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In this study, we described immediate/acute reactions to stress-related chemical cues (SC - Stress Cue) in fish. Specifically, we evaluated the effects of SC on ventilation rate (VR) and feeding behavior in Nile tilapia ( Oreochromis niloticus ), a highly relevant species for world aquaculture, therefore, to understand the diversity of stressful contexts and stress responses in this species have important practical applications (stress reduction). Stress cue was obtained from conspecifics exposed to a handling stressor. Stress was confirmed by measuring plasma cortisol levels. The responses to SC were contrasted to chemical control cues: a cue originated from non-stressed conspecifics and pure water (vehicle control). We observed that Nile tilapia exposed to SC had an increase in VR, but without effects on feeding behavior (feeding latency and ingestion). Thus, the SC is a stressor that induces readily stress response (VR increase), suggesting sympathetic activation, but did not change feeding performance. In practical terms, it is positive because although social propagation of stress via SC elicits a stress response, it did not harm appetite.

DNA methylation clocks provide information not only about chronological but also biological age, offering a high-resolution and precise understanding of age-related pathology and physiology. Attempts based on transcriptomic and epigenetic approaches arise as integrative biomarkers linking the quantification of stress responses with specific fitness traits and may help identify biological age markers, which are also considered welfare indicators. In gilthead sea bream, targeted gene expression and DNA methylation analyses in white skeletal muscle proved sirt1 as a reliable marker of age-mediated changes in energy metabolism. To complete the list of welfare auditing biomarkers, wide analyses of gene expression and DNA methylation in one- and three-year-old fish were combined. After discriminant analysis, 668 differentially expressed transcripts were matched with those containing differentially methylated (DM) regions (14,366), and 172 were overlapping. Through enrichment analyses and selection, two sets of genes were retained: 33 showing an opposite trend for DNA methylation and expression, and 57 down-regulated and hypo-methylated. The first set displayed an apparently more reproducible and reliable pattern and 10 multifunctional genes with DM CpG in regulatory regions ( sirt1 , smad1 , ramp1 , psmd2 -up-regulated; col5a1 , calcrl , bmp1 , thrb , spred2 , atp1a2 -down-regulated) were deemed candidate biological age markers for improved welfare auditing in gilthead sea bream.

Aquafeed's contamination by microplastics can pose a risk to fish health and quality since they can be absorbed by the gastrointestinal tract and translocate to different tissues. The liver acts as a retaining organ with the consequent triggering of oxidative stress response. The present study aimed to combine the use of natural astaxanthin with natural-based microcapsules to counteract these negative side effects. European seabass juveniles were fed diets containing commercially available fluorescent microplastic microbeads (1-5 μm; 50 mg/kg feed) alone or combined with microencapsulated astaxanthin (AX) (7 g/kg feed; tested for half or whole feeding trial-30 or 60 days, respectively). Fish from the different dietary treatments did not evidence variations in survival and growth performance and did not show pathological alterations at the intestinal level. However, the microplastics were absorbed at the intestinal level with a consequent translocation to the liver, leading, when provided solely, to sod1 , sod2 , and cat upregulation. Interestingly, the dietary implementation of microencapsulated AX led to a mitigation of oxidative stress. In addition, the microcapsules, due to their composition, promoted microplastic coagulation in the fish gut, limiting their absorption and accumulation in all the tissues analyzed. These results were supported by in vitro tests, which demonstrated that the microcapsules promoted microplastic coagula formation too large to be absorbed at the intestinal level and by the fact that the coagulated microplastics were released through the fish feces.

The principles of three Rs-REPLACEMENT, REDUCTION, and REFINEMENT-govern the protection and use of animals, including fish, for research purposes in the European Union and Norway. In this paper, we discuss some straightforward steps to simplify the delivery of these principles at the idea stage and adapt some of these examples for conducting fish trials related to health and welfare. Although some of the approaches are well established in other animal science arenas, we believe there can be a timely recap of their key facets. We discuss a number of simple strategies to emphasize how a reduction in fish numbers can be achieved from initial project conception to implementation, highlighting not only their advantages but also their limitations. We also highlight the role that funding agencies can play in the implementation of the 3R principles in aquaculture research. These simple points can be used in frameworks to initiate a broader and dynamic intersectoral dialogue among stakeholders of aquaculture research on how to promote ethics and embrace opportunities for this within the tenets of the 3Rs., (© 2024 The Authors. Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles.)

The aquaculture industry frequently employs various slaughter methods that subject fish to inhumane conditions, resulting in significant suffering. This study examined the effects of electrical method on the welfare of farmed fish ( Mesopotamichthys sharpeyi ) compared to air asphyxiation. Fish captured with electricity exhibited calm behavior until death, in contrast to fish treated with air asphyxiation, which exhibited violent responses within 4 minutes of capture. The electrical method achieved a complete state of unconsciousness 7 minutes faster than air asphyxiation. Our results show that air asphyxiation raised cortisol levels more than the electrical method, with no significant difference in glucose and lactate concentrations. Electrically treated fish had higher superoxide dismutase and liver-reduced glutathione levels, while suffocated fish showed elevated GSH and liver catalase levels. Radiographs revealed no fractures or skeletal changes. Electrical stunning had no effect on gill tissue, but caused brain tissue hemorrhage, whereas air asphyxiation caused less damage. Air asphyxiation caused gill tissue issues but less brain damage. Consciousness loss is crucial for humane practices. Specific electrical currents (110V for 30s) could improve aquaculture and fish welfare.

Environmental enrichment has the potential to improve the welfare and post-release survival of hatchery fish stocked for conservation purposes. However, the effectiveness of environmental enrichment is partly dependent on the fish species, life stage, and specific enrichment structure used. To enhance the effectiveness of environmental enrichment, it is crucial to focus on characteristic differences in enrichment structures, such as type and level. This study investigated how differences in enrichment type and level affected physiological and behavioral aspects of the welfare of pre-release juvenile rock bream Oplegnathus fasciatus by evaluating growth performance, basal and stressed cortisol levels, antioxidant enzyme activities, and exploratory behaviors regarding anxiety and flexibility. Fish were reared for 4 weeks in different enrichment treatments: barren, low-level cover structure, high-level cover structure, low-level interference structure (LI), and high-level interference structure (HI). The results revealed that fish reared with the LI treatment showed less anxiety and greater flexibility with respect to exploratory behaviors, without oxidative damage being detected. Despite exhibiting less anxiety as well, fish reared in the HI treatment had oxidative damage, indicated by lower superoxide dismutase activity, compared to those in the barren treatment. In addition, none of these enrichment structures enhanced growth performance or mitigate chronic and acute stress responses. Overall, the low-level interference structure may be more favorable in promoting the behavioral welfare of the fish. Application of this type and level of enrichment may increase the survival of the hatchery fish after release, which is critical to stocking success., (© 2023 Fisheries Society of the British Isles.)

Finfishes are caught from the wild for food, feed (often in the form of fishmeal and oil) and bait. According to the Food and Agriculture Organisation of the United Nations (FAO), between 74 and 83 million tonnes (averaging 77 million tonnes) were caught annually in 2000-2019. Although fishes are now widely recognised as sentient beings, capture is still quantified as biomass rather than number of individuals (in contrast to wild-caught marine mammals and crocodiles; and farmed mammals and birds). Here, we estimate global numbers of wild-caught finfishes using FAO capture production (landing) tonnages (2000-2019 data) and estimates of mean individual weight at capture, based on internet-sourced capture and market weights. We estimate that between 1,100 and 2,200 billion (1.1-2.2 × 10 12 ), or 1.1-2.2 trillion, wild finfishes were caught annually, on average, during 2000-2019. Anchoveta ( Engraulis ringens ) comprised 28%, by estimate midpoint. Estimated numbers in 2019, totalling 980-1,900 billion, were lower due to reduced anchoveta landings, but still represented 87.5% of vertebrate numbers killed for food or feed, as obtained or estimated from FAO data. These figures exclude unrecorded capture such as illegal fishing, discards and ghost fishing. Estimated finfish numbers used for reduction to fishmeal and oil represented 56% of the total 2010 estimate (1,000-1,900 billion), by midpoint. It is recommended that the FAO reports fish capture numbers. The welfare of wild-caught fishes, which is generally very poor during and after capture, should be addressed as part of sustainable utilisation of aquatic resources., Competing Interests: None., (© The Author(s) 2024.)

Ballan wrasse ( Labrus bergylta ) are commonly used as cleaner fish in salmon farms as a biological treatment to mitigate sea lice infestation. Improved welfare for cleaner fish both during production of these fish and when in sea-cages with salmon is crucial for the industry's development. A common operational procedure in ballan wrasse production is transporting juveniles from one land-based farm to another for further on-growing. Episodes of increased mortality have been reported after such transportations. In this study, the relationship between transport stress and post-transport mortality at the on-growing facility was examined. It was also investigated if light sedation with AQUI-S ® can mitigate stress during transport. Stress was quantified by measuring cortisol release rate to the tank water during transport. This was investigated in 10 commercial live carrier truck transports (6 without AQUI-S ® sedation and 4 with sedation during loading and transport). The total time of transport varied between 12 and 21 h. In general, mortality was significantly higher (1.0 ± 0.6% day -1 ) the first five days post-transport compared to 15-20 days post transport (0.5% day -1 ). There was also a strong relationship between fish weight at transport and post-transport mortality, where higher mean weight at transport reduced mortality. In contrast to what was expected, AQUI-S ® treatment during transport procedures increased cortisol excretion rate, suggesting a stimulating effect of AQUI-S ® on the stress axis in ballan wrasse. Considering these results, the value of using AQUI-S ® to reduce stress during transport of juvenile ballan wrasse might be questioned. However, there was no relationship between cortisol release rate during transport and post-transport mortality. Furthermore, this study emphasizes that water cortisol measurements can be used as a none-invasive tool for monitoring stress and can be integrated into the welfare evaluation during commercial fish transports., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Calabrese, Jonassen, Steigum, Åsnes, Imsland, Saude, Wergeland and Höglund.)

The liver health of Gilthead sea bream and European sea bass, fed with fish meal-free diets, including various proportions of plant proteins, as well as insect and poultry by-product meals, was investigated through biochemical and histological analyses using a new liver index (LI) formula. Four isoproteic (45% Dry Matter, DM) and isolipidic (20% DM) diets were compared, including a plant-based control diet (CV) and three other test diets, in which 40% of a plant protein-rich ingredient mixture was replaced with meals from Hermetia illucens (H40) or poultry by-product (P40) alone, or in combination (H10P30). The trials lasted 12 and 18 weeks for sea bream and sea bass, respectively. The results obtained thus far highlighted species-specific differences in the physiological response to dietary changes. In sea bream, the biochemical and histological responses suggest favorable physiological and liver health statuses, with higher serum cholesterol (CHO) and triglyceride (TAG) levels, as well as moderate hepatocyte lipid accumulation, with the H10P30 diet compared to the CV ( p < 0.05). In sea bass, all diets resulted in elevated serum TAG levels and lipid accumulation in the liver, particularly in fish fed the P40 one ( p < 0.05), which resulted in the highest LI, coupled with a higher frequency of severe lipid accumulation, hypertrophy, cord loss, peripheral nuclei displacement, and pyknosis. In conclusion, sea bream adapted well to the test diets, whereas sea bass exhibited altered hepatic lipid metabolism leading to incipient liver steatosis, likely due to the high lipid contents of the diets, including the insect and poultry meals. The LI formula developed in this study proved to be a reliable tool for assessing the effects of dietary changes on the liver health of sea bream and sea bass, consistent with biochemical and histological findings.

While the inclusion of synthetic polymers such as primary microplastics within personal care products have been widely restricted under EU/UK Law, water-soluble polymers (WSPs) have so far slipped the net of global chemical regulation despite evidence that these could be polluting wastewater effluents at concentrations greatly exceeding those of microplastics. Polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP) represent WSPs with common industry and household uses, down-the-drain disposal and a direct route to wastewater treatment plants, conveying high risk of environmental leaching into freshwater ecosystems. The current study is the first investigating the impacts of predicted environmental concentrations of these WSPs on life-history traits of two freshwater species also constituting a disease model (fish - Poecilia reticulata and parasite - Gyrodactylus turnbulli). Single effects of WSPs on fish as well as their interactive effects with infection of the ectoparasite were determined over a 45-day exposure. Generally, WSPs reduced fish growth and increased routine metabolic rate of fish implying a depleted energetic budget, however these effects were dose, exposure time and polymer dependent. Parasitic infection alone caused a significant reduction in fish growth and enhanced fish routine metabolic rate. In contrast, a non-additive effect on metabolic rate was evident in fish experiencing simultaneous infection and WSP exposure, suggesting a protective effect of the two WSPs for fish also exposed to a metazoan ectoparasite. Off-host parasite survival was significantly lowered by both WSPs; however, parasite counts of infected fish also exposed to WSP were not significantly different from the control, implying more complex mechanisms may underpin this stressor interaction. Distinct detrimental impacts were inflicted on both organisms implying environmental leaching of WSPs may be causing significant disruption to interspecies interactions within freshwater ecosystems. Additionally, these results could contribute to sustainable development in industry, as we conclude PVA represents a less harmful alternative to PVP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Cardiomyopathy syndrome (CMS) poses a significant threat to farmed Atlantic salmon ( Salmo salar ), leading to high mortality rates during the seawater phase. Given that controlled experimental challenge trials with PMCV do not reproduce the mortality observed in severe field outbreaks of CMS, field trials on natural CMS outbreaks are warranted. This field study explored the impact of a clinical nutrition intervention, specifically a diet enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on a severe CMS outbreak in a commercial sea farm. CMS was diagnosed in a single sea cage with high mortality rates. Histopathological analysis, RT-qPCR in situ hybridization for virus detection, and fatty acid composition analysis were used to monitor the impact of disease and the inclusion of EPA and DHA in heart tissue. Following the implementation of clinical nutrition, a decline in mortality rates, regression of CMS-associated changes, and a significant reduction in piscine myocarditis virus (PMCV) RNA load were observed within the salmon population. Fatty acid composition analysis of heart samples demonstrated increased levels of EPA and DHA, reinforcing the association between dietary factors, viral load dynamics, and overall fish health. Although further validation is needed in future studies, as field trials may not be sufficient to establish causation, our results indicate that optimizing the EPA + DHA levels may prove beneficial in severe CMS outbreaks.

In aquaculture, the transportation of live fish is a crucial but stress-inducing practice, necessitating a thorough understanding of its impact on fish welfare. This study aimed to assess the physiological stress response of meagre ( Argyrosomus regius ) juveniles during a 24 h commercial transport by quantifying muscle cortisol levels using a specific radioimmunoassay. Additionally, an immunohistochemical approach was used to detect and localize the cellular distribution of oxidative-stress-related biomarkers within various tissues and organs. The results demonstrated a significant increase in muscle cortisol levels following the loading procedure, remaining elevated above basal levels throughout the 24 h transport period. This effect may be attributed to either insufficient time for recovery from the loading stress or prolonged transportation-related stress. Immunostaining for all the antibodies we examined was observed in multiple tissues and organs, but we found no notable variations among the various transport phases. In conclusion, the observed stress response appears to be mainly linked to loading stress and the transport process itself, emphasizing the importance of implementing appropriate operational procedures to safeguard fish well-being during transport. Nonetheless, the unaltered distribution of oxidative stress markers between the control and transported groups suggests that the experienced stress might be within tolerable limits.

The primary aim of this study was to comprehensively evaluate the welfare of Nile tilapia (Oreochromis niloticus) throughout their entire life cycle within aquaculture, spanning from reproduction to slaughter. The methodology was structured to identify welfare indicators closely aligned with the principles of animal freedoms defined by the Farm Animal Council, encompassing environmental, health, nutritional, behavioral, and psychological freedom. Notably, psychological freedom was inherently considered within the behavioral and physical analyses of the animals. To accomplish this, an integrative systematic literature review was conducted to define precise indicators and their corresponding reference values for each stage of tilapia cultivation. These reference values were subsequently categorized using a scoring system that assessed the deviation of each indicator from established ideal (score 1), tolerable (score 2), and critical (score 3) ranges for the welfare of the target species. Subsequently, a laboratory experiment was executed to validate the pre-selected health indicators, specifically tailored for the early life stages of tilapia. This test facilitated an assessment of the applicability of these indicators under operational conditions. Building on the insights gained from this experimentation, partial welfare indices (PWIs) were computed for each assessed freedom, culminating in the derivation of a general welfare index (GWI). Mathematical equations were employed to calculate these indices, offering a quantitative and standardized measure of welfare. This approach equips tilapia farmers and processors with the tools necessary for the continuous monitoring and enhancement of their production systems and stimulate the adoption of more sustainable and ethical practices within the tilapia farming., Competing Interests: AP was employed by Wai Ora Aquaculture and Environmental Technology Ltd. MQ was employed by FAI Farms from Brazil. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pedrazzani, Cozer, Quintiliano, Tavares, Biernaski and Ostrensky.)

The aim of this study was to investigate the physiological response of rainbow trout ( Oncorhynchus mykiss ) before slaughtering in the last phase of farming analyzing skin mucus and plasma. Two groups of rainbow trout were considered: Group UN ("unstressed"), represented by fish randomly captured from raceways, in the last phase of a standard fattening cycle; Group S ("stressed"), collected at the end of the pre-slaughtering tank, soon after slaughtering. The fish skin mucus was swabbed from head to tail using a sterile plastic spatula and the blood was collected through an endocardial puncture. qRT-PCR was used to study the gene expression in skin mucus. The mRNA expression levels of the IL-6 and IgD genes were higher in the S than in the Group UN. The plasma analysis showed an only a decrease in the glucose plasma levels in the Group S when compared to the Group UN. The present results indicated that the procedures adopted after slaughtering only affected changes in plasma glucose and skin mucus activity in rainbow trout suggesting that management protocol was compatible with non-stressful farming conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fiordelmondo, Magi, Friedl, El-Matbouli, Roncarati and Saleh.)

Welfare assessments have risen to prominence in the aquaculture industry, with increasing awareness of their significance among stakeholders in Thailand. In this study, we conducted a welfare assessment of tilapia ( Oreochromis spp.) farms in Thailand, focusing on health, environmental, behavioural, and nutritional indicators. Comparing semi-intensive (earthen ponds) and intensive farming practices (cage culture), we found significant differences in the overall health score, particularly at farm F due to a disease outbreak (Kruskal-Wallis, p = 0.01). Skin and fin scores varied across farms, indicating their potential as indicators of tilapia health. Environmental assessments revealed differences in transparency between the two culturing systems (Mann-Whitney, p = 0.02). During the harvesting process, tilapia behaviours indicated poor welfare across all farms. However, no statistically significant difference in overall welfare scores was found between the two culturing systems. Correlations were observed between nutritional, environmental, and health indicators, with negative correlations between fish density and water transparency ( r = -0.87, p = 0.02), presence of inhabitants ( r = -0.78, p = 0.04), feeding behaviours ( r = -0.78, p = 0.04), and swimming behaviours during capture ( r = -0.98, p = 0.001). These findings provide valuable insights to enhance tilapia-farming practices and welfare in Thailand., Competing Interests: The authors declare that there are no financial and non-financial conflicts of interest. The indicators and methodology for assessing fish welfare is already in public domain, as per previous publication, with Ana Silvia being the main author, as part of her Ph.D. Murilo Quintiliano and Pau Badia are FAI employees and contributed for the paper. Ana Silvia is a WAI employee and has also contributed for the paper. The data belong to the corresponding author. The purpose of the project was to evaluate the status of tilapia welfare in Thailand without any financial support from any private company.

The trade of aquarium organisms is growing worldwide. This market depends on a continuous supply of healthy and colorful aquatic animals, but this sector has few initiatives. However, in the last decade, there has been a growing interest in researching captive breeding of these animals, aiming to develop a more sustainable aquarium hobby. Larviculture is an important phase in the cultivation process because the larvae are more sensitive to stress and variations in the bulk of variables, such as temperature, salinity, nutritional management, light intensity and spectrum, and environmental background colors. Because background color could be a promoter variable of proper welfare, we tested whether it affects the endocrine response of tomato clownfish Amphiprion frenatus larvae to an acute stress challenge. We show that background color influences the endocrine stress axis responsiveness in tomato clownfish. When fish were subjected to a standard acute stressor of 61 days after hatching, only fish adapted to white walls increased the whole-body cortisol levels. From the results presented herein, we recommend that white tanks be avoided for A. frenatus larviculture. Both, the less stress level and the good welfare condition of larvae reared in colored tanks may have robust, practical applications since almost all clownfish in the ornamental aquarium trade come from captive breeding., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

As fish welfare becomes a growing concern, it is important to ensure humane treatment during slaughter. This study aimed to assess the onset of unconsciousness in Atlantic halibut immersed in CO 2 -saturated seawater through electroencephalography (EEG). Of the 29 fish studied, 10 exhibited escape attempts, indicating aversion to CO 2 -saturated water despite its oxygenation. EEG signals showed four distinct phases: transitional, excitation (high amplitude-high frequency), suppressed, and iso-electric phases. The onset of the suppressed phase, indicative of unconsciousness, occurred on average 258.8 ± 46.2 s after immersion. The spectral analysis of the EEG signals showed a progressive decrease in median frequency, spectral edge frequency, and high frequency contribution, which corresponded to the gradual loss of consciousness. The study concludes that CO 2 -saturated water is not recommended for pre-slaughter handling of halibut due to the extended time required for the onset of unconsciousness and the observed aversive behaviour. Ensuring humane treatment during slaughter is important for addressing public concern and safeguarding fish welfare in all stages of production.

Sea lice represent a persistent and growing problem, challenging the resilience and growth of the salmon aquaculture industry. In this Norwegian case study, we studied and discuss how the absence of policy instruments directed at stimulating breeding for lice resistance (LR) might be explained. We found well-documented opportunities for selection progress for LR. Hence, breeding on LR appears with an untapped potential. We discuss how market-based, legal, institutional and interest-based factors can explain the absence of policy instruments stimulating LR breeding. Methodologically, we obtained data from document and literature studies and interviews with key players (salmon breeders, farmers, nongovernmental organizations (NGOs) and governmental bodies in Norway). First, LR is a polygenic trait, which makes it poorly suited for patenting. Furthermore, if only a small proportion of fish farmers choose seeds with higher LR, other operators can easily take on the free-rider role because they will not suffer from reduced gain in growth performance as a result of a much stronger emphasis on LR in the breeding goal. The market is thus not expected to stimulate stronger selection for LR in Norwegian salmon breeding. Second, neither genetic engineering (e.g., gene editing), still struggling with consumer acceptance, nor the uncertainty associated with possible changes in the Norwegian Gene Technology Act stimulate investment in LR via, for example, CRISPR technology. Thirdly, public policy instruments in their entirety have targeted other types of innovations against salmon lice, and none have so far been used to stimulate breeding companies to emphasize LR more strongly in their breeding programmes. From a political point of view, it seems that breeding has been left to the market and the private sector. However, neither the NGOs nor the public seem to be aware of, or pay significant attention to, the breeding potential to improve LR and fish welfare. Fragmented management of the aquaculture sector can camouflage the close ties between political and business interests. The industry is hesitant to invest significantly in long-term breeding targets such as significantly higher genetic LR. This may strengthen the assumption that strong economic interests will reduce the role of science in knowledge-based management. As farmed salmon are increasingly being exposed to stressful delousing treatments, mortality and associated welfare problems have increased significantly. For instance, large fish die more often from cardiomyopathy syndrome (CMS), resulting in growing demand for CMS-resistant salmon. This gives rise to a paradoxical situation: increasing treatments with high mortality and fish welfare issues in farmed salmon, while the lice threat to wild salmon persists., (© 2023 NOFIMA and The Authors. Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles.)

Medicinal plants are powerful antioxidants which can improve well-being and suppress oxidative stress caused by environmental toxins in aquatic animals. In this regard, the present research was designed to show the potential effects of psyllium (Plantago ovata) seed extract (PSE) on the growth, and immune responses of common carp Cyprinus carpio exposed to acute ammonia toxicity. To perform the study, fish were fed with diets containing 0 (T0), 0.25 (T1), 0.5 (T2), and 1% (T3) PSE for 60 days, and then exposed to ammonia (0.5 mg L -1 ) for 3 h. The findings showed that fish given the T1 diet outperformed the T3 and control groups in terms of ultimate weight, weight increase, and food conversion ratio. Additionally, the T1 group showed a significantly higher level of total protein and serum lysozyme activity than the other treatment groups. Moreover, the highest serum total immunoglobulin values were recorded in T1 and T2 groups. The results showed that PSE, especially at moderate levels, could successfully upregulate the transcription of immune-related genes (IFN-γ, Hsp70, TNF-ɑ, IL-1ß, IL-10, and IgE) compared to the control group after exposure to ammonia. Furthermore, improving ammonia-induced down regulations of antioxidant-related gene expressions (CYP1A, SOD, and GPX) was observed in fish fed with PSE-included diets compared to the control one. However, PSE-supplemented diets did not affect the mRNA expression level of CAT. Regarding tight junction-associated genes, the higher mRNA expression level of occludin was observed in the T1 group, whereas the downregulation of CLD3 gene occurred in all experimental groups. Conversely, significant upregulation of osmoregulation-associated gene (NKA) was recorded in all experimental groups compared to the control one. Therefore, the administration of PSE (0.25% of the diet) for 60 days is recommended to increase growth performance, improve health, and increase the resistance of common carp to oxidative stress caused by ammonia., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

In recent years, flathead grey mullets ( Mugil cephalus ) cultured in Eilat (Israel) have been highly affected by Vibrio harveyi , showing neurological signs such as uncoordinated circular swimming followed by high mortality rates. Despite the advances in and different approaches to control vibriosis associated with Vibrio harveyi , including commercial vaccines, most of them have not succeeded in long-term protection. In this study, we evaluated the effectiveness, long-term protection, and antibody production of three vaccine preparations: heat-killed bacteria (HKB), membrane proteins denaturation (BME PROT), and internal proteins (INT PROT) developed specifically against Vibrio harveyi for grey mullets. Our results show that fish immunized with heat-killed bacteria emulsified with adjuvant presented the most effective and long-lasting protection against the bacterium, and a cross-protection against other bacteria from the harveyi clade. The effectiveness of each immunization treatment correlated with the levels of specific antibody production against Vibrio harveyi in the serum of the immunized fish.

Crowded (stressed) and unstressed Atlantic mackerel with or without pre-freezing holding in refrigerated sea water (RSW) were stored at -19 °C for ∼12 months and analysed for nucleotide degradation (K value), muscle pH, water holding capacity (WHC), fillet firmness, cathepsin B/L like activity, lipid oxidation and fillet colour. The frozen storage showed the largest and most consistent direct effects on the quality metrics leading to increased lipid oxidation, discolouration (yellowing) and reduction on WHC and cathepsin activity. RSW treatment promoted nucleotide degradation and reduced WHC and fillet firmness in interaction with frozen storage and affected fillet colour lightness and saturation. Although showing only marginal main effects, crowding stress modified WHC, cathepsin activity and fillet firmness and colour through significant interactions with the frozen storage and RSW treatment. Further studies with larger sample sizes would be needed to elucidate their complex effects and interactions on the quality and storage stability of mackerel., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In a non-linear system, such as a biological system, the change of the output (e.g., behaviour) is not proportional to the change of the input (e.g., exposure to stressors). In addition, biological systems also change over time, i.e., they are dynamic. Non-linear dynamical analyses of biological systems have revealed hidden structures and patterns of behaviour that are not discernible by classical methods. Entropy analyses can quantify their degree of predictability and the directionality of individual interactions, while fractal dimension (FD) analyses can expose patterns of behaviour within apparently random ones. The incorporation of these techniques into the architecture of precision fish farming (PFF) and intelligent aquaculture (IA) is becoming increasingly necessary to understand and predict the evolution of the status of farmed fish. This review summarizes recent works on the application of entropy and FD techniques to selected individual and collective fish behaviours influenced by the number of fish, tagging, pain, preying/feed search, fear/anxiety (and its modulation) and positive emotional contagion (the social contagion of positive emotions). Furthermore, it presents an investigation of collective and individual interactions in shoals, an exposure of the dynamics of inter-individual relationships and hierarchies, and the identification of individuals in groups. While most of the works have been carried out using model species, we believe that they have clear applications in PFF. The review ends by describing some of the major challenges in the field, two of which are, unsurprisingly, the acquisition of high-quality, reliable raw data and the construction of large, reliable databases of non-linear behavioural data for different species and farming conditions.

Microplastics have been found in all surveyed ecosystems and in the diet of multiple species. Detrimental health impacts of microplastic consumption include reduced growth and fecundity, metabolic stress and immune alterations for both invertebrates and vertebrates. Limited information exists, however, on how disease resistance may be affected by microplastic exposure and consumption. Here, the impact of microplastic (0.01 and 0.05 mg l-1 of polypropylene) on fish host susceptibility to disease and mortality was assessed using the guppy Poecilia reticulata-gyrodactylid Gyrodactylus turnbulli system. Fish exposed to and/or consuming microplastic at both concentrations demonstrated significantly higher pathogen burdens over time compared with fish fed a plastic-free diet. Furthermore, microplastic (at both tested concentrations) was associated with increased mortality events for fish within all treatments, regardless of host infection status. This study adds to the growing body of evidence showing that microplastic pollution can be detrimental to fish welfare by reducing disease resistance.

Physical complexity adds physical enrichment to rearing conditions. This enrichment promotes fish welfare and reduces detrimental characteristics that fish develop in captivity. Senegalese sole ( Solea senegalensis ) is an important species for European aquaculture, where it is reared in intensive conditions using fibreglass tanks. However, reproductive dysfunctions present in this species do not allow it to complete its life cycle in captivity. Recently, dominance behaviour has been studied to try to solve this problem. The present study aimed to assess the effect of sand as environmental enrichment in the dominance behaviour and brain mRNA abundance of Senegalese sole juveniles. Four tanks of sole ( n = 48 fish in total) were established in two different environments (with and without sand). Juveniles were subjected to dominance tests of feeding and territoriality. Behaviours analysed by video recordings related to the distance from the food delivered and harassment behaviour towards other individuals (e.g., resting of the head on another individual). In both environments, dominant sole were the first to feed, displayed more head-resting behaviour and dominated the area close to the feeding point, where the events were reduced in fish maintained in the sand. mRNA expression related to differentiation of dopamine neurons ( nr4a2 ) and regulation of maturation ( fshra ) were significantly upregulated in dominant fish in the sand environment compared to dominants maintained without sand. The use of an enriched environment may affect Senegalese sole dominance, enhance welfare and possibly advance future maturation.

Curiosity-the motivation to seek out information-has been studied widely across the animal kingdom. To investigate curiosity in zebrafish we presented 30 novel objects to groups of zebrafish housed in semi-naturalistic tanks (6 tanks; 10 fish/tank; 10-min presentations). During the first 100 s and final 100 s of each object's 10-min presentation period, we recorded each group's: (i) latency to approach the object, (ii) attraction to the object, (iii) social dynamics: agonistic behavior and group cohesion and coordination, and (iv) diving behavior, a stress response in zebrafish. Comparing these behaviors to a 100 s baseline period when no object was present, we tested for neophobia (avoidance of novelty), neophilia (overall attraction to novelty), sustained interest (prolonged attraction to at least some presentations), discriminant interest (certain objects eliciting more attention than others), habituation (loss of interest over time), and alterations to social and stress behaviors. Zebrafish groups readily approached all objects (1 s median latency), were neophilic throughout all object presentations, and showed systematic sustained interest only for some object presentations at the beginning of the study (object presentations 1-10). Over the course of the study, zebrafish also showed signs of habituation such that by the final ten object presentations (21-30), there were no signs of overall sustained interest. During the beginning of the study (object presentations 1-10), we also found evidence for specific object-driven interest, with object ID accounting for 11% of the variability in interest scores ( p < 0.01), and object-driven interest corresponding to alterations in social behavior: decreased aggression ( p < 0.02), increased group cohesion ( p < 0.02), and increased group coordination ( p < 0.05). By explicitly investigating curiosity in fish, this work reveals that under certain conditions, zebrafish voluntarily engage in cognitive stimulation opportunities. More work is needed to clarify what types of information zebrafish find most rewarding and how long-term exposure to such opportunities may affect fish welfare., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Franks, Gaffney, Graham and Weary.)

Global farmed finfish production increased from 9 to 56 million tonnes between 1990 and 2019. Although finfishes are now widely recognised as sentient beings, production is still being quantified as biomass rather than number of individuals (in contrast to farmed mammals and birds). Here, we estimate the global number of farmed finfishes slaughtered using FAO aquaculture production tonnages (1990-2019 data) and estimates of individual weight at killing (determined from internet searches at species and country level where possible). We relate these numbers to knowledge on humane slaughter, animal welfare law, and certification schemes. Since 1990, farmed finfish numbers killed annually for food have increased nine-fold, to 124 billion (1.24 × 10 11 , range 78-171 billion) in 2019. This figure does not represent the total number farmed (due to mortalities during rearing and non-food production) and is expected to increase as aquaculture expands. Our estimates indicate that farmed finfishes now outnumber the 80 billion farmed birds and mammals killed globally each year for food. The majority are produced in Asia. Inhumane slaughter practices cause suffering for most farmed finfishes. Most, 70-72%, have no legal welfare protection, and less than 1% have any fish-specific legal protection, at slaughter. The main global certification schemes in 2013-2015 accounted for 2% of slaughtered farmed finfishes. Fishes for which species-specific parameters for automated humane stunning are published comprise 20-24%. As the dominant taxa of farmed vertebrates, finfishes would benefit from better welfare if species-specific humane slaughter was defined and incorporated into laws and certification schemes., Competing Interests: None., (© The Author(s) 2023.)

Fish health personnel have limited tools in combatting viral diseases such as heart and skeletal muscle inflammation (HSMI) in open net-pen farmed Atlantic salmon. In this study, we aimed to predict HSMI by intensified health monitoring and apply clinical nutrition to mitigate the condition. We followed a commercial cohort (G1) of Atlantic salmon that was PRV-1 naïve when transferred to a sea cage at a location where HSMI outbreaks commonly occur. The fish in the other cages (G2-G6) at the location had a different origin than G1 and were PRV-1 positive prior to sea transfer. By continuous analysis of production data and sequentially (approximately every fourth week) performing autopsy, RT-qPCR (for PRV-1 and selected immune genes), blood and histological analysis of 10 fish from G1 and G2, we identified the time of PRV-1 infection in G1 and predicted the onset of HSMI prior to any clinical signs of disease. Identical sequences across partial genomes of PRV-1 isolates from G1 and G2 suggest the likely transfer from infected cages to G1. The isolates were grouped into a genogroup known to be of high virulence. A commercial health diet was applied during the HSMI outbreak, and the fish had low mortality and an unaffected appetite. In conclusion, we show that fish health and welfare can benefit from in-depth health monitoring. We also discuss the potential health value of clinical nutrition as a mean to mitigate HSMI., (© 2023. The Author(s).)

The aim of this study was to evaluate the level of neuronal depression in juvenile tambaqui, Colossoma macropomum , exposed to geraniol (GRL) and citronellol (CTL) in immersion baths. A total of 36 juveniles weighing 35.2 ± 9.4 g were used, organised into six experimental groups: I-control (clean water); II-ethanol (water containing the highest volume of ethanol used in the anaesthetic pre-dilution); III-GRL induction (70 µL·L -1 ); IV-CTL induction (90 µL·L -1 ); V-GRL recovery; VI-CTL recovery. Electroencephalographic (EEG) recordings were performed for 300 s in each group. EEG tracings of the control and ethanol groups showed regular and similar activity. Upon exposure to the anaesthetics, irregularities were observed in the tracings showing neuronal excitability and increased amplitudes, mainly in the case of CTL. Overall, GRL-exposed fish showed depression of the central nervous system with low and regular tracings throughout induction, presenting a gradual recovery and stable tracings, which were consistent with an adequate general anaesthetic effect. On the other hand, fish exposed to CTL showed altered EEG activity during induction, that could be considered incompatible with an appropriate anaesthetic effect and smooth recovery, presenting high and irregular EEG tracing amplitudes.

Knowledge about fish welfare and its impact on fish fillet quality is still insufficient. Therefore, the influence of two aspects of fish welfare (slaughtering method: bled and unbled fish; fish stock densities: 90, 120, and 150 kg·m -3 ) on African catfish fillet quality during postmortem conditions was investigated. The aim of study was to determine (i) the efficiency of bleeding on oxidation progress and (ii) the influence of stock density on fillet quality. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a higher protein loss in the unbled than in the bled groups, especially in the heavy myosin chain (MHC) band. However, density did not show any influence on protein profile. Western blot analysis showed fewer oxidized carbonyls in the bled than in the unbled groups; higher oxidation development, microbial growth, and lower hardness were observed in unbled fillets. Additionally, hardness was higher at 90 and 120 kg·m -3 densities in bled fillet compared to 150 kg·m -3 . The first three days of storage showed a higher oxidation rate in unbled fillets than in bled fillets, confirming the contribution of hemoglobin to oxidation development with different mechanisms of protein oxidation. The obtained results revealed the same fillet quality in all aspects at either 90 or 120 (kg·m -3 ) stock densities, which would suggest 120 kg·m -3 for the fishery industry. However, higher stocking density in this study would not be appropriate for fish welfare.

The study of the molecular mechanisms of stress appraisal on farmed fish is paramount to ensuring a sustainable aquaculture. Stress exposure can either culminate in the organism's adaptation or aggravate into a metabolic shutdown, characterized by irreversible cellular damage and deleterious effects on fish performance, welfare, and survival. Multiomics can improve our understanding of the complex stressed phenotype in fish and the molecular mediators that regulate the underlying processes of the molecular stress response. We profiled the stress proteome and metabolome of Sparus aurata responding to different challenges common to aquaculture production, characterizing the disturbed pathways in the fish liver, i.e., the central organ in mounting the stress response. Label-free shotgun proteomics and untargeted metabolomics analyses identified 1738 proteins and 120 metabolites, separately. Mass spectrometry data have been made fully accessible via ProteomeXchange, with the identifier PXD036392, and via MetaboLights, with the identifier MTBLS5940. Integrative multivariate statistical analysis, performed with data integration analysis for biomarker discovery using latent components (DIABLO), depicted the 10 most-relevant features. Functional analysis of these selected features revealed an intricate network of regulatory components, modulating different signaling pathways related to cellular stress, e.g., the mTORC1 pathway, the unfolded protein response, endocytosis, and autophagy to different extents according to the stress nature. These results shed light on the dynamics and extent of this species' metabolic reprogramming under chronic stress, supporting future studies on stress markers' discovery and fish welfare research.

An estimated 1.5-2.7 billion wild fish are caught by UK commercial sea fishing fleets annually. Most are subjected to severe stressors during capture; stunning is rare and gutting alive is common practice. Fish are recognised in UK law as sentient beings, but commercially caught wild fish are excluded from welfare protections. Animal welfare impacts in wild capture sea fisheries are therefore on a massive scale, with major potential for legislative and market-based reforms. Interviews were conducted with 18 experts working within the fishing and seafood industry, fisheries management, scientific research and animal/fish advocacy organisations. The findings reveal a significant welfare gap between societally acceptable ways to treat sentient animals and the reality of capture fisheries. The participants pointed to harms caused to fish throughout different stages of the capture process caused by combinations of variables from fishing gear and methods to biological, environmental and other factors, noting that all require mitigation. Interviews revealed that the nature of harms may be exacerbated by conservative attitudes towards brutal practices in the industry, driven by profit and efficiency and free from legal restraint. To address the welfare gap, stakeholders favour engagement with the industry to improve understanding of harms and to identify mutually beneficial and shared objectives through alleviating stressors to fish in the capture process. This empirical research is focused on UK wild capture sea fisheries. However, given the dearth of welfare legislation globally, it has significance for fishing nations and the many billions of wild sea fish captured each year around the world.

Environmental enrichment (EE) improves the growth rate and welfare of some cultured fishes. However, most cultured fish species are raised in non-enriched housing conditions. Clarias gariepinus is an important commercial fish species, but little is known about the effect of EE on their welfare. This study examined the effect of different EE on the survival rate (SR), growth [mean weight gain (MWG), specific growth rate (SGR) and feed conversion ratio (FCR)], behavioral (feed response, aggressive acts and shoaling time) and physiological responses (blood glucose) of C. gariepinus. One hundred and twenty juveniles of C. gariepinus (31.65 ± 0.69 g) were randomly allocated at 10 fish/tank and subjected to either Plant Enriched (PE), Substratum Enriched (SE), Plant and Substratum Enriched (PSE) and Non-Enriched (NE) tanks in triplicates for 56-days. Behavioral acts were observed for 10 min twice daily, and glucose level in blood samples was evaluated. Data were checked for normality using the Shapiro-Wilk test before being analyzed with the Kruskal-Wallis test. SR and MWG were significantly higher in Clarias gariepinus exposed to SE, with no significant differences among PE, PSE and NE treatments. There was no significant difference between the SGR of PSE and NE. FCR was similar between treatments. The highest condition factor (k) was recorded in SE tanks. Duration of feed response was shorter in SE, but there was no significant difference between the feed response of C. gariepinus exposed to PE and PSE. C. gariepinus exposed to PE, SE and PSE displayed a similar frequency of aggressive acts. African catfish reared in NE (barren) tanks had the least duration of shoaling period. The experiment consistently found the highest and least glucose values in PSE and SE. In conclusion, environmentally enriched housing tanks with SE resulted in the best MWG with a reduced level of aggression in C. gariepinus under laboratory conditions. Thus, EE might be applicable to boost fish productivity on a commercial scale., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ojelade, Durosaro, Akinde, Abdulraheem, Oladepo, Sopein, Bhadmus and Olateju.)

Welfare is an individual attribute. In general, providing captive nonhuman animals with conditions conducive to good welfare is an idea more easily applied when dealing with few individuals. However, this becomes much harder-if not impossible-under farming conditions that may imply high numbers of animals living in large group sizes. Although this is a problem inherent to intensive animal farming, it is possibly best exemplified in fish farming, for these practices often rely on extremely high numbers. In this paper we review evidence supporting the notion that fishes are individuals and fish welfare should thus also be considered at the individual level, examine the current state of welfare assessment in the aquaculture industry, evaluate these practices in light of individualized terrestrial animal welfare assessment methods, and make recommendations regarding research that could lead to a better understanding of how to provide each individual fish with good welfare in captivity.

An international survey was conducted regarding species used for research, methods of euthanasia, health monitoring, and biosecurity in fish laboratories. A total of 145 facilities from 23 countries contributed. Collectively, over 80 different species (or groups of species) were reported to be used for research, of which zebrafish ( Danio rerio ) was the most common by far. About half of the participating laboratories used multiple species. Anesthetic overdose was the preferred method for euthanasia for adult, fry (capable of independent feeding), and larval (not capable of independent feeding) fish. For all developmental stages, the most popular anesthetic compound was tricaine (MS-222), a substance associated with distress and aversion in several species. Moreover, around half of the respondents did not perform a completion method to ensure death. One-quarter of the responding facilities did not have a health monitoring system in place. While most respondents had some form of quarantine process for imported fish, only a small fraction reported quarantine routines that ensure reliable biological barriers. Furthermore, less than one in five screened fish for pathogens while in quarantine. In sum, there was little consensus amongst facilities in how to perform biosecurity measures. Regarding euthanasia, health monitoring, and biosecurity processes, there is a need for updated and universal guidelines and for many laboratories to adjust their practices.

Peracetic acid (PAA) is an organic peroxide that produces free radicals, which contribute to its potent disinfection power. At therapeutic doses, PAA is considered a mild stressor that can trigger transient local and systemic oxidative stress in fish, but the resulting consequences in the brain have yet to be identified. Therefore, we report the brain transcriptome of Atlantic salmon ( Salmo salar ) smolts that have been periodically exposed to PAA. Fish were treated three times (every 15 days) with PAA with either short (15 min) or long (30 min) exposure periods. After the third treatment, the whole brain was collected and subjected to biochemical and transcriptomic analyses. The level of reactive oxygen species in the brain was not significantly affected by recurrent PAA treatments. Microarray analysis was performed on the whole brain and revealed 205 differentially expressed genes (DEGs), regardless of the duration of the treatment. The short exposure duration had a more considerable impact on the brain transcriptome, correlating with 70% more DEGs than the long exposure. Strikingly, the brain transcriptome was characterised by the downregulation of gene expression, especially in the short exposure group, and around 82% of the identified DEGs were downregulated. Some of the highly affected genes were key molecules of the vasotocinergic and isotocinergic systems and the corticotropin-releasing factor signalling system, indicating interference of the stress axis but could also suggest an anxiolytic effect. In addition, there were alterations in genes involved in cellular metabolism and processing, signalling and trafficking, and innate immunity, which underscores the physiological changes in the brain following recurrent PAA treatment. Overall, the transcriptomic data reveal that recurrent oxidant treatment could influence brain functions, and although the magnitude was marginal, the alterations suggested neurological adaptations of fish to PAA as a potential chemical stressor. The results identify the risks of PAA, which would be valuable in drafting a framework for its empirically driven use in fish farming., Competing Interests: No conflict of interest., (© 2022 The Authors.)

Chemical pollutants are a major factor implicated in freshwater habitat degradation and species loss. Microplastics and glyphosate-based herbicides are prevalent pollutants with known detrimental effects on animal welfare but our understanding of their impacts on infection dynamics are limited. Within freshwater vertebrates, glyphosate formulations reduce fish tolerance to infections, but the effects of microplastic consumption on disease tolerance have thus far not been assessed. Here, we investigated how microplastic (polypropylene) and the commercial glyphosate-based herbicide, Roundup®, impact fish tolerance to infectious disease and mortality utilising a model fish host-pathogen system. For uninfected fish, microplastic and Roundup had contrasting impacts on mortality as individual stressors, with microplastic increasing and Roundup decreasing mortality compared with control fish not exposed to pollutants. Concerningly, microplastic and Roundup combined had a strong interactive reversal effect by significantly increasing host mortality for uninfected fish (73% mortality). For infected fish, the individual stressors also had contrasting effects on mortality, with microplastic consumption not significantly affecting mortality and Roundup increasing mortality to 55%. When combined, these two pollutants had a moderate interactive synergistic effect on mortality levels of infected fish (53% mortality). Both microplastic and Roundup individually had significant and contrasting impacts on pathogen metrics with microplastic consumption resulting in fish maintaining infections for significantly longer and Roundup significantly reducing pathogen burdens. When combined, the two pollutants had a largely additive effect in reducing pathogen burdens. This study is the first to reveal that microplastic and Roundup individually and interactively impact host-pathogen dynamics and can prove fatal to fish., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Globally, it is estimated that around 10% of the fish that are caught are discarded. This is considered to be a wasteful human marine activity since these fish are often dead or dying. To reduce the high discard rates of commercial fisheries, the European Union (E.U.) has enacted a landing obligation that includes the ability to exempt "species for which scientific evidence demonstrates high survival rates". Therefore, discard survival studies (henceforth DSSs) have become one of the most politically prioritized fisheries research areas in European fisheries. International expert groups have produced guidance reports to promote best practices and to harmonize the methodologies. Nevertheless, there has not been any focus on how to implement animal welfare (AW) regulations experimentally. Discard survival studies are "frontrunners" in fisheries science research areas that are embedded by animal research welfare requirements and are expected to be more restrictive in the future because of an increased public focus on fish welfare. This paper focuses on AW regulations in relation to conducting DSSs, but the outreach is much broader. We investigate experimental procedures by bringing in relevant examples, using output results, and relating this information to relevant AW guidelines and regulations by focusing on implementing 3R principles.

Sampling protocols and water quality sensors have been developed to assess fish health and welfare in recirculating aquaculture systems (RASs). Still, the use of fish-based non-invasive welfare indicators, reflecting the physiological state of the fish, is limited in this type of system. Cortisol, the major stress-coping hormone in fish, diffuses through the gills. Consequently, waterborne cortisol is a potential fish-based non-invasive welfare indicator in RAS. However, its use in commercial rearing systems is sparse. In this study, we evaluated water cortisol levels and feeding behavior as welfare indicators of newly inserted smolt in a commercial RAS for harvest size Atlantic salmon. The RAS consisted of two parallel fish rearing raceways, raceways 1 and 2, sharing the same water treatment with common outlets and inlets. The smolts were inserted in raceway 1 while salmon that have been in the system for 6 months or more were kept in raceway 2. The smolt insertion period was 3 days. Samples for water cortisol levels were withdrawn the day before, 1 and 3 days after the smolt insertion period. Smolt insertion resulted in elevated water cortisol concentrations in the entire system, with the highest values in raceway 1, one day after smolt insertion. Estimated cortisol production in newly inserted smolt decreased over time, was similar to what has been reported in salmon adapting to experimental tanks. Feeding behavior indicated that the appetite was not fully resumed in the newly inserted smolts, while the appetite of fish in raceway 2 was unaffected by smolt insertion. These results, obtained in a highly intensive commercial RAS, suggest that waterborne cortisol together with feeding behavior can be used as indicators for adaptive processes associated with stress resilience in farmed fish. Thus, they are promising non-invasive indicators for assessing the impact of potential stressors on fish welfare in this type of rearing system., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Höglund, Fernandes, Rojas-Tirado, Rundberget and Hess-Erga.)

Aquaculture is a growing industry worldwide and Canadian finfish culture is dominated by marine salmonid farming. In part due to increasing public and stakeholder concerns around fish welfare protection, the first-ever Canadian Code of Practice for the Care and Handling of Farmed Salmonids was recently completed, following the National Farm Animal Care Council's (NFACC) rigorous Code development process. During this process, both the Scientific (responsible for reviewing existing literature and producing a peer-reviewed report that informs the Code) and Code Development (a diverse group of stakeholders including aquaculture producers, fish transporters, aquaculture veterinarians, animal welfare advocates, food retailers, government, and researchers) Committees identified research gaps in tandem, as they worked through the literature on salmonid physiology, health, husbandry, and welfare. When those lists are combined with the results of a public "top-of-mind" survey conducted by NFACC, they reveal several overlapping areas of scientific, stakeholder, and public concern where scientific evidence is currently lacking: (1) biodensity; (2) health monitoring and management, with a focus on sea lice infection prevention and management; (3) feed quality and management, particularly whether feed restriction or deprivation has consequences for welfare; (4) enclosure design, especially focused on environmental enrichment provision and lighting design; and (5) slaughter and euthanasia. For each of these five research areas, we provide a brief overview of current research on the topic and outline the specific research gaps present. The final section of this review identifies future research avenues that will help address these research gaps, including using existing paradigms developed by terrestrial animal welfare researchers, developing novel methods for assessing fish welfare, and the validation of new salmonid welfare indices. We conclude that there is no dearth of relevant research to be done in the realm of farmed salmonid welfare that can support crucial evidence-based fish welfare policy development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gaffney and Lavery.)

Seaweeds still possess a large undisclosed potential, mainly due to their constituent's richness, which may have several uses for society. In aquaculture, they may play a role as an ecological sustainable aquafeed supplement to increase overall health and fight pathogenic outbreaks. This study aimed to evaluate the general health modulation that the inclusion of Gracilaria gracilis could accomplish in the diet of Dicentrarchus labrax. Dried algae at 2.5% and 5% and algal extract at 0.35% inclusion levels were supplemented to seabass diet to evaluate possible growth, haematological, immunological, antioxidant, metabolic, and intestinal morphological modulations. The supplementations did not impact growth or feed utilization, and barely affected the haematological profile and some metabolic parameters. Nevertheless, it caused a marked outcome on lysozyme, some oxidative stress biomarkers, and intestine morphology, suggesting beneficial consequences from the algal inclusion. Dried algae powder, with a 2.5% inclusion, boosted immune response, with higher plasmatic lysozyme and intestinal acid goblet cells and protected against oxidative damages by improved enzymatic and non-enzymatic responses. Thus, we provide evidence that dietary seaweed application may be a path towards a more sustainable aquaculture industry., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Advances in tag technology now make it possible to monitor the behavior of small groups of individual fish as bioindicators of population wellbeing in commercial aquaculture settings. For example, tags may detect unusual patterns in fish heart rate, which could serve as an early indicator of whether fish health or welfare is becoming compromised. Here, we investigated the use of commercially available heart rate biologgers implanted into 24 Atlantic salmon weighing 3.6 ± 0.8 kg (mean ± SD) to monitor fish over 5 months in a standard 12 m × 12 m square sea cage containing ∼6,000 conspecifics. Post tagging, fish established a diurnal heart rate rhythm within 24 h, which stabilized after 4 days. Whilst the registered tagged fish mortality over the trial period was 0%, only 75% of tagged fish were recaptured at harvest, resulting in an unexplained tag loss rate of 25%. After 5 months, tagged fish were approximately 20% lighter and 8% shorter, but of the similar condition when compared to untagged fish. Distinct diurnal heart rate patterns were observed and changed with seasonal day length of natural illumination. Fish exhibited lower heart rates at night [winter 39 ± 0.2 beats per min (bpm), spring 37 ± 0.2 bpm, summer 43 ± 0.3 bpm, mean ± SE] than during the day (winter 50 ± 0.3 bpm, spring 48 ± 0.2 bpm, summer 49 ± 0.2 bpm) with the difference between night and day heart rates near half during the summer (6 bpm) compared to winter and spring (both 11 bpm). When fish experienced moderate and severe crowding events in early summer, the highest hourly heart rates reached 60 ± 2.5 bpm and 72 ± 2.4 bpm, respectively, on the day of crowding. Here, if the negative sublethal effects on fish that carry tags (e.g., growth rate) can be substantially reduced, the ability to monitor diurnal heart rate patterns across seasons and detect changes during crowding events, and using heart rate biologgers could be a useful warning mechanism for detecting sudden changes in fish behavior in sea cages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Warren-Myers, Hvas, Vågseth, Dempster and Oppedal.)

China is the largest food fish producer in the world. Chinese consumers normally purchase fish that are still alive to ensure freshness. Therefore, the live transport of fish is important in China's aquaculture, although it carries potential risks for animal welfare. This study investigated the attitudes and knowledge of stakeholders within Chinese aquaculture towards the live transport and welfare of fish. Semi-structured interviews were conducted with 12 participants who were involved with the aquaculture industry in China. Most participants self-rated their transport-related knowledge as moderate and had some understanding of animal welfare, although this term was generally considered only relevant to terrestrial animals. Participants' responses indicated that the live transport of fish occurs frequently in China, generally using sealed tanks, plastic bags, and foam boxes, in purpose-built vehicles. Seasonal changes, such as changes in ambient and water temperature, are considered to be important contributors to successful live transport, as well as sufficient oxygen supplies and stocking density. The use of anesthetics was not commonly reported, particularly in food fish, and fish capture is predominantly by conventional dipnets. The health status of transported fish is determined mostly by morphology (body injury, body or eye color, and fin condition), as well as vigor and swimming ability. Our results indicate that live transport poses a number of welfare risks to fish but that participants in the process associated welfare concerns more with terrestrial animals, not fish.

Growing research effort has shown that physical enrichment (PE) can improve fish welfare and research validity. However, the inclusion of PE does not always result in positive effects and conflicting findings have highlighted the many nuances involved. Effects are known to depend on species and life stage tested, but effects may also vary with differences in the specific items used as enrichment between and within studies. Reporting fine-scale characteristics of items used as enrichment in studies may help to reveal these factors. We conducted a survey of PE-focused studies published in the last 5 years to examine the current state of methodological reporting. The survey results suggest that some aspects of enrichment are not adequately detailed. For example, the amount and dimensions of objects used as enrichment were frequently omitted. Similarly, the ecological relevance, or other justification, for enrichment items was frequently not made explicit. Focusing on ecologically relevant aspects of PE and increasing the level of detail reported in studies may benefit future work and we propose a framework with the acronym DETAILS (Dimensions, Ecological rationale, Timing of enrichment, Amount, Inputs, Lighting and Social environment). We outline the potential importance of each of the elements of this framework with the hope it may aid in the level of reporting and standardization across studies, ultimately aiding the search for more beneficial types of PE and the development of our understanding and ability to improve the welfare of captive fish and promote more biologically relevant behaviour., (© 2021 The Authors. Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of Fisheries Society of the British Isles.)

Background: In humans the stress response is known to be modulated to a great extent by psychological factors, particularly by the predictability and the perceived control that the subject has of the stressor. This psychological dimension of the stress response has also been demonstrated in animals phylogenetically closer to humans (i.e. mammals). However, its occurrence in fish, which represent a divergent vertebrate evolutionary lineage from that of mammals, has not been established yet, and, if present, would indicate a deep evolutionary origin of these mechanisms across vertebrates. Moreover, the fact that psychological modulation of stress is implemented in mammals by a brain cortical top-down inhibitory control over subcortical stress-responsive structures, and the absence of a brain cortex in fish, has been used as an argument against the possibility of psychological stress in fish, with implications for the assessment of fish sentience and welfare. Here, we have investigated the occurrence of psychological stress in fish by assessing how stressor controllability modulates the stress response in European seabass (Dicentrarchus labrax)., Results: Fish were exposed to either a controllable or an uncontrollable stressor (i.e. possibility or impossibility to escape a signaled stressor). The effect of loss of control (possibility to escape followed by impossibility to escape) was also assessed. Both behavioral and circulating cortisol data indicates that the perception of control reduces the response to the stressor, when compared to the uncontrollable situation. Losing control had the most detrimental effect. The brain activity of the teleost homologues to the sensory cortex (Dld) and hippocampus (Dlv) parallels the uncontrolled and loss of control stressors, respectively, whereas the activity of the lateral septum (Vv) homologue responds in different ways depending on the gene marker of brain activity used., Conclusions: These results suggest the psychological modulation of the stress response to be evolutionary conserved across vertebrates, despite being implemented by different brain circuits in mammals (pre-frontal cortex) and fish (Dld-Dlv)., (© 2021. The Author(s).)

Intraspecific aggression is detrimental to body/fin damage, physiological stress, and other problems in aquaculture. Environmental enrichment has been proposed to have positive effects on fish aggressive behavior, physiological stress, and fish welfare, but there are mixed results. Here, we examine the impact of physical enrichment levels (i.e., the intensity of physical enrichment) on aggression in black rockfish ( Sebastes schlegelii ) and fat greenling ( Hexagrammos otakii ). Generally, with the increase in the enrichment level, the frequency of the aggressive behavior of black rockfish gradually decreased. In contrast, a non-monotonous effect of the enrichment level on aggression was observed for fat greenling, with low and intermediate levels leading to no or more aggression, while a high enrichment level reduced aggression. After three days, the high-level enrichment groups in both rockfish and greenling reached social stability (i.e., a relatively stable social structure indicated by lower aggression), while aggression in the other groups continued increased. These results show the significant regulatory effect of enrichment levels on the aggressive behavior in both black rockfish and fat greenling. This study may promote the development of environmental enrichment measures, and it provides useful information for reducing fish aggression and improving fish welfare in aquaculture.

In this study, Atlantic salmon post smolts (~250 g, ~29 cm) were fasted for four weeks at 12 °C in full strength seawater. During this period, the critical swimming speed (U crit ) was measured after 1, 2 and 4 weeks of fasting, as well as in a fed control group. Furthermore, blood samples were taken in each treatment group prior to the swim test, at fatigue, and following 3 h and 24 h of subsequent recovery. Four weeks of fasting gradually reduced the condition factor from 1.03 to 0.89. However, the U crit remained statistically unaffected at 3.5 body lengths s -1 . Exhaustive exercise stress caused large increases in plasma osmolality, [Cl - ], [Na + ], [Ca 2+ ], [lactate] and [cortisol], while haematocrit and [haemoglobin] also increased. Plasma ions and lactate had increased further after 3 h recovery, and osmolality, [Cl - ] and [Na + ] were still elevated above control levels after 24 h while other blood parameters were fully recovered. Osmotic disturbances may therefore be considered the most challenging stressor during strenuous exercise in seawater. Only minor effects of fasting period on blood parameters in response to exhaustive exercise were detected, which included slightly higher osmotic disturbances and a repressed response in red blood cell recruitment at fatigue in fasted fish. Furthermore, the 4-week fasting group had a reduced cortisol response following fatigue compared to the other treatment groups. In conclusion, these results show that Atlantic salmon maintain their full swimming capacity as well as their ability to respond and recover from acute stress during an extended period of food deprivation., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

As a first attempt to assess bone health in cleaner fish production, wild and cultured ballan wrasse Labrus bergylta and lumpfish Cyclopterus lumpus were examined by radiology. In C. lumpus, wild fish (57%) had more vertebra deformities (≥1 deformed vertebrae) than cultured fish (2-16%). One wild C. lumpus had lordosis and another was missing the tail fin. In L. bergylta, wild fish (11%) had fewer vertebra deformities than cultured individuals (78-91%). Among the cultured L. bergylta, 17-53% of the fish had severe vertebra deformities (≥6 deformed vertebrae) with two predominate sites of location, one between vertebra 4 and 10 (S1) in the trunk, and one between 19 and 26 (S2) in the tail. Fusions dominated S1, while compressions dominated S2. Although wild L. bergylta had a low vertebra deformity level, 83% had calluses and 14% had fractures in haemal/neural spines and/or ribs. The site-specific appearance and pathology of fracture and callus in wild L. bergylta suggests these are induced by chronic mechanical stress, and a possible pathogenesis for fish hyperostosis is presented based on this notion. In conclusion, good bone health was documented in cultured C. lumpus, but cultured L. bergylta suffered poor bone health. How this affects survival, growth, swimming abilities and welfare in cultured wrasse should be further investigated. SIGNIFICANCE STATEMENT: Skeletal deformities were studied in ballan wrasse and lumpfish of both wild and cultured origin for the first time to identify potential welfare issues when deploying them as cleaner fish in salmon sea cages. While cultured lumpfish showed good bone health, cultured wrasse had a high occurrence of vertebra deformities, which is expected to impact lice eating efficiency and animal welfare negatively. These deformities are most likely induced early in development., (© 2020 The Authors. Journal of Fish Biology published by John Wiley & Sons Ltd on behalf of The Fisheries Society of the British Isles.)

Thanks to its small size, external fertilization and fecundity, over the past four decades, zebrafish ( Danio rerio ) has become the dominant fish model species in biological and biomedical research. Multiple lines of evidence, however, suggest that the reliance on only a handful of genetic model organisms is problematic, as their unique evolutionary histories makes them less than ideal to study biological questions unrelated to their historically contingent adaptations. Therefore, a need has emerged to develop novel model species, better suited for studying particular problems. The paradise fish ( Macropodus opercularis ) has a much more complex behavioral repertoire than zebrafish and has been a favored model animal in ethological research during the last decades of the previous century. We believe that with currently available, easily adaptable genetic toolkits, this species could be easily developed into a popular model of behavioral genetics. Despite its earlier popularity, however, the description of a detailed housing and husbandry protocol for this species is still missing from scientific literature. We present here a detailed description of how to raise and breed paradise fish successfully under laboratory conditions, and also discuss some of the challenges we faced while creating a stable breeding population for this species in our facility.