8th RESAMA Days

As research progresses, ethical questions arise. Indeed, the knowledge that research has provided, particularly regarding animal sentience, demonstrates the need to respect animals and their well-being. This results in the need for research itself to take animal welfare into account in its practices. This presentation outlines current knowledge regarding sentience in marine animals, and recalls the notions of animal welfare and good treatment.

We will then present the practical guide created at Ifremer (French Research Institute for the Exploitation of the Sea), intended for all staff using animals within the institute. This document aims to record the regulatory obligations relating to the use of marine animals for scientific purposes, and, for species and/or activities that the law does not concern, to highlight the ethical questions raised by researchers, and the answers already provided to these questions by the Institute. Finally, it sets out avenues for reflection that could further improve the well-being of the animals used.

While under controlled laboratory conditions (temperature and physicochemical quality of water), anesthesia of fish is rather easy, its application in the field, in a natural environment, can sometimes be complicated. On the one hand, the physicochemical conditions vary greatly from one site to another, during the season or during the day, and this can increase the risk of mortality of anesthetized fish, even if the prescribed doses are respected. On the other hand, field anesthesia often involves working with several species and several individuals of different sizes that may respond differently to recommended doses. In 2025, we carried out a study as part of a veterinary thesis whose objective was to carry out a comparative analysis of the different products available to anesthetize fish in order to evaluate their benefits and disadvantages for the fish, the environment, the operator and the consumer in the context of possible re-capture a posteriori. The study also made it possible to clarify the regulatory compliance of the various methods used. A survey was carried out among field operators working on various species of freshwater fish and using several different products to collect their feedback, both positive and negative. The results of the survey will be formalized in the form of a practical guide in order to share and enrich collective know-how (sharing of good anesthesia practices, concentrations/species, precautions to take). The presentation will mainly focus on the restitution of the survey carried out.

In behavioral aquatic ecology, individual monitoring represents an important part of the methods used. With technological advances, a large number of technologies have emerged in recent years: acoustic telemetry, radio, satellite, RFID, etc. The common point between all these methods is that they must 1- capture the individuals to be studied and 2- equip them with a transmitter.

With the regulations on UAFS, advances in practices, the evolution of mentalities, many refinement methods have been added and are now used. These additional methods, although interesting in principle, have the main impact of adding handling time.

Through a concrete example, we propose to discuss the benefit/cost balance of new refinement methods in individual marking on aquatic species.

As human lifespan increases, this increased longevity is accompanied by an increasing incidence of diseases and alterations associated with aging, often linked to metabolic dysregulation. However, the evolution of organ-specific metabolism during aging remains poorly characterized. The African turquoise Killifish (Nothobranchius furzeri, hereafter referred to as ATK), an emerging short-lived vertebrate model, provides a unique opportunity to study systemic aging processes. Here we study age-related metabolic reprogramming in this model using in vivo stable isotope tracing with uniformly labeled 13C glucose and targeted quantitative metabolomics. The experiments were conducted comparing young and old adult male ATK, with glucose administration by water infusion. Mass spectrometry analysis of the incorporation of 13C into various metabolites made it possible to map the absorption and use of glucose in various organs (liver, muscles, brain) but also to formulate hypotheses on metabolic dialogues via plasma analysis. Metabolite profiling revealed distinct age-related changes in content in all tissues. Despite age, a low dependence of glucose metabolism was consistently observed, suggesting that other substrates, such as amino acids and fatty acids, may play a dominant role. Interestingly, our results also suggest a lactate-mediated metabolic crosstalk between oxidative and glycolytic tissues.

This work establishes a methodological framework for metabolic studies in ATK, or other aquatic models, and provides insights into age- and tissue-specific metabolic remodeling. These results contribute to the understanding of age-related metabolic evolution in this emerging model of vertebrate aging.

With the constant increase in human lifespan, the prevalence of pathologies linked to aging follows the same trend, which is becoming a real public health problem. To study aging, a very innovative preclinical model was implemented at the RESTORE institute: the African Turquoise Killifish (ATK). It spontaneously presents a short lifespan of only 8 months and a very rapid multi-organ holistic aging mirroring that of humans. Despite the growing use of this model generating significant new discoveries in the field of aging, a burning question regarding ATK remains unanswered: “what are the main causes of Killifish death?” » Only a dedicated anatomopathology study can answer this question. To meet this objective, the biobanking of a large number of individuals is underway at LabHPEC (experimental and comparative histopathology laboratory of the National Veterinary School of Toulouse), a stakeholder in the RESTORE institute. In this context, an international consortium involving 20 laboratories spread across 4 continents is being structured to collect samples very broadly and quickly, and organize a network of veterinary pathologists involved in the study of this species! The virtual slides of tissues and whole fish will be grouped together on a virtual microscopy platform allowing annotations shared by the consortium and the creation of a digital atlas of physiological and pathological tissues on a website. This international collaborative scientific work will give students, teachers, aquarists, veterinarians or scientists the opportunity to have access to numerous original data, in order to better understand the trajectories of aging and the associated pathologies of this fascinating model.

The gray Bichir, Polypterus senegalus, presents a key taxonomic position for understanding the evolutionary mechanisms behind the colonization of the terrestrial environment. The only representative of the actinopterygians capable of breathing in the open air using its primitive lungs, it demonstrates a remarkable capacity for adoption when forced into terrestrial life. Remarkable research work carried out by the laboratory of Emily Standen (1) (University of Ottawa) has demonstrated musculoskeletal plasticity in response to moderate terrestrial conditions. This musculoskeletal plasticity combined with the key phylogenetic position of this species as the closest living relative of the group of vertebrates that initiated the evolutionary transition to land, opens the possibility within our laboratory to use P. senegalus as a unique model to understand the origins and basic physiological processes involved in the plasticity of cardiac automaticity in the transition between aquatic and terrestrial life. Involved from the start of the collaboration between the University of Ottawa (E. Standen) and the Institute of Functional Genomics of Montpellier (M. Mangoni), the ZebKO aquatic platform has broadened its field of expertise and is now developing this exceptional new aquatic animal model in order to offer it to the IGF teams but also to the entire scientific community.

The FRESHCO team, attached to the INRAE ​​RECOVER unit in Aix-en-Provence, has been studying the effects of global warming on freshwater ecosystems for more than ten years. To do this, it relies on an experimental platform based on the breeding of medakas in aquariums, in temperature-controlled rooms. This device makes it possible to analyze the impact of temperature on numerous parameters at the individual and population scale, such as life history traits, physiology, metabolism and even behavior.

At the same time, a set of 25 mesocosms of 1000 liters each makes it possible to reconstruct trophic networks including these fish, with the aim of better understanding how warming modifies the structure of communities and influences the ecological trajectories of lakes.

The particularity of our work also implies a specific approach to animal welfare. Indeed, our breeding constitutes in itself a long-term experiment, having to meet both regulatory requirements and scientific objectives. Also, mesocosms have many advantages in terms of animal welfare, by offering a more natural and less restrictive environment for fish. This is the direction in which we wish to direct our work in the future, by developing the use of these devices as much as possible. Nevertheless, aquarium breeding remains essential to respond to certain specific scientific issues, requiring precise control of experimental conditions.

The National Museum of Natural History has two marine stations. Among them, the Dinard marine station has an aquatic animal facility approved as a User Establishment Using Animals for Scientific Purposes.

This animal facility is a key tool, both for experimental laboratory studies and for in-situ studies.

Through several examples, we propose to illustrate how an experimental animal facility can help research in a natural environment.

This presentation is devoted to research carried out at EPFL using zebrafish as a model. We will first introduce the working framework: the presentation of the school, the infrastructure of the animal facility, the team as well as our activities.

We will then present two projects:

PROJECT 1: Observe without disturbing: automated tracking

PROJECT 2: Egg retention: a wellness challenge

The objective is to demonstrate how EPFL exploits this model for scientific and technological advances.

Presentation of the UAR 3514 CNRS Stella Mare

Structural enrichment represents an essential lever for improving the well-being of model animals bred in captivity. However, the devices currently available on the market for the species Danio rerio (zebrafish) present several limitations: health risk, accidental ingestion of materials, difficult maintenance or even high costs. The zebrafish, a social species, sometimes displays dominance behaviors, notably when feeding. These behaviors can go as far as aggressive interactions, sources of stress for the weakest individuals. It is by observing the social behavior of other species housed in our institute (xenopus, rodents, non-human primates) that the idea of developing enrichment was born simple and effective structural: a partial visual barrier, installed in aquariums making it possible to modulate social interactions (the movements of fish and therefore their interactions), particularly at the time of food intake.

This device allows: • free movement of fish from one side of the aquarium to the other, • to modify their perception of the environment, • encourage exploratory behavior, • reduce aggressive behavior, • to limit stress, particularly among dominated individuals.

Developed collaboratively within our Animal Welfare Structure (SBEA), this project mobilized the complementary skills of platform staff and zootechnics, the RnD Unit platform (fablab), and the research teams from the Institute of Brain. This work is fully in line with the 3R approach, and more particularly in the approach Refinement of zebrafish housing conditions. Satisfied with the first beneficial effects observed with the implementation of this enrichment in our pet store, we therefore wish to share it with the aquatic community, in order to that each pet store can access a simple, economical and effective solution for us we think, refine the accommodation of fish and thus improve their well-being.

The European Union's Marine Strategy Framework Directive (MSFD) identifies underwater noise as a significant environmental concern, emphasizing the need to maintain noise levels that do not harm marine ecosystems (MSFD 2008/56/EC). Shipping has significantly increased noise levels on major shipping routes over the past 50 years. Given the rapidly changing ocean soundscape, there is an urgent need for further research into the effects of anthropogenic noise on marine life. This research is essential for developing effective policies and protecting ocean health. Currently, most studies focus on the effect of sounds on vertebrates (cetaceans and pinnipeds). To fully understand and mitigate the effects of noise pollution, it is essential to broaden the scope of research to include a wider range of sound species and frequencies. Despite their ecological and economic importance, the impact of increased anthropogenic noise on cephalopods has been relatively little studied. This study attempts to provide some answers.

Sound and noise pollution in pet stores are often overlooked issues. The impact on animals can be significant. In order to present the new Vibrasson working group dedicated to the management of sound and vibrations in aquatic animal facilities, we are offering a round table on these themes and their importance in the management of animal welfare of aquatic species.

Approval under article R214-99 of the CRPM and context of official controls. Priority checkpoints and follow-up to checks.

An overview of the numbers of animals used in research in France and the EU until 2023 will be presented, with a focus on aquatic species as well as their evolution over recent years.

Sardina pilchardus, a pelagic and gregarious bony fish, feeds mainly on plankton. Its distribution area extends in the North-East Atlantic from Iceland to Senegal, and also includes the Mediterranean Sea and the Black Sea. Maintaining this species in captivity remains a real challenge: to date, neither its complete life cycle nor the associated zootechnical practices are fully mastered. In laboratory and aquarium conditions, the survival of sardines is often compromised by significant stress and high mortality. As part of the OMEGA project, we collaborated with Océanopolis to identify key parameters likely to improve the well-being and survival of S. pilchardus in captivity. This presentation aims to share our feedback on maintaining sardines in experimental conditions and to expose the action levers, developed jointly with the Océanopolis aquarium, to optimize zootechnical practices for this particularly sensitive species.

In this brief communication, we will present the ExpeFS training, co-led by the National Museum of Natural History, the CNRS and the OFB.

This training aims to train scientists to work on Unhoused Wild Fauna within the framework of the Use of Animals for Scientific Purposes.

We will present the current training, its taxa and the different training modalities proposed (initial training, continuing training / maintenance of skills, extension to other taxonomic groups), as well as taxa being added (aquatic models).

AFSTAL is a French association under the law of 1901, founded in 1972, serving people involved in the use of animals for scientific purposes to help them train, obtain information and disseminate their know-how in this field, the aim being to adopt ethical conduct and improve in vivo projects.

Within this association, COMAQUA is the commission dedicated to the use of aquatic animals for scientific purposes. COMAQUA, made up of field experts representing public and private sector organizations, aims to disseminate recent advances in the field of animal welfare, to offer a network of exchange and communication between professionals in the field, whether they work in the laboratory or in the natural environment, and to participate in updating technical and regulatory skills through numerous actions.

The GIS FC3R regularly organizes calls for tenders for research projects dedicated to the implementation of the 3Rs (Refine, Reduce, Replace) in animal experimentation. These calls for tenders can significantly improve the welfare of animals used for scientific purposes. The presentation will quickly introduce the GIS FC3R tenders and provide guidance on how to respond effectively to these tenders.

The National Stranding Network (RNE) managed by PELAGIS was structured in response to the need to scientifically supervise and promote accidents involving cetaceans which wash out on the French maritime coasts. Although not as frequent, this type of incident also involves an (apparently) increasing prevalence of elasmobranchs (sharks or rays) which are increasingly observed in very shallow depths, particularly on beaches. However, unlike cetaceans which can breathe in the open air, their aquatic respiration seems to limit their total exposure. This criterion suggests the use of the term “beachage” from the English “beach” which means “beach”, which animals approach without necessarily stranding themselves. This process - most often ending with the death of the animal - presents challenges:

- safety: with risks of bites - in the case of sharks -, in particular when witnesses try to help the animal; - epidemiological risk: with the possibility of involvement of pathogens causing zoonoses; - conservation: regarding animals with highly vulnerable status (which their legal status does not reflect); - practical management: particularly concerning the supervision and management of rendering.

To date, no structured and efficient network exists and therefore deserves to be developed, in order to capitalize on scientific information allowing us to better understand and potentially better manage this type of incident. The structuring of this network intends - as far as possible - to be carried out in synergy with the RNE and relying on individuals and associations already involved in elasmobranch beaches (such as AILERON, APECS, Groupe Phocéen des Requins, Shark Education, Shark Mission France). The recent emergence of the Rays & Sharks Technical Group of the Veterinarians for Biodiversity association (VPB) can be an asset in this context by mobilizing veterinarians who have proven expertise and legal legitimacy on the management of beaching and the scientific documentation of potential autopsies. They could contribute to the improvement and implementation (via the publication of tools and training) of beach management protocols.

Concerning the term structuring of the B2R network, the approach which has proven itself for the RNE seems the most relevant, with a triptych composed of:

- a data acquisition system implemented by trained and competent workers - scientific coordination which could be assumed by UAR3278 CRIOBE (Perpignan) - governance ensured by a steering committee made up of members from management institutions (notably MTE, OFB), the scientific sphere (notably CRIOBE, MARBEC, MNHN) and the associative world (AILERON, APECS, Groupe Phocéen des Requins, Shark Education, Shark Mission France, VPB, etc.)

Bringing together the different stakeholders around the same project can sometimes be a real challenge, particularly when it comes to the conservation of protected species. At the interface between researchers, design offices and associative structures, the Herpetological Society of France, the national reference association in herpetology, will present the key stages of the success of a conservation project. Bringing together all the stakeholders and skills around the same issue makes it possible to increase the power of the resulting project results. However, it is often difficult to bring together all the actors from different sectors, with divergent issues and requirements, sometimes leading to incomprehension, difficult relationships and opposing prioritization of actions. Knowing the global network of actors who revolve around a theme is often one of the keys to the success of such projects. Faced with the decline of species, it is crucial to de-sanctuary the different conservation actors in order to make them converge towards common projects.

Sometimes feared for their stings, sometimes admired for their hypnotic grace, jellyfish still remain largely unknown. To make these gelatinous creatures known to as many people as possible, the Biarritz Aquarium has an exhibition dedicated to them, coupled with a breeding laboratory to ensure autonomy of supplies.

In bites motivated by predation, large sharks such as the tiger shark (Galeocerdo cuvier) typically ingest bones (especially limbs). In the context of fishing implemented following a fatal bite, the use of co-responsible protocols aims to neutralize only the sharks that have bitten while sparing the others. An innovative technical solution would consist of carrying out an in situ x-ray centered on the shark's stomach in order to determine the presence of bones indicating the suspected responsibility of the shark. In this context, a collaboration with a company marketing large devices suitable for equine radiography was undertaken to finalize a prototype adapted to sharks, by i) waterproofing the printing plate and ii) developing a cradle allowing effective restraint of the animal which must remain submerged (at least partially) during manipulation. In addition to the (restricted) shark risk management market, such a device could be of interest to scientists involved in the study of large sharks.

In the fields of ecophysiology and functional ecology relating to Teleost fish populations, it is important to observe these organisms in the complex, heterogeneous reality of their ecosystem living environments. The ethics associated with the requirements of developing long-term population observation projects require the use of methodologies for obtaining physio-pathological data that are as stress-free as possible for the animal. These involve simple, rapid and non-invasive sampling strategies.

A collaborative project allowed us to define a protocol for urine sampling in two species of teleosts, the sea bass (Dicentrarchus labrax) and the rainbow trout (Oncorhynchus mykiss). The urine samples collected were analyzed and validated by the combination of two techniques, namely osmolality measurements by nano-osmometry associated with Fourier transform infrared spectroscopy observations. Developed in the laboratory, the urinary sampling and analysis method was then tested in a study carried out in a natural environment on the rainbow trout species.

This technique was able to provide molecular fingerprints reflecting the stressed state of fish. It should help improve our knowledge of the ecophysiology of fish stress in the natural environment.

Over the last ten years, the use of zebrafish as a model organism has increased considerably, which is why it is necessary to improve lineage management in our structures by implementing cryopreservation.

This will involve presenting the implementation of the cryopreservation service within the ZEFIX platform in Montpellier.

Feedback on Zebrafish cryopreservation in a research team that has been in existence for more than 20 years.

The cryopreservation of biological material for the purposes of conservation of genetic resources requires that the frozen cells retain their ability to regenerate intact individuals at the end of the procedure of freezing, cold storage, thawing and development. This assumes that the damage induced by cold shock and by extra and intracellular crystallization is controlled by a freezing speed and suitable permeant and impermeant cryoprotectants. Adopting a freezing method by crystallization (slow freezing) or by vitrification (ultra-rapid freezing) is one of the strategic choices to be made depending on the equipment available and the resistance of the cells. Then, storing the material in liquid nitrogen (at -196°C) is to date the most reliable method for stabilizing the dormant state of biological material, even if nitrogen-free solutions exist.

The choice of the cell type to cryopreserve is also of strategic importance when considering the preservation of a given genetic heritage and ensuring the restoration of a lineage. In fish, spermatozoa, somatic cells and germline stem cells are able to support cryopreservation and are reliable cellular candidates for integration into cryopreservations (cryobanks). On the other hand, ova and whole fish embryos cannot be cryopreserved due to the complexity of the yolk compartment. This prevents direct restoration of individuals upon thawing, and different restoration methods must then be adapted to the cryopreserved cell type: fertilization from sperm only allows 50% of the nuclear genome to be restored at each generation if the eggs used come from another lineage, and the mitochondrial genome of interest carried by the sperm is lost; somatic cells can support the development of a new individual thanks to their injection into a recipient egg, using nuclear transfer technology, but this method is currently of little use in practice due to significant reprogramming defects in embryonic chromatin; germinal stem cells, for their part, are currently the only cellular support that allows the restoration of the entire genome of interest (nuclear and mitochondrial), as well as obtaining eggs from cryopreserved biological material. Indeed, these stem cells, once thawed, can initiate complete gametogenesis and produce functional gametes following their transplantation into the gonads of immature recipient individuals, preferably sterile. However, restoration by transplant is more technically demanding than fertilization with cryopreserved sperm.

Thus, a set of cutting-edge technologies and practices have been developed and are still being improved or adapted to new species of fish, to enable the genetic heritage of lines produced for research, or for aquaculture purposes, to be secured in a cryobank.

Workshop n°8: Emmanuel Meunier, Laurent Legendre

The health management of aquatic animals used in scientific research is based on an integrated set of organizational, technical and scientific measures aimed at guaranteeing their well-being, the experimental validity of the work and the safety of personnel. It is based on three main pillars: environmental monitoring, clinical monitoring and daily biosecurity.

Water quality parameters, early detection of behavioral anomalies and rigorous maintenance of installations constitute the first lines of prevention. The concept of epidemiological unit (EU), defined as all aquariological systems sharing the same water or presenting a risk of cross-contamination, is central for the implementation of coherent biosecurity and monitoring measures.

A harmonized health surveillance program, developed in international consultation (FELASA, AALAS), distinguishes pathogens to be screened frequently (SMOP – Screen More Often Pathogens) from those requiring epidemiological monitoring (SLOM – Screen Less Often Microorganisms), in order to adapt the frequency of controls to the real risk. Two complementary tools facilitate the standardization and sharing of information: the animal facility description sheet (“Fish Health Monitoring Description”) and the health monitoring report (“Health Monitoring Report”), allowing uniform traceability of breeding and screening data.

Finally, the 4D approach — Detect, Diagnose, Decide, Document — illustrates the integrated and continuous health management approach, essential to transparency and improvement of practices.

This workshop aims to promote a common culture of health management in aquatic research animal facilities, in support of international recommendations and animal welfare requirements.

Since its creation in 2012, the Health Study Network for Aquatic Model Animals (RESAMA) has contributed to the harmonization of health monitoring of aquatic research animal facilities in France and Europe.

This work presents the synthesis of analyzes carried out between 2012 and 2025 on a total of 2,926 animals from 37 fish and 18 amphibian pet stores, representing more than 30 species, mainly Danio rerio and Xenopus laevis.

The examinations carried out include complete autopsies, histology, bacteriology, antibiograms and PCR analyzes targeting the main pathogens (Mycobacterium spp., Pseudoloma neurophilia, Batrachochytrium dendrobatidis, Ranavirus, Chlamydiaceae, etc.).

Despite the expansion to new species and the identification of a few emerging agents (Mycobacterium stomatepiae, M. szulgai), infection rates have remained generally stable since 2019.

This assessment, supported by the GIS FC3R with Inserm funds (project 22FC3R-003), illustrates the relevance of a long-term collaborative system bringing together veterinarians, zootechnicians and researchers. Beyond technical aspects, it highlights the need for continuous coordination to consolidate health surveillance, harmonize screening protocols and promote improvement in animal welfare.

Latest knowledge on different amphibian pathogens and precautions to take when working with amphibians in the field.

The host's symbiotic microbial communities play a major physiological role. For example, the intestinal microbiota is involved in many functions such as energy production, growth and protection against pathogens. The skin microbiota is much less studied and yet it is just as important, particularly in immunity. Based on the latest developments in research carried out at the interface of microbial ecology and disease ecology, we will use the example of amphibians to present the Anna Karenina principle and the adaptive microbiome hypothesis. We will show that environmental factors are capable of modulating the interactions between the host, the pathogen and the host microbiota, and therefore of modulating the expression of the disease. Captivity is an environmental factor of particular interest for promoting the health and well-being of individuals, as it can alter the diversity, composition and therefore functionality of the host microbiota.

Ultrasound constitutes a non-invasive imaging method widely used in human and veterinary medicine, and which is experiencing increasing development in the field of animal experimentation.

Applied to fish, it notably allows the determination of sex, the monitoring of gonadal maturation and the assessment of the physiological state of individuals, while reducing the use of invasive methods.

It also constitutes an essential support, particularly in the research sector, for the selection of broodstock and the management of conservation or aquaculture programs, by providing rapid and precise information directly in the field.