The rapid and intensive growth of aquaculture over the last decade, poses a tremendous challenge to this industry in order to comply with the latest guidelines, established to minimise its negative effects on the environment, animal welfare and public health. Farmed fish welfare has become one of the main priorities towards sustainable aquaculture production with several initiatives launched by the European Union within the framework of the 2030 agenda. It is clear that an unbiased and reliable way to access farmed fish welfare needs to be implemented due to the lack of reliable indicators and standardised methods that are used at present. In this review, we start by addressing the status quo of animal and fish welfare definition in particular, describing the methods and assays currently used to measure it. We then explain why we believe these methods are unreliable and why there is a need to establish new ones that will promote productivity and consumer's acceptance of farmed fish. The establishment of a new type of welfare biomarkers using cutting‐edge technologies like proteomics and other omics technologies is proposed as a solution to this issue. Therefore, we provide a brief description of these new methodologies, describing for each one how they can improve our scientific knowledge and the role they can play in farmed fish welfare biomarker discovery.

Background: Aquaculture is a fast-growing industry and therefore welfare and environmental impact have become of utmost importance. Preventing stress associated to common aquaculture practices and optimizing the fish stress response by quantification of the stress level, are important steps towards the improvement of welfare standards. Stress is characterized by a cascade of physiological responses that, in-turn, induce further changes at the wholeanimal level. These can either increase fitness or impair welfare. Nevertheless, monitorization of this dynamic process has, up until now, relied on indicators that are only a snapshot of the stress level experienced. Promising technological tools, such as proteomics, allow an unbiased approach for the discovery of potential biomarkers for stress monitoring. Within this scope, using Gilthead seabream (Sparus aurata) as a model, three chronic stress conditions, namely overcrowding, handling and hypoxia, were employed to evaluate the potential of the fish protein-based adaptations as reliable signatures of chronic stress, in contrast with the commonly used hormonal and metabolic indicators. Results: A broad spectrum of biological variation regarding cortisol and glucose levels was observed, the values of which rose higher in net-handled fish. In this sense, a potential pattern of stressor-specificity was clear, as the level of response varied markedly between a persistent (crowding) and a repetitive stressor (handling). Gel-based proteomics analysis of the plasma proteome also revealed that net-handled fish had the highest number of differential proteins, compared to the other trials. Mass spectrometric analysis, followed by gene ontology enrichment and protein-protein interaction analyses, characterized those as humoral components of the innate immune system and key elements of the response to stimulus. Conclusions: Overall, this study represents the first screening of more reliable signatures of physiological adaptation to chronic stress in fish, allowing the future development of novel biomarker models to monitor fish welfare.

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Hepatic metabolic adjustments are key adaptive mechanisms to stress in fish targeting at increasing energy availability for the animal to efficiently cope with a stressor. Teleosts exhibit a broad variety of these metabolic responses, depending on the species biology, individual experiences and the challenge’s characteristics. Nevertheless, the molecular response to a prolonged stress can be more heterogeneous and far more complex to interpret than that to an acute stress. A comparative proteomics analysis was employed to discover the set of liver proteins involved in the adaptive processes that tune the physiological response of Sparus aurata to different suboptimal rearing conditions and physical challenges. Three separated trials were established where fish were submitted to different conditions (overcrowding, net handling and hypoxia). The response at the transcript level of 13 genes was also assessed. Mass spectrometric analysis revealed 71 differential abundant proteins distributed among the trials. Prolonged exposure to stress seems to have induced widespread changes in amino acid, carbohydrate, and lipid metabolisms, antioxidant response and protein folding, sorting and degradation processes. Two genes corresponding to heat-shock proteins were found to be differently expressed in net handled fish. These results shed light on the dynamics and extent of this species’ metabolic reprogramming under different challenges, supporting future studies on stress markers’ discovery and fish welfare research.

Stress triggers a battery of physiological responses in fish, including the activation of metabolic pathways involved in energy production, which helps the animal to cope with the adverse situation. Prolonged exposure to stressful farming conditions may induce adverse effects at the whole-animal level, impairing welfare. Fourier transform infrared (FTIR) spectroscopy is a rapid biochemical fingerprinting technique, that, combined with chemometrics, was applied to disclose the metabolic alterations in the fish liver as a result of exposure to standard stressful practices in aquaculture. Gilthead seabream (Sparus aurata) adults exposed to different stressors were used as model species. Spectra were preprocessed before multivariate statistical analysis. Principal components analysis (PCA) was used for pattern recognition and identification of the most discriminatory wavenumbers. Key spectral features were selected and used for classification using the k-nearest neighbour (KNN) algorithm to evaluate whether the spectral changes allowed for the reliable discrimination between experimental groups. PCA loadings suggested that major variations in the hepatic infrared spectra responsible for the discrimination between the experimental groups were due to differences in the intensity of absorption bands associated with proteins, lipids and carbohydrates. This broad-range technique can thus be useful in an exploratory approach before any targeted analysis.

One of the main constraints in aquaculture production is farmed fish vulnerability to diseases due to husbandry practices or external factors like pollution, climate changes, or even the alterations in the dynamic of product transactions in this industry. It is though important to better understand and characterize the intervenients in the process of a disease outbreak as these lead to huge economical losses in aquaculture industries. High-throughput technologies like proteomics can be an important characterization tool especially in pathogen identification and the virulence mechanisms related to host-pathogen interactions on disease research and diagnostics that will help to control, prevent, and treat diseases in farmed fish. Proteomics important role is also maximized by its holistic approach to understanding pathogenesis processes and fish responses to external factors like stress or temperature making it one of the most promising tools for fish pathology research.

Background In the verge of getting a greater understanding of fish welfare, sensitive technological tools, such as proteomics, may assist the aquaculture industry as it allows an unbiased approach for the discovery of potential biomarkers for stress monitoring. Stress is characterized by a cascade of physiological responses that end-up inducing further changes at the whole-animal level that might either increase fitness or impair welfare. Monitorization of this dynamic process, up till now relies on indicators that are only a snapshot of the stress level experienced. Within this scope, using gilthead seabream ( Sparus aurata ) as a model, three chronic stress conditions, namely overcrowding, handling and hypoxia, were employed to evaluate the potential of the fish protein-based adaptations as reliable signatures of chronic stress, in contrast with the commonly used indicators of primary and secondary stress responses.Results A large spectrum of biological variation regarding cortisol and glucose levels was observed, which values rose higher in net handled fish. In this sense, a potential pattern of stressor-specificity was evidenced since the magnitude of response and tolerance varied markedly from a permanent (crowding) to a repetitive stressor (handling). Gel-based proteomics analysis of the plasma proteome also revealed that net handled fish had the highest number of proteins with significantly altered abundance, compared to the other trials, whereas mass spectrometric analysis, followed by gene ontology enrichment and protein-protein interaction analyses, characterized those as humoral components of the innate immune system and key elements on the response to stimulus.Conclusions Overall, this study represents the first screening of more reliable signatures of physiological adaptation to chronic stress in fish, allowing the future development of novel biomarker models to monitor fish welfare.