile Atlantic salmon [31–33]. However, to ensure that the potential impacts of PIT tagging on mortality and growth were entirely isolated from any negative effects stemming from color marking, all PIT-tagged fish were also given dye-marks on the caudal peduncle region. Excess dye was carefully flushed off the fish with water as recommended by Hart and Pitcher [34]. The duration of the procedures for the control, sham-operated, 23 mm PIT tag, 23 mm PIT tag with suture closure, and 32 mm PIT tag treatment groups took on average 33, 38, 43, 77 and 44 s, respectively. Fish were not fed within 24 hours of surgery and the same surgeon performed all surgeries. All surgery equipment was disinfected (96% ethanol) prior to use and scalpel blades were changed frequently to avoid tearing of the tissue.
After treatment, fish were placed in an aerated barrel (60 L) until they recovered from anesthesia (3 to 4 min) and then transferred to the experimental tanks. The tanks (200 × 200 cm) were supplied with fresh water from a flow-through filtration system at a flow rate of 30 L min-1 ensuring well-oxygenated water. The water depth was adjusted to 35 cm, resulting in a water volume of 1,400 L. The average water temperature in the tanks was 6.9°C (range: 5.5°C to 7.5°C) and the photoperiod followed natural day-light cycles. Fish were fed during light hours with commercial trout pellets (Aller Performa, Aller Aqua, Denmark) at a ratio of 2% body mass per day using automatic feeders. The tanks were inspected daily for dead fish and rejected PIT tags. Dead fish were removed from the tanks, individually identified, measured, weighed, and examined externally and internally. The codes of rejected PIT tags found at the bottom of the tanks were recorded using a handheld reader (Agrident GmbH, APR350, Barsinghausen, Germany). After 35 days, the surviving Atlantic salmon were killed with an overdose of benzocaine, measured, weighed, and identified. The weights of fish tagged with 23 and 32 mm PIT tags were corrected by subtracting the weight of the tag from the final body mass (0.6 g and 0.8 g, respectively). The tagging incisions were inspected for infection, inflammation, and healing.
Data analysis
Fish that died or lost their PIT tag during the experiment were only used in analyses of survival and rates of tag retention. The percent tag loss was calculated as number of lost tags divided by the total number of fish tagged multiplied by 100. At the end of the experiment, the survival rate was calculated and a Chi-square test was used to compare survival among treatment groups. Tag-to-body mass ratio (%) in air was calculated with the formula:
where Mi is the mass of fish prior to tagging.
The SGR (g day-1) was calculated for each individual fish according to the following equation:
where Mi and Mf are the initial and final mass (g), respectively, and t is the time in days. A one-way ANOVA was used to determine if there were differences in SGR among size classes of treatment groups. Tukey HSD multiple comparisons were subsequently performed to determine which treatment groups had significantly different SGR among size classes. Tank number was included as a random effect variable in the analyses.
An analysis of covariance was conducted to elucidate the effect of experimental treatments, length, and the interaction between these two variables on the body mass of the Atlantic salmon at the end of the experiment. The fish length-mass relationship was used as a proxy for body condition. Analyses were carried out on log-transformed length-mass data.
All statistical analyses were performed in SPSS 20.0 (Statistical Package for the Social Sciences; SPSS Inc, Chicago, IL, USA). Prior to analysis, data exploration was carried out as per Zuur et al.
[35]. Assumption of homogeneity of variance and normal distribution for the models were ensured by visual inspection of residual plots. Variation in association with recorded mean values is given as standard error throughout. Statistical significance for all analyses was set at α = 0.05.