Ethics
This experiment was approved by Virginia Tech’s Institutional Animal Care and Use Committee (20-074) and was conducted in the Department of Food Science and Technology’s aquaculture facility at Virginia Tech’s Human, Agriculture, and Biosciences Building I from August to December 2020. This experiment was performed in accordance with the Institutional Animal Care and Use Committee’s relevant guidelines and regulations.
Subjects and housing
Rainbow trout (n = 108; F1 generation Shasta strain) were bred and hatched in January 2020 and cultured at the Wytheville State Fish Hatchery (Wytheville, VA). In August 2020, fingerlings were transported to the research facility in 2 tanks of 14.1°C water. Upon arrival, fingerlings were acclimated to the recirculating aquaculture system water conditions (13.3°C) and facility management for 4 weeks. No mortalities were observed during or 24 hours post-transportation. On day 1 of the experiment, fish were distributed across 5 tanks (45.7 x 73.7 x 21.6 cm; water volume = 0.0726m3) under commercial conditions with 21, 22, or 23 fish in each tank. Temperature, dissolved oxygen level, ammonia-N, nitrite-N, nitrate-N, pH, and alkalinity were monitored at least once per week. All water quality parameters remained within suitable ranges64,65, with the exception of alkalinity, which dropped below the optimal range on day 3. Sodium bicarbonate was added to the system throughout the trial to maintain optimal alkalinity values. Fish were fed a commercial trout diet (3 mm Finfish Gold, Zeigler Bros Inc., Gardner, PA, USA) once daily ad libitum. A subsample of fish (n = 72) were tagged with T-bar tags (Floy Tag, WA, USA) after sedation with sodium bicarbonate buffered MS-222 (Syndel, Ferndale, WA, USA) on days 1-366.
Treatments
This experiment involved a 2´2 factorial design using environmental complexity and stocking density as factors, resulting in 4 treatment groups: HC/HD, HC/LD, LC/HD, and LC/LD. All fish (n = 108) were kept under the same commercial conditions (5 tanks) until day 64 of the experiment. During this time, all fish were trained on a judgement bias task at tank level. After the judgement bias training was completed and on day 64, 40 fish from the 2 tanks that were successfully trained were allocated to 8 treatment tanks (5 successfully trained fish/treatment tank). Tags of successfully trained fish were marked with black marker (n = 40) to differentiate them from fish that were not successfully trained, most importantly in HD tanks. Three arbitrarily selected fish from the 2 successfully trained tanks were excluded from the experiment to achieve an even distribution of successfully trained fish across the 8 treatment tanks. Twenty-eight fish from the remaining 3 tanks that did not meet the learning criterion were arbitrarily selected and evenly distributed across 4 of the 8 tanks for the HD treatment (n = 7 randomly selected fish/HD tank). The remaining fish (n = 37) were excluded from the experiment. Thus, from days 1-64 all fish were kept under commercial conditions in 5 tanks, then a subsample of fish (n = 68) were redistributed over 8 treatment tanks in which they remained until day 96 (Figure 4).
Four tanks provided a complex environment (HC), while the other four tanks provided a simple environment similar to commercial standards (LC). HC tanks contained one PVC shelter structure (cut in half, 10.2cm diameter, 15.2cm long), which was placed at the bottom of the tank, two artificial floating lily pads (17.5cm x 17.0cm, Amazon.com, Inc., WA, USA), and two artificial cabomba plants (17.8cm, AquaTop, CA, USA). Enrichment objects were removed and disinfected daily. LC tanks contained no enrichment objects.
Based on the mean fish weight of 287g at day 96 and the Food and Agriculture Organization of the United Nations guidelines1, 4 tanks (HC = 2; LC = 2) were stocked at a high-density (HD) of 12 fish (165 fish/m3). The other 4 tanks (HC = 2; LC = 2) were stocked at a low-density (LD) of 5 fish (69 fish/m3).
Judgement bias test
The judgement bias process followed a 7-step approach (habituation; training: phase 1, phase 1A, phase 2, phase 3; reminder training; and testing; Table 2) and took place in the home tanks, at tank-level. All steps of the judgement bias test were performed using a removable plexiglass arena with 5 equally-distanced opaque blue chambers and sliding doors (Figure 5). Sliding doors allowed for the experimenter to provide or deny access to each of the 5 chambers in the arena.
Habituation to the judgement bias arena without sliding doors was performed with all fish within a tank (n = 21-23 fish/tank), including the subsample of tagged fish (Table 2). A single experimenter gently placed the arena in the tanks from days 13-31 for 9 habituation sessions. For the first 4 sessions, the arena was placed into each tank for 5min with the experimenter out of sight. For the following 3 sessions, the arena was placed and the experimenter remained in line of sight for 15min per session. During the last 2 sessions, the arena was placed and the experimenter sprinkled feed into the tank, remaining in line of sight for 15min per session.
Training phases 1-3, reminder training, and testing involved placing the arena into each tank with the sliding doors closed for 3min to allow fish to acclimate. If a fish entered the POS chamber within any 15s attempt during phases 1-3, feed was immediately placed into the chamber and fish were allowed 10s to feed. After every attempt, the chamber door was closed and remaining feed was removed. Habituation and phases 1-3 were performed while fish were housed in commercial conditions (n = 21-23 fish/tank), while reminder training and testing took place while fish were housed under treatment conditions (Table 2).
Phase 1 of training was performed at tank-level (Table 2). Fish in each tank (n = 21-23 fish/tank) were trained to associate a chamber on either the far left (3 tanks) or far right (2 tanks) side of the arena with a reward (POS; approximately 30 feed pellets). If no fish entered the chamber within 15s, feed was placed into the chamber and 10s was allowed for fish to enter and feed. The learning criterion for phase 1 was met when at least 1 fish entered the POS chamber within 15s for 9 out of 12 attempts during two consecutive phase 1 training sessions. Two tanks passed the learning criterion for phase 1 of training between days 38 and 58, the other three tanks moved on to phase 1A of training, as the fish were not close to meeting the learning criterion after 5 sessions. Training phase 1A was similar to phase 1, however, fish were rewarded with feed if they oriented towards or swam within 15cm of the POS chamber opening. Tanks returned to phase 1 of training once at least 1 fish entered the POS chamber within 15s during two consecutive phase 1A training sessions. Only one tank met the learning criterion for phase 1A and returned to phase 1 on day 46, meeting the phase 1 learning criterion on day 47. The other two tanks remained in phase 1A until day 57, when they were excluded from the judgement bias task due to unresponsiveness.
Phase 2 of training with the remaining three tanks was aimed to habituate fish to being in a smaller group within their tank (approximately 10 fish/group; 2 groups/tank; Table 2). Half of the fish (group 1) were gently herded to the side of the tank containing the arena and separated from their conspecifics (group 2) by placing a blue opaque plexiglass separator into the middle of the tank. Then, the POS chamber door was opened. If no fish entered the chamber within the first 15s attempt, feed was placed into the chamber and fish were allowed 10s to enter and feed. If no fish entered during any of the following 15s attempts, the chamber door was immediately closed and the next attempt began. If a fish entered the chamber within any of the 15s attempts, feed was immediately placed into the chamber and fish were allowed 10s to feed. After group 1, group 2 underwent the same training session. Phase 2 learning criterion was met when at least one fish in both groups 1 and 2 entered the POS chamber within 15s and consumed feed in 9 out of 12 attempts during two consecutive phase 2 sessions. All three tanks passed the learning criterion for phase 2 of training between days 45 and 59.
In training phase 3, the negative chamber was introduced (NEG; net placed in water for 1s; Table 2). NEG and POS cue presentations were pseudorandomized according to a predetermined order, never allowing more than two consecutive presentations of the NEG or POS cue, and began and ended with the POS cue. Half of the fish (group 1; approximately 10 fish) were gently herded to the side of the tank containing the arena and separated from their conspecifics (group 2; approximately 10 fish) by placing the blue opaque plexiglass separator into the middle of the tank. Then, the POS chamber door was opened. If a fish did not enter within 15s, the door was immediately closed. When the NEG chamber was opened, fish were allotted 15s to enter the chamber. If a fish entered within 15s, a green net was placed into the water for approximately 1s, then the chamber door was closed. If no fish entered within 15s, the chamber door was immediately closed. After 6 attempts (1 session) for group 1, group 2 was trained. Phase 3 learning criterion was met when at least one fish from both groups enter the POS chamber 100% of the time it was accessible and neglected to enter the NEG chamber 100% of the time it was accessible during two consecutive phase 3 training sessions. Two tanks passed the learning criterion for phase 3 on day 61. The third tank was excluded from the judgement bias task due to time constraints.
The trained subsample of fish (HD) or all fish (LD) underwent weekly reminder training sessions identical to phase 3 of training (Table 2). In HC tanks, enrichment objects were removed prior to a session.
Each of the 8 tanks (n = 38 fish) were tested for judgement bias three times on days 92, 93, and 95 (Table 2). In addition to the POS and NEG cues, three ambiguous cues (near positive, NP; middle, MID; near negative, NN) were individually presented at intermediate locations within the arena (near left, middle, and near right; Figure 2). Each ambiguous cue was presented once per session according to a pre-determined order. Fish were given 20s to enter an opened chamber. Testing always began and ended with the POS cue, which was presented 8 times with fish receiving a feed reward if they entered the chamber. The NEG cue was presented 4 times and if a fish entered the chamber, it was chased by a net for 1s. All ambiguous cues were neither rewarded nor punished. For HC tanks, enrichment objects were removed prior to testing. In HD tanks, the tagged subsample of fish was separated from the other 7 fish with a blue plexiglass separator throughout testing. Fish were allowed 3min to acclimate. Latency for the first fish to enter each chamber (s) was recorded. A maximum latency score of 20s was appointed to attempts during which no fish entered the chamber.
Body weight and standard length
On days 64 (n = 68) and 96 (n = 66), individual weights and lengths were recorded. On day 96, only 66 fish were weighed and measured due to 2 mortalities. Fish were individually netted and placed into a buffered MS-222 water bath (sedation strength of 75-100mg/L) for approximately 5min or until sedated66. After sedation, the fish was placed on a scale (PTS3000, 0.1g precision, PESOLA, CH, USA) and then beside a ruler to measure standard length (from the tip of the nose to the base of the caudal fin). Then, the fish was immediately placed in a fresh water recovery tank where it was monitored until normal swimming behavior resumed. At this point, the fish was placed back into the home tank.
Feeding behavior
Latency to begin feeding was assessed at tank level on days 79-95 (observation n = 128). During daily feeding, the observer sprinkled a handful of feed into the tank and immediately started a timer. Latency until the first fish began feeding (s) was recorded for each tank once daily.
Plasma cortisol
On day 96, blood was collected from the 38 fish trained on the judgement bias task (n = 4-5/tank). Fish (n = 38) were individually netted and placed into a buffered MS-222 water bath (sedation strength of 75-100mg/L) for approximately 5min or until sedated66. After sedation, the fish was placed on a wetted work table and bled from the caudal tail vessel using a 23-gauge needle and syringe. Approximately 0.5-1.0mL of blood was obtained, then the fish was immediately euthanized by an overdose of buffered MS-222 (euthanasia strength of 250mg/L). Blood was placed in heparinized tubes and kept on ice until centrifugation. Samples were centrifuged at 3000 x g for 10min at 12°C, then plasma was stored at -80°C until ELISA analysis using a commercial cortisol express ELISA kit (Cayman Chemical, Ann Arbor, MI, USA). The ELISA was performed following the manufacturer protocol. Four of 38 samples were excluded from the statistical analysis due to unreliable assay results.
Statistical analysis
All data were analyzed in JMP pro 15 (SAS Institute Inc., Cary, NC, USA). Judgement bias data residuals were deemed normally distributed based on visual examination of normal quantile plots. Mixed models were used with complexity, stocking density, and test session as fixed factors, tank as a random factor, and latency to enter each chamber as the response variable. There was an effect of stocking density on latencies, so we blocked the analysis by chamber type (POS, NP, MID, NN, NEG) to assess the effect of density on latencies for each separate chamber. Tukey HSD post-hoc comparisons were used to evaluate pairwise differences. Length and weight data residuals were normally distributed and analyzed using mixed models, with environmental complexity and stocking density as fixed factors and tank as a random factor. Latency to begin feeding data were log10 transformed to obtain a normal distribution of data residuals and are presented as raw means. Then, mixed models were used with environmental complexity and stocking density as fixed factors and tank as a random factor. Data are presented as least squares means ± standard error unless otherwise noted.