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In fish, as well as in other vertebrates, contrasting suites of physiological and behavioral traits, or coping styles, are often shown in response to stressors. However, the magnitude of the response (i.e. stress responsiveness) has been suggested to be independent of stress coping style. One central neurotransmitter that has been associated with both stress responsiveness and differences in stress coping styles is serotonin (5-hydroxytryptamine, 5-HT). In this study, we investigated to what extent stress responsiveness reflects differences in stress coping, and the potential involvement of the 5-HT system in mediating such differences in farmed Gilthead seabream. Initially, fish were classified as proactive or reactive based on their behavioural response to net restraint. Following 1.5 months, fish classified as proactive still showed a higher number of escape attempts and spent longer time escaping than those classified as reactive. These differences were reflected in a generally higher brain stem 5-HT concentration and a lower telencephalic 5-HT activity, i.e. the ratio of 5-hydroxyindoleacetic acid (5-HIAA) to 5-HT, in proactive fish. Independent of stress coping styles, stress responsiveness was reflected in elevated 5-HIAA concentrations and 5-HIAA/5-HT ratios in telencephalon and brain stem together with increased plasma cortisol concentrations at 0.5 and 2 h following the last net restraint. The current results show that 5-HT signaling can reflect different behavioural output to a challenge which are independent of neuroendocrine responses to stress and lend support to the hypothesis that stress coping styles can be independent of stress responsiveness.

Metabolic programming refers to the induction, deletion, or impaired development of a somatic structure or "setting" of a physiological system by an early life stimulus operated at a critical period during development. Ghrelin is the only known orexigenic gut hormone, is an acylated peptide that acts as an endogenous ligand specific for growth-hormone secretagogue-receptor. The aim of the present work was to evaluate if an in ovo ghrelin administration could positively influence the zebrafish performance in the long-term and to gain insight on the mechanisms associated to ghrelin regulation of food intake during the larval phase. Food intake, growth potential, protein metabolism, expression of target genes involved in ghrelin, feeding behaviour regulation and locomotor activity were assessed in zebrafish (Danio rerio) larvae at 25 days post-fertilization. Elevated levels of acylated ghrelin in zebrafish eggs did not result in increased growth or food intake. Differences in mRNA expression between larvae fasted for 16 h before and 1 h after feeding were found for igf1ra, gh1 and pomca. Moreover, ghrelin treated larvae showed higher swimming activity, indicating that the peptide may have an important role on foraging activity. The present study addressed for the first time the effects of an early stimulus of ghrelin during the embryonic stage of zebrafish, however, further studies are needed to clarify the metabolic pathways affected by the early stimulus as well as focus on the effects on metabolic regulation of energy balance through lipid and carbohydrate metabolism. FCT/MCTES (Portugal)Portuguese Foundation for Science and Technology [PTDC/CVT/102481/2008, UID/Multi/04326/2013] FCT - European Social Fund [IF/00482/2014/CP1217/CT0005] Operational Programme Human Potential Foundation for Science and Technology of Portugal (FCT)Portuguese Foundation for Science and Technology

Evolution has resulted in behavioural responses to threat which show extensive similarities between different animal species. The reaction to predator cues is one example of such prevailing responses, and functional homologies to mammalian limbic regions involved in threat-sensitive behaviour have been found in the teleost telencephalon. The dorsolateral (Dl) and dorsomedial (Dm) regions of the pallium are thought to perform hippocampus and amygdala-like functions respectively. To what degree these regions are involved in the neuroendocrine responses to stress and predator cues however remains largely unknown. In the present study the involvement of Dl and Dm in such responses was investigated by exposing Nile tilapia (Oreochromis niloticus) to a standardized confinement stress and to skin extract from conspecifics. Nile tilapia develops a characteristic anticipatory behaviour to hand feeding, and effects of skin extract on this behaviour and locomotor activity were studied to characterise threat sensitive behaviour. Nile tilapia responded behaviourally to conspecific alarm cues by reducing feeding anticipatory behaviour. This may reflect a general elevation of alertness, and further studies combining skin extract with other challenges are needed to reveal neuroendocrine effects associated with this predator cue. Confinement stress resulted in an elevation of cortisol and serotonin (5-hydroxytryptamine, 5-HT) metabolism in both Dl and Dm. A similar tendency was observed in fish exposed to chemical alarm cues, but this effect did not reach the level of statistical significance. Hence, limbic responses to stress and fear, akin to those seen in extant mammals, are also present in the teleost lineage.

The accumulation of steroids released by fish in recirculating aquaculture systems (RAS) may potentially influence their physiology and behavior. The present study examined the release rate of cortisol and testosterone by Nile tilapia, Oreochromis niloticus, and their accumulation in six identical lab scale RAS operated at different water exchange rates (150 L/kg feed/day, (LowRAS) and 1500 L/kg feed/day, (HighRAS)) and how steroid accumulation is affected by grading and weighing induced stress. Water cortisol and testosterone concentrations during the experimental period ranged between 1.0 and 5.1 ng/L and between 1.4 and 9.4 ng/L, respectively. Water cortisol concentration was 34% and 43% significantly higher in LowRAS when compared with HighRAS at week 3 and week 4, whereas for water testosterone concentration the two treatments did not differ. Overall steroid release rates were 0.02 +/- 0.02 and 0.04 +/- 0.02 ng/g body weight/h for cortisol and testosterone, respectively. Two hours after grading and weighing water cortisol concentration increased 30% in the LowRAS whereas water testosterone concentration remained unchanged. Calculated cortisol and testosterone discharge from the system were, respectively, 87% and 89% lower in LowRAS than in HighRAS. These findings show that reduced water usage and acute stressors can induce significant accumulation of cortisol in the rearing water at levels close to olfactory detection. Accumulation of metabolites, in particular steroids, should be taken in consideration when designing and managing RAS to prevent exceeding allowable concentrations. Statement of relevance: RAS containing Nile tilapia, cortisol and testosterone are released to the culture water. Water cortisol concentration depends on the water exchange rate that is used and higher concentrations are expected in lower water exchange rates. Fish grading and weighing has a short-term effect on the water cortisol concentrations, after which concentrations return to the basal levels. These findings show that reduction in water usage and fish handling may lead to a significant increase of hormones in the rearing water, which emphasizes the importance of accounting for steroids in the design and management of RAS. (C) 2016 Elsevier B.V. All rights reserved. Portuguese institution FCT - Fundacao para a Ciencia e Tecnologia (QREN-POPH - Advanced Training - European Social Fund) [SFRH/BD/65673/2009] Portuguese institution FCT - Fundacao para a Ciencia e Tecnologia (QREN-POPH - Advanced Training - national funds MEC) [SFRH/BD/65673/2009]

Steroids accumulate in recirculating aquaculture system (RAS), although explanatory factors for such accumulation are still poorly explored. This study investigated the effect of water exchange rate and pH in six replicated RAS on the concentration of the stress hormone cortisol in rainbow trout blood plasma and in the holding water and of the sex steroids testosterone, 11-ketotestosterone (11-KT) and 17,20-dihydroxypregn-4-en-3-one (17,20-P) over a 70-day experimental period. Three combinations of water exchange rate and pH were used each treatment, with two replications: (i) high water exchange rate (+/- 1700Lkg(-1) feed) and neutral pH (+/- 7.3), (ii) low water exchange rate (+/- 500Lkg(-1) feed) and neutral pH (+/- 7.3) and (iii) low water exchange rate (+/- 500Lkg(-1) feed) and low pH (+/- 5.8). Plasma cortisol concentrations at day 70 were higher (24.4 +/- 9.5ngmL(-1)) for fish kept at low pH when compared to fish kept at neutral pH (12.0 +/- 0.1 and 8.7 +/- 0.2ngmL(-1)). Water cortisol and testosterone concentrations at day 35 were higher at low pH than at neutral pH, whereas water 11-KT and 17,20-P did not differ among treatments. At day 70, there were no significant differences between low and high pH. These results demonstrate that low pH contributes to increased plasma cortisol concentrations and to its accumulation in water, possibly indicating a stress response to low pH. The higher concentration of testosterone but not of the other sex hormones point to unspecified reproductive effects that need further investigation. PhD scholarship - FCT - the National Foundation for Science and Technology of Portugal through QREN-POPH Advanced Training European Social Fund and National funds (MEC) [SFRH/BD/65673/2009] info:eu-repo/semantics/publishedVersion

Risk taking and avoidance responses during confinement have been identified in a variety of species, including fish. In addition, differences in metabolic rate have also been attributed to divergent personalities, coping styles or behavioral types. In this study we disclose for the first time a link between risk taking and the behavioral responses and oxygen consumption under confinement stress in the gilthead seabream Sparus aurata. Fish were submitted to a risk-taking test twice to determine the consistency of behavioral responses. Afterwards fish were transferred to individual confinement chambers where their swimming activity and oxygen consumption were measured. Latency to take risks was negatively correlated to both movement and oxygen consumption rates, indicating that risk-avoiders (long latency) were less active and, hence, did not consume so much oxygen as risk-takers. In conclusion, this work reports the first data on the links between risk-taking and the behavioral responses and oxygen consumption during a confinement stress in fish. The relationships between behavioral and physiological variables are significant, suggesting the existence of divergent coping styles in gilthead seabream.

Tryptophan (TRP) supplemented diets have been shown to have therapeutic effects in farmed animals including fish by modulating the activity of the neurotransmitter serotonin (5-hydroxytryptamine; 5-HT). The effects reported in fish have been obtained using individually-housed fish and include a reduction in stress response, aggression and stress-induced anorexia. In land farmed animals, TRP supplemented diets have also been shown to improve meat quality as a result of reduced stress during slaughter while in fish no data is currently available. This study aims at investigating whether short-term supplementation with TRP supplemented diets changes brain serotonergic activity and the stress response associated with slaughter handling in grouped-housed Nile tilapia Oreochromis niloticus. Adult fish (n = 108, 490.6 ± 4.0 g, 12 individuals per tank) were exposed to one of the three treatments (triplicates per treatment were used): control (0.48 g/100 g), TRP 4 × (1.87 g/100 g) and TRP 10 × (4.45 g/100 g) diets during 7 days. Afterwards, half of the fish in each tank were subjected to an acute stressor consisting of a combination of crowding and chasing, just prior to slaughter. The other half of the fish represented undisturbed conditions. Blood and brain samples were collected for cortisol and serotonergic activity analyses, respectively. Flesh quality was also assessed in both undisturbed and stressed fish for all treatments by measuring muscle pH and rigor mortis over a 72 h period. Results showed that the highest TRP supplemented diet (TRP 10 ×) induced a significant reduction in undisturbed plasma cortisol (10.57 ± 2.71 ng/ml) as compared to TRP 4 × (24.93 ± 3.19 ng/ml) and control diets (18.69 ± 2.94 ng/ml) and no effect on post-stress cortisol levels. After stress, the major 5-HT metabolite (5-hydroxyindoleacetic acid, 5-HIAA) was higher in the TRP 10 × (471.31 ± 60.95 ng/g) as compared to the other diets (TRP 4 ×: 313.52 ± 30.12 ng/g; control: 260.36 ± 19.65 ng/g). Stress before slaughter induced a significant increase in plasma cortisol (from 18.40 ± 1.76 ng/ml under undisturbed conditions to 80.34 ± 7.16 ng/ml), however, it was not sufficient to cause a faster deterioration of flesh quality. TRP supplement diets had also no effect on muscle pH and rigor mortis during the 72 h observation period. In conclusion, this study showed that only the highest levels of supplementation (10 × the control diet) affect serotonergic activity. However, these levels did not result in reduced stress responsiveness or improved flesh quality when an acute stressor is applied before slaughter. Therefore, these results underline the fact that effects of TRP on cortisol production are dose- and context-dependent, and further experiments are needed to determine under which conditions the optimal effect is obtained.

Individual differences in behaviour and physiological responses to stress are associated with evolutionary adaptive variation and thus raw material for evolution. In farmed animals, the interest in consistent trait associations, i.e. coping styles, has increased dramatically over the last years. However, one of limitations of the available knowledge, regarding the temporal consistency, is that it refers always to short-term consistency (usually few weeks). The present study used an escape response during a net restraining test, previously shown to be an indicative of coping styles in seabream, to investigate long-term consistency of coping styles both over time and during different life history stages. Results showed both short-term (14 days) consistency and long-term (8 months) consistency of escape response. However, we did not found consistency in the same behaviour after sexual maturation when the restraining test was repeated 16, 22 and 23 months after the first test was performed. In conclusion, this study showed consistent behaviour traits in seabream when juveniles, and a loss of this behavioural traits when adults. Therefore, these results underline that adding a life story approach to data interpretation as an essential step forward towards coping styles foreground. Furthermore, a fine-tuning of aquaculture rearing strategies to adapt to different coping strategies may need to be adjusted differently at early stages of development and adults to improve the welfare of farmed fish.

As a consequence of water re-use and high stocking densities, Recirculating Aquaculture Systems (RAS) may lead to an accumulation of substances released by the fish into the water, e.g. cortisol and alarm pheromones. This study investigated the effect of stressing fish on the feeding motivation of other fish not subjected to stress but sharing the same water of stressed fish. Two identical RAS were used (operated at 30 L kg feed-1 day-1) and contained grouped (stressed fish) and individually (receiving water from stressed fish) housed Nile tilapia. A stress test was applied in grouped housed fish on days 17 and 55. Feeding behaviour (intake and latency) was recorded in the individually housed fish 3 days before, during and 3 days after the stress test. The results showed no effect on feeding behaviour in fish receiving the water from stressed fish. These results could be a consequence of insufficient cortisol/alarm cues' release by the stressed fish into the water or inactivity of such substances, either due to a trapping effect of humic acids or due to degradation in the nitrification and denitrification processes. Future research is needed to clarify how these processes may affect the water concentration of cortisol and alarm pheromones and should be extended by measuring other behavioural and physiological traits

Individual variation in stress physiology and behaviour has been previously reported in several fish species. As seen in other vertebrates, existence of stress coping styles seems to be reflected by the presence of individual variation. Aggressive behaviour, amongst others, is one of the most commonly used parameters to characterize coping styles. However, not all fish species exhibit aggressive behaviour, such as the flatfish Senegalese sole, Solea senegalensis (Kaup, 1858). Therefore, the goal of this study was to determine the magnitude of individual variation in behavioural parameters other than aggression (feeding motivation and activity during stress) as well as in growth and stress response in Senegalese sole. The relationship between these variables was investigated to determine whether they could be used as indicators of coping styles. Thirty-six juvenile fish (9.9 2.2 g) were individually housed for 73 days. Feeding motivation, measured as the time (in s) taken by each fish to react to feed, was determined on days 10, 17, 24 and 31. Blood samples for plasma cortisol were collected on days 51 and 71 for determination of undisturbed and stress levels, respectively. The stress test consisted of holding each fish individually in a net, outside the water, for 3 min. Duration of escape attempts, i.e. the time taken by each fish to stop struggling (in an attempt to escape) in the net, was quantified. The results showed a pronounced individual variation in both control (CV = 54%) and acute stress (CV = 71%) cortisol levels. Senegalese sole also exhibited high coefficient of variation in the behavioural parameters: 75% in feeding latency and 96% in duration of escape attempts. Growth (RGR = 1.17 0.38) showed to be the parameter with lower variation of only 32% and was not correlated with any of the measured parameters. A significant correlation between undisturbed cortisol levels and duration of escape attempts was found. Undisturbed cortisol levels (8.08 4.36 ng/ml) were negatively correlated with duration of escape attempts (P = 0.009, rs =0.503). Correlations between plasma cortisol levels after stress (398.45 282.67 ng/ml) and the behavioural parameters were not found. The observed individual variation in behaviour and stress physiology as well as their relationship suggests the existence of coping styles in Senegalese sole where proactive individuals exhibit shorter feeding latency, higher duration of escape attempts and lower undisturbed cortisol levels than passive individuals.

The accumulation of substances in Recirculating Aquaculture Systems (RAS) may impair the growth and welfare of fish. To test the severity of contaminants accumulated in RAS, early-life stages of fish were used. Ultrafiltered water from two Recirculating Aquaculture Systems (RAS), one RAS with a high accumulation of substances (water exchange rate 30 L/kg feed/day) and one RAS with a low accumulation of substances (water exchange rate 1500 L/kg feed/day), was used to incubate eggs and rear larvae of common carp Cyprinus carpio . A broad range of read-out parameters was used to determine the effect of accumulation level on the development of the early-life stages; from hatching dynamics to larvae length and dry weight. The water quality (temperature, pH, dissolved O 2 , conductivity, total bicarbonate, ortho-phosphate-P, TAN, NO 2 − –N, NO 3 − –N and minerals) was compared between the 2 treatments. Carp eggs developing in the high-accumulation water had higher mortality percentages (both for eggs and larvae), reduced hatching percentages, delayed hatching dynamics and reduced larvae length and body weight. However, these larvae exhibited fewer deformities than larvae incubated in the low-accumulation water. Furthermore, an accelerated development both of the embryo (appearance of heart beat, pectoral fin bud and tail movement) and yolk-sac larvae (depletion of the yolk sac) was observed in the high-accumulation water. The high-accumulation water had significantly lower pH and higher conductivity, NO 2 − –N, NO 3 − –N and ortho-phosphate-P. Most of the minerals (As, Cu, Mn, Ni, Zn, K, Mg, Na, P and S) including heavy metals, were present at a higher concentration in the high-accumulation water. The influence of these parameters on the embryonic and larval development of fish is discussed. It is suggested that in the high-accumulation water, the concentration of ortho-phosphate-P, nitrate and of the heavy metals arsenic and copper is likely to have impaired the embryonic and larval development and therefore deserves further research as potential growth inhibiting factors in RAS.

It has been suggested that fish cultured in recirculating aquaculture systems (RAS) grow less as compared with fish cultured in flow-through systems due to the accumulation of substances. In the Netherlands, the commercial culture of Nile tilapia Oreochromis niloticus in 300 and 600 MT's systems is done exclusively in RAS operated at water exchange rates as low as 30 L/kg feed/day due to nitrate control by single-sludge denitrification reactors. The use of such nearly closed RAS raises the question whether growth retardation (GR) is present in Nile tilapia. This study is the first to investigate the existence of growth retardation in Nile tilapia by comparing the growth, feeding behaviour and stress response of Nile tilapia cultured in RAS with different levels of substances accumulated. Three RAS, operated at 30 L/kg feed/day (HIGH accumulation), 70 L/kg feed/day (MIDDLE accumulation) and 1500 L/kg feed/day (LOW accumulation) were used. Each RAS contained 24 glass aquaria with individually housed fish. To determine whether GR is size-dependent, per RAS 3 fish size categories were tested in the 57 day experimental period: large (288.7 ± 34.2 g; N = 8), medium (162.4 ± 23.4 g; N = 8) and small (81.4 ± 21.0 g; N = 8). Experimental fish were fed ad libitum , twice per day. Feeding behaviour was determined once per week and was measured as the time taken by each fish to eat the first pellet (latency, LAT) and the total time spent feeding (total feeding time, TFT). Temperature, pH, conductivity, alkalinity, dissolved oxygen, dissolved CO 2 , nitrogen compounds (TAN, NO 2 -N and NO 3 -N), chemical oxygen demand and orthophosphate-P were measured over time. At day 57 fish were weighed, blood sampled and returned to their tanks for an extra experimental period of 15 days, and subjected at day 72 to an acute stress followed by blood sampling. Blood was analysed for glucose and cortisol. Results showed that the water quality parameters measured in the 3 RAS (with the exception of alkalinity) were still within the optimum range for growth of Nile tilapia. Large individuals showed a tendency to grow more in the LOW treatment (2.66 ± 1.35 g/kg 0.8 /day) as compared with the MIDDLE treatment (0.93 ± 1.63 g/kg 0.8 /day). On the contrary, small individuals grew significantly less in the LOW treatment (3.60 ± 1.74 g/kg 0.8 /day) as compared with the HIGH (7.22 ± 1.58 g/kg 0.8 /day) and MIDDLE (6.82 ± 4.54 g/kg 0.8 /day) treatments. Small fish were more motivated to eat (lower latency) in the MIDDLE (4.63 ± 5.24 min) as compared with the LOW treatment (8.94 ± 6.41 min). In the HIGH accumulation treatment higher glucose levels were observed in the small fish, before and after acute stress, as compared with the LOW accumulation treatment. In conclusion, this study showed that the extent to which the accumulation of substances in RAS affects growth depends on fish size: large individuals show a trend towards growth retardation in the highest accumulation RAS while small individuals, on the contrary, seem to grow better in such systems.

Individual differences in maintenance levels have been related with energetically expensive processes such as aggression. This relationship is not fully understood as on one hand individuals with higher maintenance requirements seem to be more aggressive but on the other hand have smaller metabolic scopes and therefore less scope for activity. In this study the relationship between individual differences in maintenance requirements and aggression of African catfish is investigated using the residual feed intake as an approximation of maintenance requirements. Residual feed intake, defined as the difference between actual feed intake and that predicted from mean observed requirements for growth and maintenance was determined in individually housed African catfish. Afterwards, the aggressive behaviour of each individual was tested using pairwise contests. The results suggest that individuals with lower RFI (i.e. more efficient) are more aggressive. The most metabolically efficient individuals may have advantage during short and intense aggressive encounter, such as the one used in this study. Being more efficient in terms of nutrient use may therefore allow a faster but shorter term metabolic response in order to fulfil the energetic demands imposed by aggression.

One of the challenges that Recirculating Aquaculture Systems (RAS) are still facing is the risk that in RAS fish grow less than in flow-through systems due to the accumulation of substances originating from feed, fish or bacteria associated with the water re-use. The present study investigated whether RAS with high and low accumulation levels of these substances affect feed intake and growth of Nile tilapia Oreochromis niloticus, African catfish Clarias gariepinus, and European eel Anguilla Anguilla. One-hundred and twenty individuals of each species were used (start body weights: Nile tilapia 264.8 ± 8.3 g; African catfish 253.2 ± 2.1 g and European eel 66.6 ± 1.3 g). For a period of 39 days, growth and feed intake were compared between high and low accumulation RAS. HIGH accumulation RAS was designed for maximal accumulation of substances in the water by operating the system at nearly-closed conditions (30 L/kg feed/d), using mature biofilters and high feed loads; and (2) LOW accumulation RAS was designed to be a proxy for flow-through systems by operating at high water exchange rates (1500 L/kg feed/d), new biofilters and low feed load. HIGH accumulation RAS induced a reduction in feed intake (42%) and growth (83%) of Nile tilapia, as compared to systems that are a proxy for flow-through conditions. This effect was not observed in European eel and African catfish. The cause of this reduced feed intake and growth rate of Nile tilapia is still unclear and should be addressed in further studies.

This study shows that in African catfish Clarias gariepinus feed intake (IF) is stimulated by the presence of conspecifics resulting in higher IF and growth rate. The feeding duration and the frequency of feeding acts were lower in isolated fish as compared with non-isolated fish, indicating a decreased feeding motivation when fish are held individually. Isolation per se, however, seems not to act as a stressor in the short-term or to affect the stress response, probably because periods of isolation are part of the African catfish lifestyle

Within fish farming grading, i.e. the process of sorting to approximate size, changes the group composition from heterogeneous to homogeneous. Although this procedure is considered an acute stressor, the long-term consequences of grading on aggression and stress levels have not yet been investigated. The objective of this study was to quantify the consequences for welfare of rearing African catfish ( Clarias gariepinus ) in groups of homogeneous low-, middle- and heavy-weight fish as compared with a heterogeneous group. Two thousand fish were grown for 8 weeks. Afterwards, fish were individually weighed, tagged and manually selected. Four treatments were established according to weight (mean ± S.D.): homogeneous low- (L, 83.0 ± 8.2 g), homogeneous middle- (M, 140.2 ± 8.7 g), homogeneous heavy-weight (H, 198.0 ± 8.1 g) and a heterogeneous group (HET, 139.7 ± 48.4 g). Fish were sampled three times (days 13, 27 and 34) to determine aggression levels (number of skin lesions and percentage of fish bitten). Blood samples (for cortisol and glucose analysis) were taken at the end of the experiment before and after a stress test (netting). Despite all treatments exhibited high aggression levels (>50%), the H treatment exhibited the lowest aggression levels (58.34 ± 0.92% of fish bitten as compared with 74.13 ± 2.52% in L; 69.70 ± 4.38% in M and 69.94 ± 3.29% in HET, P = 0.002). Aggression significantly decreased over time in the M and H treatment but not in the L and HET treatments. In addition, heavy fish reared in heterogeneous groups exhibited higher number of skin lesions in the body (2.0 ± 0.1) than fish with similar weight reared in homogeneous groups (1.4 ± 0.1) ( P = 0.002). Also, middle-weight fish showed lower cortisol values (before the stress test) in the homogeneous group (45.7 ± 7.5 ng/ml) as compared with middle-weight fish in heterogeneous groups (73.5 ± 13.3 ng/ml). The stress response in low-weight fish was also affected by the group composition. After a stress test, the glucose levels were higher for low-weight fish reared in homogeneous (7.1 ± 0.4 mmol/l) than in heterogeneous (5.1 ± 0.7 mmol/l) groups. This study suggested that the group composition affects welfare in African catfish, particularly in the L treatment where aggression levels were as high as 74%. Middle-weight and heavy fish seemed to benefit from living in homogeneous while light fish from heterogeneous groups.

Understanding the major causes of growth variation is crucial for the success of fish farming since its reduction contributes to maximize production efficiency, reduce food waste and improve water quality. The growth variation observed in aquaculture has been associated with the establishment of social hierarchies. However, some studies suggest that this variation may not be mainly a consequence of social hierarchies but mainly a result of inherent (genetic) differences. This study investigates the magnitude of individual responses, independently of group effects (fish housed individually), in growth efficiency and feeding behaviour of African catfish Clarias gariepinus (Burchell 1822). Despite the low variation in initial body weight (6.5%) and cumulative feed consumption (7.5%) over the experimental period, catfish exhibited high variation in final body weight (18.1%), specific growth rate (17.2%) and feed conversion ratio (27.9%), suggesting that individual variation in growth efficiency is important in determining growth rate. This individual variation may be related with individual differences in protein/fat deposition since faster growing fish deposited more protein and less fat than slower growing fish. Pronounced individual differences in feeding behaviour (reaction towards feed and time spent eating) were also observed and correlated with individual differences in growth efficiency. Fast eaters were the fast growers. We suggest that the growth variation observed in African catfish may be inherent and that the use of grading to increase uniformity should be further investigated

Individual variation in growth, feed intake and feeding behaviour has been previously recognized in several fish species. However, there is a lack of information regarding the consistency of such individual differences, which is important to understand the probability of a certain individual trait to be inherent, i.e. genetically linked. The goal of this study is to quantify the consistency of individual differences in growth, feed intake/efficiency and feeding behaviour in African catfish Clarias gariepinus (Burchell 1822). Forty-eight juvenile fish (58.9±0.4 g) were housed individually for 47 days and weighed every second week. The consistency of growth, feed efficiency (residual feed intake (RFI)) and feeding behaviour (total feeding time (TFT)) was determined using repeatability estimates. Fish exhibited pronounced individual variation in growth (CV=52.8%), feed intake (34.3%) and in the TFT (>100%). The repeatability estimates were 0.55 for growth, 0.70 for feed intake, 0.49 for RFI and 0.81 for TFT. Individual differences in growth were mainly explained by individual differences in feed intake (80%). Individual differences in feeding behaviour contributed to explain differences in feed intake by affecting the RFI. With increasing TFT, the maintenance levels also increased suggesting that slow eaters were less efficient in feed/energy utilization. The results of this study indicate that individual differences in growth, feed intake/efficiency and feeding behaviour are consistent over time and therefore probably inherent. Moreover, this study may have implications on the use of feeding behaviour as a predictor of feed efficiency in juveniles of African catfish.

Little information is available on steroid concentrations in the rearing water of aquaculture systemsand whether they accumulate in recirculating aquaculture systems (RAS). Therefore this study aimedat determining (1) the concentrations and variation of cortisol and sex steroids in RAS, (2) the con-tribution of fish rearing conditions to steroid concentrations in seven commercial RAS. Each RAS wassampled twice at three different points: (1) make-up water; (2) influent and (3) effluent of the rear-ing unit. The results showed significant higher steroid concentrations in the influent and effluent whencompared with the make-up water. On average cortisol concentration was 15.7% higher in the efflu-ent when compared with the influent. Mean steroid concentrations in the rearing unit effluent variedbetween: 3.8–217.0 ng/L for cortisol, 3–12.5 ng/L for testosterone, 0.9–7.1 ng/L for 11-ketoteststerone and1.8–12.8 ng/L for 17,20 -dihydroxypregn-4-en-3-one. Stocking density, Total Ammonia-Nitrogen con-centration and orthophosphate-P concentration (a measure of make-up water usage) showed a positivecorrelation with sex steroids in the water. The steroid concentrations from the present study were ordersof magnitude lower than initial estimations indicating a water treatment efficiency of >99%. The resultssuggest that an intensification of fish production through decrease of make-up water use and increaseof stocking density will lead to a build-up of steroids in the water. Although intensification is critical forthe economical success of RAS, this ultimately could affect fish performance as steroids accumulates inthe water of RAS at levels that can potentially be detected by some fish species. Institution FCT – Fundação para a Ciência e Tecnologia

Individual variation in the response to environmental challenges depends partly on innate reaction norms, partly on experience-based cognitive/emotional evaluations that individuals make of the situation. The goal of this study was to investigate whether pre-existing differences in behaviour predict the outcome of such assessment of environmental cues, using a conditioned place preference/avoidance (CPP/CPA) paradigm. A comparative vertebrate model (European sea bass, Dicentrarchus labrax) was used, and ninety juvenile individuals were initially screened for behavioural reactivity using a net restraining test. Thereafter each individual was tested in a choice tank using net chasing as aversive stimulus or exposure to familiar conspecifics as appetitive stimulus in the preferred or non preferred side respectively (called hereafter stimulation side). Locomotor behaviour (i.e. time spent, distance travelled and swimming speed in each tank side) of each individual was recorded and analysed with video software. The results showed that fish which were previously exposed to appetitive stimulus increased significantly the time spent on the stimulation side, while aversive stimulus led to a strong decrease in time spent on the stimulation side. Moreover, this study showed clearly that proactive fish were characterised by a stronger preference for the social stimulus and when placed in a putative aversive environment showed a lower physiological stress responses than reactive fish. In conclusion, this study showed for the first time in sea bass, that the CPP/CPA paradigm can be used to assess the valence (positive vs. negative) that fish attribute to different stimuli and that individual behavioural traits is predictive of how stimuli are perceived and thus of the magnitude of preference or avoidance behaviour. European Commission [265957]; Portuguese Fundacao para a Ciencia e Tecnologia (FCT) [FRH/BPD/72952/2010]; FCT [SFRH/BD/80029/2011]

Individual differences in exploratory behaviour have been shown to be consistent across contexts and suggested to be part of behavioural syndromes in a diversity of species, including fish. Exploration has also been shown to be a key factor in understanding complex ecological processes such as sexual selection and cooperation. Another important question in ecology is why animals breed in colonies. Exploration syndromes, by affecting prospecting behaviour, dispersal and public information use may also contribute to our understanding of coloniality. This study aims at investigating whether an exploration syndrome exists in a colonial fish species, Neolamprologus caudopunctatus. Individuals of this species were subjected to two consecutive tests, a novel environment and a novel object test. Results show that more explorative individuals in a novel environment are also less neophobic in the presence of a novel object, suggesting that the tendency to engage with novelty per se is a consistent trait and part of an exploration syndrome. These results are discussed in light of the contribution of an exploration syndrome to explain colony formation in animals.

Little research has been done in optimizing the nitrogenous fraction of the fish diets in order to minimize welfare problems. The purpose of this review is to give an overview on how amino acid (AA) metabolism may be affected when fish are under stress and the possible effects on fish welfare when sub-optimal dietary nitrogen formulations are used to feed fish. In addition, it intends to evaluate the current possibilities, and future prospects, of using improved dietary nitrogen formulations to help fish coping with predictable stressful periods. Both metabolomic and genomic evidence show that stressful husbandry conditions affect AA metabolism in fish and may bring an increase in the requirement of indispensable AA. Supplementation in arginine and leucine, but also eventually in lysine, methionine, threonine and glutamine, may have an important role in enhancing the innate immune system. Tryptophan, as precursor for serotonin, modulates aggressive behaviour and feed intake in fish. Bioactive peptides may bring important advances in immunocompetence, disease control and other aspects of welfare of cultured fish. Fishmeal replacement may reduce immune competence, and the full nutritional potential of plant-protein ingredients is attained only after the removal or inactivation of some antinutritional factors. This review shows that AA metabolism is affected when fish are under stress, and this together with sub-optimal dietary nitrogen formulations may affect fish welfare. Furthermore, improved dietary nitrogen formulations may help fish coping with predictable stressful events.

Consistent individual differences in cognitive appraisal and emotional reactivity, including fearfulness, are important personality traits in humans, non-human mammals, and birds. Comparative studies on teleost fishes support the existence of coping styles and behavioral syndromes also in poikilothermic animals. The functionalist approach to emotions hold that emotions have evolved to ensure appropriate behavioral responses to dangerous or rewarding stimuli. Little information is however available on how evolutionary widespread these putative links between personality and the expression of emotional or affective states such as fear are. Here we disclose that individual variation in coping style predicts fear responses in Nile tilapia Oreochromis niloticus, using the principle of avoidance learning. Fish previously screened for coping style were given the possibility to escape a signalled aversive stimulus. Fearful individuals showed a range of typically reactive traits such as slow recovery of feed intake in a novel environment, neophobia, and high post-stress cortisol levels. Hence, emotional reactivity and appraisal would appear to be an essential component of animal personality in species distributed throughout the vertebrate subphylum. Foundation for Science and Technology, Portugal [SFRH/BPD/42015/2007, SFRH/BD/38697/2007, SFRH/BD/44103/2008] info:eu-repo/semantics/publishedVersion

Feed intake is commonly used as one of the most important performance indicators in fish. However, very little is known about the behavioural processes involved in ingesting food such as meal duration, feeding frequency and latency to start eating. This study aims at the characterization of feeding behaviour in Nile tilapia Oreochromis niloticus in terms of consistency of individual differences over time and correlation with performance traits. Seventy-two individuals were individually housed for 27 days. These fish had three different weight (age) categories: low (initial body weight: 61.32 ± 5.97 g), middle (151.19 ± 4.26 g) and high (265.90 ± 19.13 g), each containing 24 fish. Daily feed intake was recorded per fish. On days 8, 15 and 21 feeding behaviour was assessed by measuring the latency to start feeding (LAT, min), the total feeding time (TFT, min) and the number of feeding acts (NFA). In the end of the experimental period, fish were weighed and individual growth rate and feed efficiency (measured as residual feed intake) determined. Results show that feeding latency and the number of feeding acts have significant repeatability estimates for all weight classes. These estimates were, however, low in magnitude and decreased as the weight class increased. The correlation between the different feeding behaviours showed that individuals that are more active during feeding (higher NFA) on average, start to consume their feed earlier (lower LAT) and for longer periods (higher TFT). The number of feeding acts was significantly correlated with both feed intake and growth in all size classes. The residual feed intake was correlated with feeding latency only in the small weight class. These results showed that individual differences in feeding behaviour have low repeatability estimates and can partly explain differences in performance traits in juveniles of Nile tilapia.

Previous reviews of stress, and the stress hormone cortisol, in fish have focussed on physiology, due to interest in impacts on aquaculture production. Here, we discuss cortisol in relation to fish welfare. Cortisol is a readily measured component of the primary (neuroendocrine) stress response and is relevant to fish welfare as it affects physiological and brain functions and modifies behaviour. However, we argue that cortisol has little value if welfare is viewed purely from a functional (or behavioural) perspective—the cortisol response itself is a natural, adaptive response and is not predictive of coping as downstream impacts on function and behaviour are dose-, time- and context-dependent and not predictable. Nevertheless, we argue that welfare should be considered in terms of mental health and feelings, and that stress in relation to welfare should be viewed as psychological, rather than physiological. We contend that cortisol can be used (with caution) as a tractable indicator of how fish perceive (and feel about) their environment, psychological stress and feelings in fish. Cortisol responses are directly triggered by the brain and fish studies do indicate cortisol responses to psychological stressors, i.e., those with no direct physicochemical action. We discuss the practicalities of using cortisol to ask the fish themselves how they feel about husbandry practices and the culture environment. Single time point measurements of cortisol are of little value in assessing the stress level of fish as studies need to account for diurnal and seasonal variations, and environmental and genetic factors. Areas in need of greater clarity for the use of cortisol as an indicator of fish feelings are the separation of (physiological) stress from (psychological) distress, the separation of chronic stress from acclimation, and the interactions between feelings, cortisol, mood and behaviour.

Despite the importance of feed efficiency and stress response in fish production, the number of studies focusing on the relationship of their individual differences has never been investigated. This study tests whether individual differences in glucose and cortisol responsiveness after an acute stressor can explain differences in feed efficiency of African catfish Clarias gariepinus. Experimental fish were juveniles of African catfish, housed individually. Fish were fed until apparent satiation with a commercial diet once per day. Individual feed consumptions were registered and the residual feed intake used as a measure of feed efficiency. Blood samples for plasma cortisol, glucose and lactate were taken at the start of the experiment (control, indicative of basal levels) and after a stress test (netting) at the end of the experiment. There was a pronounced individual variation in both basal and post-stressor levels. Basal levels of glucose, lactate and cortisol did not contribute significantly to explain differences in feed efficiency. However, glucose levels obtained after the stress test could explain differences in feed efficiency by 12% in experiment 1 and 32% in experiment 2. In experiment 2, the cortisol levels obtained after the stress test also contributed to explain differences in feed efficiency by 17%. These results suggest that high glucose- and cortisol-responders are less efficient fish, suggesting that individual differences mainly in glucose responsiveness explain part of the differences in feed efficiency by explaining variance in maintenance levels.

The goal of this study is to evaluate the effect of size distribution on growth performance and feeding behaviour in juveniles of African catfish. Two thousand sibling fish were grown for 8 weeks until the start of the experiment. Afterwards fish were individually weighed, manually selected and tagged. Four treatments were established according to similarity in weight ( n = 108 per treatment, mean ± S.D.): homogeneous low-weight (L, 83.0 ± 8.2 g), homogeneous medium-weight (M, 140.2 ± 8.7 g), homogeneous heavy-weight (H, 198.0 ± 8.1 g) and a heterogeneous group (HET, 139.7 ± 48.4 g). During 27 experimental days, fish were fed ad libitum twice per day. Fish behaviour (resting and swimming activity, waiting-in-feeding area, total feeding time, TFT and feeding rate, FR) was studied using video cameras and by direct observation on days 2, 4, 8, 11, 15, 18 and 26. Growth rate, feed intake and feed conversion ratio did not differ significantly between the treatments. There were also no significant differences between fish of similar weight reared in homogeneous and heterogeneous groups. The H treatment had significantly higher swimming activity (82.8 ± 6.4%) and waiting-in-feeding-area behaviour (51.5 ± 11.2%) than the L treatment (70.3 ± 9.3% and 26.9 ± 5.7%, respectively). The TFT (min) and FR (g min − 1 ) were lower in L (3.2 ± 0.2 min and 23.8 ± 1.05 g min − 1 ) than in M (4.7 ± 0.1 min and 29.4 ± 0.82 g min − 1 ) and H (5.5 ± 0.1 min and 29.7 ± 2.3 g min − 1 ) treatments. Overall, this study indicates that size distribution does not affect growth performance. Low-weight fish do not exhibit increased growth rates in the absence of heavier fish. The use of grading in juveniles of African catfish should therefore be re-considered. The differences in weight observed in this study seem not to be a direct consequence of social hierarchies where the larger fish suppress the growth of smaller fish. Instead, feeding behaviour seems to be an important factor, with heavier fish exhibiting feeding behaviours that may give advantage when feed is limited.

The interest in animal personality, broadly defined as consistency of individual behavioural traits over time and across contexts, has increased dramatically over the last years. Individual differences in behaviour are no longer recognised as noise around a mean but rather as adaptive variation and thus, essentially, raw material for evolution. Animal personality has been considered evolutionary conserved and has been shown to be present in all vertebrates including fish. Despite the importance of evolutionary and comparative aspects in this field, few studies have actually documented consistency across situations in fish. In addition, most studies are done with individually housed fish which may pose additional challenges when interpreting data from social species. Here, we investigate, for the first time in fish, whether individual differences in behavioural responses to a variety of challenges are consistent over time and across contexts using both individual and grouped-based tests. Twenty-four juveniles of Gilthead seabream Sparus aurata were subjected to three individual-based tests: feed intake recovery in a novel environment, novel object and restraining and to two group-based tests: risk-taking and hypoxia. Each test was repeated twice to assess consistency of behavioural responses over time. Risk taking and escape behaviours during restraining were shown to be significantly consistent over time. In addition, consistency across contexts was also observed: individuals that took longer to recover feed intake after transfer into a novel environment exhibited higher escape attempts during a restraining test and escaped faster from hypoxia conditions. These results highlight the possibility to predict behaviour in groups from individual personality traits. European Commission [265957 COPEWELL]; European Social Fund of Andalusia; Foundation for Science and Technology, Portugal [SFRH/BPD/77210/2011] info:eu-repo/semantics/publishedVersion