Acute stress response on Atlantic salmon: a time-course study of the effects on plasma metabolites, mucus cortisol levels, and head kidney transcriptome profile.

Farmed Atlantic salmon (Salmo salar) welfare and performance can be strongly influenced by stress episodes caused by handling during farming practices. To better understand the changes occurring after an acute stress response, we exposed a group of Atlantic salmon parr to an acute stressor, which involved netting and transferring fish to several new holding tanks. We describe a time-course response to stress by sampling parr in groups before (T0) and 10, 20, 30, 45, 60, 120, 240, 300, and 330 min post-stress. A subgroup of fish was also subjected to the same stressor for a second time to assess their capacity to respond to the same challenge again within a short timeframe (ReStressed). Fish plasma was assessed for adrenocorticotropic hormone (ACTH), cortisol, and ions levels. Mucus cortisol levels were analyzed and compared with the plasma cortisol levels. At 5 selected time points (T0, 60, 90, 120, 240, and ReStressed), we compared the head kidney transcriptome profile of 10 fish per time point. The considerably delayed increase of ACTH in the plasma (60 min post-stress), and the earlier rise of cortisol levels (10 min post-stress), suggests that cortisol release could be triggered by more rapidly responding factors, such as the sympathetic system. This hypothesis may be supported by a high upregulation of several genes involved in synaptic triggering, observed both during the first and the second stress episodes. Furthermore, while the transcriptome profile showed few changes at 60 min post-stress, expression of genes in several immune-related pathways increased markedly with each successive time point, demonstrating the role of the immune system in fish coping capacity. Although many of the genes discussed in this paper are still poorly characterized, this study provides new insights regarding the mechanisms occurring during the stress response of salmon parr and may form the basis for a useful guideline on timing of sampling protocols.

Increasing Dip Net Mesh Size Results in More Fin Splits in Post-Smolt Atlantic Salmon (Salmo salar).

Dip nets are widely used to retrieve fish from the water but can cause injuries to the fish. The objective of this study was to document the potential effects of dip net mesh size with respect to external injuries, more specifically fin splits and scale losses, in Atlantic salmon (Salmo salar). The study included 273 post-smolt Atlantic salmon from two fish batches referred to as the Tank group (w- = 178 ± 36 g, n = 198) and the Cage group (w- = 1319 ± 334 g, n = 75). Four identical dip nets with mesh sizes of 5, 10, 15, and 20 mm, respectively, were used to net fish out of their enclosures before the external injuries were assessed by visual inspection. The results showed that the number and severity of fin splits increased with dip net mesh size, but no effect of mesh size on the percentage of scale losses was found. Dip-netting of five fish at a time instead of one with the 20 mm dip net, had no statistically significant effect on the examined injuries. It is concluded that the use of dip nets with smaller mesh sizes can be positive for fish welfare by reducing fin splitting.

Warm water treatment increased mortality risk in salmon.

Thermal treatment is a controversial method to control sea lice in the Atlantic salmon farming industry. This study aimed to complement the growing evidence base to document the impact of thermal treatments on salmon welfare, behaviour, physiology and health. Here, fish were treated two times (four weeks apart) for 30 s in either 27, 30, or 33 °C warm water, and parameters were compared to a procedural control (exposed to their holding temperature of 14 °C) or a negative control (where no treatments were applied). The fish had a clear behavioural response to the warm water, despite low difference between treatment and holding temperature (Δt = 13, 16 or 19 °C). Eye damages were more prevalent in the warm water treated groups than in the controls. Little difference was recorded between treatment groups in their growth and condition factor, blood plasma values, organ health, and long-term coping ability. There was, however, a significant increase in mortality as a function of temperature after the first treatment (14 °C: 6.5%, 27 °C: 5.3%, 30 °C: 12.4% and 33 °C: 18.9% mortality). The first treatment was performed only two weeks after the fish had been tagged and moved into the experimental holding tanks, while the fish had been allowed to recover for four weeks without any handling before the second treatment. The group of fish that were not subjected to any treatments (the negative control) had no mortality throughout the entire experimental period.

Sudden exposure to warm water causes instant behavioural responses indicative of nociception or pain in Atlantic salmon.

Thermal treatment has become the most used delousing method in salmonid aquaculture. However, concerns have been raised about it being painful for the fish. We studied the behavioural response of Atlantic salmon acclimated to 8 °C when transferred to temperatures in the range 0-38 °C. Exposure time was 5 min or until they reached the endpoint of losing equilibrium and laying on their side, a sign of imminent death. At temperatures below 28 °C, none of the fish reached endpoint within the 5-min maximum. At 28 °C four of five fish reached endpoint, and fish reached endpoint more rapidly as temperature increased further. Fish transferred to temperatures above 28 °C had higher swimming speed immediately after transfer and maintained a high swimming speed until just before loss of equilibrium. Their behaviour was from the start characterised by collisions into tank walls and head shaking. Just before loss of equilibrium they started breaking the surface of the water, swimming in a circle pattern and in some instances displayed a side-wise bending of their body. In other words, salmon transferred to temperatures above 28 °C showed instant behavioural responses indicative of nociception or pain.

Thermal injuries in Atlantic salmon in a pilot laboratory trial.

Thermal delousing is a new method for removing sea lice from farmed Atlantic salmon (Salmo salar L). We investigated thermally-related tissue injuries in Atlantic salmon in a pilot laboratory trial to describe the acute effect of high water temperatures (34-38 °C). Acute tissue injuries in gills, eyes, brain and possible also nasal cavity and thymus were seen in salmon exposed to water temperatures of 34 - 38 °C in 72 to 140 s. This implies that exposing salmon to such water temperatures is a welfare risk, not only due to the direct tissue injuries that may also be dependent on exposure time, but also due to risk of thermal pain and aversion, including flight reactions.

Effect of predictability on the stress response to chasing in Atlantic salmon (Salmo salar L.) parr.

The possibility to prepare for and respond to challenges in a proper manner is essential to cope with a changing environment, and learning allows fish to up or downregulate the stress response based on experience. The regulation of the response to predicted needs should be easier in more predictable environments. We exposed salmon parr to chasing of either 15 s (weak stressor) or 5 min (strong stressor) twice daily for a 7-day learning period, with chasing either announced by a 30 s light signal (conditioned) or not announced (unconditioned). The behavioural response to the light signal was different between the conditioned and unconditioned groups, demonstrating that conditioned groups associated the signal with chasing. We could, however, not demonstrate any effect on the stress response of anticipation. The fish habituated to repeated stress exposures with a similar decrease in oxygen hyperconsumption in all groups. Due to habituation, possible effects of predictable announcement of a stressor on the physiological stress response may not have been expressed in this study. Plasma cortisol concentrations 1h after light signal and chasing the day after the training period was moderate in all groups although higher after 5 min chasing (13 ng ml(-1)) than 15 s chasing (7 ng ml(-1)). There was no physiological stress response after exposure to the light signal only after the learning period. We argue that the benefit of predictability of stressors is limited when the fish have no way to avoid the stressor.

Innovative behaviour in fish: Atlantic cod can learn to use an external tag to manipulate a self-feeder.

This study describes how three individual fish, Atlantic cod (Gadus morhua L.), developed a novel behaviour and learnt to use a dorsally attached external tag to activate a self-feeder. This behaviour was repeated up to several hundred times, and over time these fish fine-tuned the behaviour and made a series of goal-directed coordinated movements needed to attach the feeder's pull string to the tag and stretch the string until the feeder was activated. These observations demonstrate a capacity in cod to develop a novel behaviour utilizing an attached tag as a tool to achieve a goal. This may be seen as one of the very few observed examples of innovation and tool use in fish.

Duration of effects of acute environmental changes on food anticipatory behaviour, feed intake, oxygen consumption, and cortisol release in Atlantic salmon parr.

We compared behavioural and physiological responses and recovery time after different acute environmental challenges in groups of salmon parr. The fish were prior to the study conditioned to a flashing light signalling arrival of food 30 s later to study if the strength of Pavlovian conditioned food anticipatory behaviour can be used to assess how salmon parr cope with various challenges. The effect on anticipatory behaviour was compared to the effect on feed intake and physiological responses of oxygen hyper-consumption and cortisol excretion. The challenges were temperature fluctuation (6.5C° over 4 h), hyperoxia (up to 380% O(2) saturation over 4 h), and intense chasing for 10 min. Cortisol excretion was only elevated after hyperoxia and chasing, and returned to baseline levels after around 3 h or less. Oxygen hyper-consumption persisted for even shorter periods. Feed intake was reduced the first feeding after all challenges and recovered within 3 h after temperature and hyperoxia, but was reduced for days after chasing. Food anticipatory behaviour was reduced for a longer period than feed intake after hyperoxia and was low at least 6 h after chasing. Our findings suggest that a recovery of challenged Atlantic salmon parr to baseline levels of cortisol excretion and oxygen consumption does not mean full recovery of all psychological and physiological effects of environmental challenges, and emphasise the need for measuring several factors including behavioural parameters when assessing fish welfare.

Learning and anticipatory behaviour in a "sit-and-wait" predator: the Atlantic halibut.

We studied the learning capacities and anticipatory behaviour in a "sit-and-wait" predatory fish, the Atlantic halibut, Hippoglossus hippoglossus. In Experiment 1 two groups of halibut received series of light flashes (conditioned stimulus, CS) that started before delivery of food (unconditioned stimulus, US) and persisted until after food delivery, i.e. delay conditioning. Control groups received unpaired CS and US presentations. The anticipatory behaviour of delay conditioned halibut consisted mainly of take-offs towards the surface shortly after onset of the CS. In Experiment 2 six groups of halibut were trained in three trace conditioning procedures: Two groups with 20s, two groups with 60s and two groups with 120s trace interval. Learning was evident in the 20 and 60s trace groups and in one of the 120s trace groups. In contrast to delay conditioning the anticipatory behaviour of trace conditioned halibut was characterized by subtle movements near the tank floor with orientation towards the CS. The cautious responses of halibut after trace conditioning differed markedly from what is observed in other fish species and are suggested to reflect a "sit-and-wait" foraging strategy that requires the predator to remain undetected until the prey is within lunging range.

Sign- and goal-tracking in Atlantic cod (Gadus morhua).

When animals associate a stimulus with food, they may either direct their response towards the stimulus (sign-tracking) or towards the food (goal-tracking). The direction of the conditioned response of cod was investigated to elucidate how cod read cue signals. Groups of cod were conditioned to associate a blinking light (conditioned stimulus, CS) with a food reward (unconditioned stimulus, US), with the CS and the US located at opposite sides of the tank. Two groups were trained in a delay conditioning procedure (CS = 60 s, interstimulus interval = 30 s) and two groups were trained in a trace conditioning procedure (CS = 12 s, trace interval = 20 s). The response pattern was similar for the delay- and trace-conditioned groups. The initial main response at the onset of the CS was approaching the blinking lights, i.e. sign-tracking. In the early trials, the fish did not gather in the feeding area before the arrival of food. In the later trials, the fish first approached the blinking lights, but then moved across the tank and gathered below the feeder before the food arrived, i.e. sign-tracking followed by goal-tracking within each trial. These two responses are interpreted as reflecting two learning systems, i.e. one rapid, reflexive response directed at the signal (sign-tracking) and one slower, more flexible response based on expectations about time and place for arrival of the food (goal-tracking). The ecological significance of these two learning systems in cod is discussed.

Learning in cod (Gadus morhua): long trace interval retention.

Basic knowledge about learning capacities and awareness in fish is lacking. In this study we investigated which temporal gaps Atlantic cod could tolerate between two associated events, using an appetitive trace-conditioning paradigm with blinking light as conditioned stimulus (CS) and dry fish food as unconditioned stimulus (US). CS-US presentations were either temporally overlapping (delay conditioning, CS duration 24 s, interstimulus interval 12 s) or separated by 20, 60, or 120 s (trace conditioning, CS duration 12 s) or 2 h (control, CS duration 12 s). The percentage of fish in the feeding area increased strongly during CS presentation in all delay, 20 s, and 60 s trace groups and in one out of two 120 s trace groups, but not in the control groups. In the 20 and 60 s trace procedures, the fish crowded together in the small feeding area during the trace interval, showing strong anticipatory behaviour. In all the conditioned groups, the fish responded to the CS within eight trials, demonstrating rapid learning. At 88 and 70 days after the end of the conditioning experiments, the delay and 20 s trace groups, respectively, were presented the CS six times at 2-h intervals without reward. All groups responded to the light signal, demonstrating memory retention after at least 3 months. This study demonstrates that Atlantic cod has an impressively good ability to associate two time-separated events and long time retention of learnt associations.