Currently, nutrition testing in aquaculture relies heavily on in vivo feeding trials, which involve raising fish or shrimp under controlled conditions and monitoring growth, feed utilization, immune and health-related criteria. These trials are resource-intensive, time-consuming (weeks to months), and require large numbers of animals, making them costly and ethically challenging. New Approach Methodologies (NAMs) offer an opportunity to modernise aquatic nutrition by integrating mechanistic, exposure relevant and species-specific information within a framework of validated contexts of use (CoU). NAMs include a variety of technologies, methodologies, and approaches such as in vitro tests, in silico computational models, biomarkers, and modified in vivo assays that can fill critical information gaps and build confidence for their use in proof of efficacy and regulatory decisions.
The availability of NAMs that adequately reflect fish biology remains a key gap in aquaculture research. While in vitro cell culture approaches and advanced platforms such as organoids (3D miniaturised organ���like structures derived from stem cells) and organ���on���chip systems (microfluidic devices mimicking organ functions) offer clear potential as NAMs, their application in fish and shrimp remains incipient. In this context, ex vivo fish tissue explants provide a pragmatic and biologically relevant alternative, preserving native tissue organisation and metabolic competence while supporting the principles of Replacement, Reduction and Refinement (3Rs) in animal nutrition research.
Methodological approaches
Explants are small sections of native tissue (e.g., intestine, skin, gills, scales) maintained ex vivo for short-term experiments. Explants preserve native tissue architecture and microbiome, cell-cell interactions, and biochemical signalling, offering a more physiologically relevant model than traditional cell lines. The ex vivo tissue explants approach bridges the gap between in vitro cell culture and whole-animal in vivo trials, enabling mechanistic insights into nutrient absorption, barrier function, and immune responses without the complexity of entire organisms.
Figure 1. A general view of the RIASEARCH AQUA-NAMs platform.
In higher vertebrates, intestinal mucus is also increasingly used as a key component in NAMs, to simulate the gastrointestinal barrier for testing nutrient/drug absorption, toxicity, and host pathogen interactions. Additionally, fish and shrimp faeces are also a matrix with the potential to harbour a series of biomarkers linked to intestinal inflammation and immune status.
Results
While presenting a schematic roadmap of New Approach Methodologies (NAMs) in aquaculture species is conceptually straightforward, their practical implementation is extremely challenging, requiring a lengthy setup of novel methodologies, the identification and qualification of context���specific biomarkers, and the generation and integration of large, high���quality datasets to robustly establish in vivo relevance. We are actively engaged in this process, with established experimental platforms using intestinal and skin explants and mucus host-pathogen interaction assays across Atlantic salmon, Nile tilapia and European seabass. These systems are currently being applied to evaluate raw materials, feed additives and other non-nutritional compounds, integrating multiparametric endpoint frameworks encompassing immune response, epithelial integrity, and wound healing process. A selection of results derived from these ongoing efforts will be presented to exemplify the translational potential of NAMs in aquaculture nutrition research.
Conclusions
A NAMs-based approach is highly innovative, delivering faster and ethically sound testing options while providing mechanistic insights that in vitro tests cannot achieve. Although, at this stage, we are still far from fully replacing classical in vivo efficacy and tolerance trials, NAMs can transform early-stage nutritional screening, offering a significant competitive advantage in the aquafeed innovation pipeline.
Acknowledgment
This work is part of the SKINS project, Operation No. 21638 (COMPETE2030-FEDER-02247200, ALGARVE-FEDER-02247200), co financed by the European Union through the Algarve 2030 and COMPETE 2030 Operational Programs, in the framework of Portugal 2030.