7th RESAMA Days

Changes made by Directive 2024/1262 of March 13, 2024 amending Directive 2010/63 and in particular changes in housing conditions for fish (in particular zebrafish) and cephalopods.

Vertebrates and cephalopods are protected by European regulations on animal experimentation. In France, it is therefore necessary to request project authorization from the ministry responsible for research, as long as a procedure is planned which implies that an animal is likely to experience pain, suffering or anxiety or to suffer lasting damage. The Ministry of Research, to make its decision, relies on the opinion of animal experimentation ethics committees located throughout the country. We will briefly present the missions of ethics committees, the principles of ethical evaluation as well as the functioning of an ethics committee. Emphasis will be placed on certain points of vigilance to keep in mind when preparing projects involving fish.

Often considered as a new regulatory constraint, the project authorization request should be seen as a way of questioning the need to carry out an experimental procedure on animals. Drafting the DAP is an exercise in style, certain points of which will be carefully analyzed by the ethics committee and the AFIS unit of the Ministry of Higher Education and Research. So what should be its points of attention?

Ecotoxicology remains a little-known subject despite its major role in environmental protection. Indeed, European legislation requires any manufacturer wishing to place a product on the market to carry out an assessment of the impact of said product on the environment. The company Rovaltain Research Staphyt responds to this demand by offering ecotoxicity tests on vertebrate and invertebrate organisms, whether terrestrial (i.e. bees) or aquatic (i.e. fish; amphibian).

The maintenance of vertebrate species and their well-being is one of the central points of the company: the breeding system as well as the practices put in place by the SBEA will be presented to you with a particular point on the Pimephales Promelas species.

Presentation, history, choices and perceptions for the AZR within the Rockefeller faculty. A new model for the scientific community in Lyon Est.

ACSED is a support platform for research and teaching located on the La Doua Campus of Claude Bernard Lyon 1 University in Villeurbanne. It hosts endotherm and ectotherm species which are either conventional or from wild fauna.  It has numerous accommodation and experimentation rooms spread over 620m².

In animal testing, the “end point” is defined as the moment from which the suffering and/or distress of an experimental animal must be stopped, minimized or reduced (AFSTAL definition). The 3 R rule (replace, reduce, refine) promotes the refinement of experimental procedures, in particular by determining limit points which are still little investigated and difficult to define for fish species. Failing that, several scoring grids have been developed, based essentially on the daily observations/macroscopic analyzes of the experimenters.

The IERP (Experimental Infectiology of Rodents and Fish) unit carries out work in infectiology aimed at studying pathogenic host interactions, characterizing the virulence of different pathogens or identifying diagnostic methods or treatments for infectious diseases. In standard protocols, most of which are classified as “severe procedures”, the stopping criterion is the death of the infected individual due to the absence of end points. Our commitment to the refinement of procedures and animal welfare, in accordance with the evolution of ethical protocols, leads to a finer characterization of phenotypes/behaviors determining end points such as a lack of response to external stimuli, atypical swimming or a modification of food intake.

As part of the European VetBioNet program (WP9.2 Behavior and Physiological Monitoring), IERP in collaboration with the Dutch company Noldus (www.noldus.com) has implemented an innovative approach for individual behavioral monitoring of rainbow trout infected by flavobacteriosis by bathing. Existing approaches are mainly limited to the behavioral study of the zebrafish model by individual monitoring after isolating the animal (stress factor) or for monitoring a group of individuals in global analyzes (loss of information on inter-individual variabilities in response to infectious agents). Our work makes it possible for the first time to study the individual behavior of rainbow trout within a group, for several days during an infectious process. The first results reveal the appearance of behavioral anomalies during the infection phases and then during the process in infected individuals compared to control individuals. The identification and quantification of different parameters is in progress. Their analysis and integration provide precise information on these anomalies then correlated with biological data to ultimately envisage the construction of tools for prediction and automated identification of end points in infectious diseases.

Zebrafish are increasingly used for the study of cancer. The most common model is that of melanoma, an aggressive skin cancer resulting from the transformation of melanocytes which form pigmented fish. We use this model to evaluate the influence of certain genes on the occurrence of tumors and on their biological properties, in particular their metastatic dissemination. Here we will describe some typical experiments on this type of model, mentioning in particular the specificities of monitoring fish developing tumors and the associated end points.

Presentation of our strategy, allowing the refinement of a fin harvesting procedure on adult zebrafish.

This work, carried out under joint supervision of INRAE-ANSES, aims to study the effects of rising temperatures and associated stress on 1/ the health of fish and 2/ two pathogens of salmonids, the viral hemorrhagic septicemia virus (VSHV) and the infectious hematopoietic necrosis virus (IHNV), viruses presenting a broad host spectrum and a high evolutionary potential 3/ Host/pathogen interactions. This infectious disease project is based on a multi-scale approach from in vitro to in vivo.

The first results obtained as part of this doctoral project will be presented. They will contribute to improving knowledge on the impact of warming on the cellular physiology of trout, to identifying stress markers and to evaluating the adaptation capacities of novirhabdoviruses - for better anticipation of the emergence of epidemics.

This project is carried out in collaboration with Stéphane Biacchesi's team, as part of Calvin Fauvet's thesis (Université Paris-Saclay, INRAE, UVSQ, Molecular Virology and Immunology, Jouy-en-Josas, France).

The aquatic platform of the Toulouse Integrative Biology Center brings together 3 animal facilities, zebrafish, xenopus and hemigrammus. Under the direction of the aquatic zootechnics department, these models are used for internal research but also in the context of teaching for the Paul Sabatier University of Toulouse. In 2025, external services will be offered.

Due to their unique phylogenetic position, at the boundary between invertebrates and vertebrates, tunicates offer a valuable model for the study of the origin of vertebrates. Among them, Ciona intestinalis has established itself as a reference species in chordate developmental biology since the end of the 19th century. Since the sequencing of its genome in 2002, it has also been a key model in fundamental molecular biology. Faced with growing demand, an innovative production route has been developed to provide laboratories with quality specimens in sufficient quantities throughout the year.

Based on the free CherryPy framework, the Cherryfish software is an aquatic animal monitoring software resulting from the need to follow lines in zebrafish and cichlid farms within the Ruggiero research team at the IGFL.

The software allows management and monitoring of animal facility groups, monitoring of physicochemical conditions and daily actions to be carried out within the animal facility. This software is a true extension of the handbook in paper format and is complementary to it.

What are the determining factors in the choice to design specific tools for the management of animals within a structure? This question raises the importance of adapting management systems to specific needs.

Three examples of 'in-house' designs for the management of Xenopus, Sturgeon and Danios will be explored, based on tools developed at the IBPS aquatic animal facility at Sorbonne University and at the INRAE ​​aquaculture experimentation station in Saint-Seurin-sur-l'Isle.

Presentation of the breeding and experimental software “Mykiss” from the company KAMAHU - management of stocks of fish, food and veterinary products - technical data of aquaculture breeding  - multi-species software adaptable to different structures

Presentation of a management tool developed internally on Excel allowing the monitoring of the operation of the animal facility and the use of animals to meet regulatory and Ministry of Research requirements (genetically altered lines), DDPP inspections and facilitate annual declarations.

By associating a batch of animals with a line, a protocol, and actions such as entry - exit - grouping - separation - use of drugs, etc. the tool feeds Excel files and generates sheets (Entry/exit register - Laying records - Daily monitoring - Monitoring of batches of animals - Medication register - Monitoring by protocol/lineage - Monitoring of accommodation services/Laying) in pdf format that is easily consultable and understandable.

Presentation of the results of the survey carried out among the RESAMA community on the feedback from different users of pet store management software.

The National Committee for Ethical Reflection on Animal Experimentation (CNREEA) is a multidisciplinary advisory committee placed within the National Commission for the Protection of Animals Used for Scientific Purposes (known as CNEA), responsible for advising public authorities on animal protection.

It published the National Charter on the ethics of animal experimentation and operating guides for animal experimentation ethics committees (CEEA) which supplement the legislative texts in force.

It establishes the annual report of the ethics committees and makes recommendations aimed at improving their practices.

It publishes methodological recommendations likely to improve the welfare of animals used for experimental or other scientific purposes.

The GIS FC3R (French Center for the 3Rs)'s mission is to encourage and implement the ethical principle of the 3Rs - Replace, Reduce, Refine - in research in France through education, the promotion of responsible and innovative research, and transparent communication.

It undertakes concrete actions to bring together a strong and synergistic community around the needs and objectives of researchers of today and tomorrow: project financing, 3R Prizes, training, project engineering, data sharing, transparent communication, etc.

During this first year, the members of the animal placement working group discussed the practices in place in their establishments and the practices already authorized. In order to support other structures in this process, species sheets have been prepared as well as educational kits which can be made available to establishments to simplify the transmission of information to buyers on the characteristics and needs of the animals placed.

Work will continue into 2025 to forge links with reception structures, communication and support for user establishments. Volunteers are welcome in this GT.

Practical illustration of the collaboration between academia and the Planet Ocean Montpellier aquarium which has enabled the publication of new results on the conditions of captivity, reproduction, embryonic development and growth of the spotted chimera.

This partnership is an example of the many successful collaborations that exist between public aquariums and research laboratories.

Since the creation of the Public Aquariums webpage in FishBase in the early 2000s, no collaboration could be established between the FishBase consortium and public aquariums, so this page remained unchanged for almost 20 years. At the end of 2019, we decided to try to develop a strong and lasting collaboration between FishBase and French public aquariums to promote the knowledge and data acquired within their establishments thanks to FishBase. After five years, more than 50 students from the professional degree “Continental Aquaculture and Aquariology” at IUT Nancy Brabois (University of Lorraine, France) participated in the project. The web page has been modified, including an increase in the number of public aquariums listed (from 39 to 52) and the updating or addition of new species lists for seven of them. Nearly 700 photos from three aquariums were also uploaded, some of which are now the chosen image for the species page.

Three articles describing these collaborations were published in a special issue of Cybium celebrating the 30th anniversary of FishBase and the 15th anniversary of SeaLifeBase. In 2024, 21 students completed a survey to evaluate four topics: 1. collaboration between public aquariums and research, 2. description of aquarium facilities and equipment, 3. monitoring the life cycle of fish in their aquarium, and 4. collaborations with FishBase. Overall, we found that aquariums are willing to collaborate more with researchers and want to continue sharing their data with FishBase. As a proof of concept, a pair of students spoke with 10 aquariums to learn several pieces of information for each species housed, including the origin of the fish, levels of domestication within the aquarium, number of generations, maximum age and length, and whether photos were available. This work is continuing this year in order to update each aquarium's page on FishBase in 2025.

Conservation physiology is a discipline that is increasingly at the center of common interests of researchers from several disciplines and managers.

Indeed, understanding how organisms are able to adapt to increasingly significant environmental constraints is necessary to effectively build conservation plans. A particular case is that of the Apron du Rhône, an emblematic fish of our rivers and in danger of extinction. Thanks to close collaboration with the Citadel of Besançon, studies in experimental conditions were carried out with the aim of understanding the physiology of this particular model.

Within Ifremer, the Atlantic Marine Molluscs Experimental Unit offers experimental resources for all research units working on marine molluscs, crustaceans or microalgae.

It is developing strong expertise in the field of water treatment from shellfish farms, in order to improve the safety of animals and the protection of the environment.

Marine mollusks, and more particularly the Pacific oyster, are good alternative models for studying the health of marine ecosystems and anthropogenic impacts.

The Lyon Institute of Functional Genomics (IGFL) is a mixed research unit whose themes revolve around three main axes: Development, Physiology and Evolution. In order to understand how these disciplines shape the genome, one of the particularities of the IGFL is the use of unconventional model organisms such as Gerromorphs and Parhyales.

Gerromorphs, or “water skaters,” are semi-aquatic insects. They are present on all continents and live on the surface of the water in very varied environments. Gerromorph species exhibit a number of evolutionary innovations and diverse morphologies. Their adaptation to life on the aquatic surface and their biodiversity make them a good model for identifying the genes and genetic modifications responsible for morphological changes during evolution (evo-devo).

Parhyales are small omnivorous and detritivorous crustaceans living in shallow marine waters. Like most crustaceans, they have the particularity of being capable of regenerating their limbs (legs and antennae). In addition, they are small and transparent, and can be manipulated genetically, which makes them a model of choice for studying regeneration mechanisms by microscopy and genetics. The species raised in the laboratory is Parhyale hawaiensis.

Our presentation will focus on the breeding methods developed for these organisms, as well as the research themes associated with them.

Located on the island of Embiez, the Paul Ricard Oceanographic Institute (IOPR) has never stopped for more than 50 years, mixing research and awareness, science and sharing.

The acquisition of knowledge about the marine environment is therefore strongly associated with transmission to the general public, schools, professionals, institutions and scientists.

Its research activities are versatile and linked to major climate issues and sustainability sciences. In addition to its research laboratory, last year IOPR acquired an innovative 600 m² aquaculture platform.

The presentation aims to illustrate this new tool and the main programs carried out there.

The EXAAQT platform (Animal Experimentation in Aquatic Environment) is a platform shared between three research units and is intended to host short and medium-term experiments on aquatic animal species (i) falling within the framework of regulations on animal experimentation or (ii) requiring specific experimental facilities: accommodation, use of contaminants, behavioral analyses.

The platform can accommodate invertebrate animals (mollusks, urochordates, cnidarians, crustaceans, etc.) and vertebrate animals (cartilaginous and bony fish). The subject of research projects is the biological processes at the origin of the evolution of species, their ecology, in particular their relationship to other species and the physico-chemical parameters of their biotic and abiotic environment.

Presentation of the current state of the platform for the approval request.

Role of acoustic and multimodal communication during social interactions in fish. Some impacts of anthropogenic sounds on behavior and cognition.

The Mediterranean shad, a potamotoque fish, is a species that is both particularly demanding in terms of crossing obstacles and fish passes, but also fragile and sensitive to handling. A desire emerging from the authorities for the management of migratory fish in the Rhône Méditerranée territory is to construct a study to assess the passability of structures crossing thresholds or dams built on the rivers of the territory.

Such a telemetry study was implemented in the early 2000s on the Rhône and its renewal is recommended by the MIgratory FISH MANAGEMENT PLAN (PLAGEPOMI) to assess the benefits of restoring ecological continuity. To do this, it seemed necessary to review the protocol for capturing and marking Mediterranean shad in order to better take into account their sensitivity to handling and limit the impacts of handling on the fish as much as possible. Thus, the choice was made to carry out initial experiments on the Hérault river, colonized each year by Shads.

At each stage of our protocol, thought has been given to minimizing the impact of marking on individuals. This protocol was also refined during three years of investigations thanks to regular exchanges between the project team, researchers and veterinarians.

The results obtained made it possible to initiate reflections on the management of the structure where the experiments were carried out. This protocol for capturing and marking Mediterranean shad now seems transposable and discussions on setting up a large-scale study on the Rhône are underway.

At the SIMOS Laboratory of Toxins, Receptors and Ionic Channels (CEA-Saclay), we use Xenopus (Xenopus laevis) as oocyte donors to characterize the mechanism of action of neurotoxins or synthetic molecules on their molecular target.

This communication is feedback on the use of the X. laevis oocyte model for electrophysiology. Oocytes are recovered in large numbers by laparotomy at developmental stage V-VI Dumont, i.e. immature eggs incapable of being fertilized. Indeed, the X. laevis oocyte (stage V-VI) allows the synthesis of exogenous receptors and/or ion channels after injection of genetic material (cDNA, mRNA). The proteins are correctly folded and inserted into the plasma membrane of the oocyte, making electrophysiological recordings possible without the use of a living animal.

By using an appropriate surgical technique, the animal's pain and stress are reduced to a minimum, without post-operative complications. Additionally, laparotomies are limited to six with an interval of four months between each operation per animal. The use of electronic chips allows us to monitor each xenopus operated on (weight, loss of stitches, behavior before and after the operation).

Cephalopods, like vertebrates, have been included since 2013 in Directive 2010/63/EU relating to the use of animals for research.

Refinement work on the common cuttlefish, Sepia officinalis, continues at the Marine Biological Resources Center of the Roscoff Biological Station, in association with the Natural History Museum of Paris, in order to improve the consideration of Well-Being and to offer a relevant “scoring” sheet to the scientific community using the model.

The American organization OCEARCH carries out great white shark fishing campaigns in order to improve knowledge and the ecology of this species. The campaigns rely on local researchers and mobilize the international scientific community. The campaign carried out in French waters in August 2024 is part of a European campaign stretching from Spanish waters to Irish waters. These international campaigns allow the deployment of beacons and the collection of biological material.

At the French level, a campaign of this magnitude requires the coordination of various institutional partners and the obtaining of several authorizations including a request for authorization for a project using animals for scientific purposes. This experience, which mobilized many stakeholders, is an opportunity to share on the various regulatory aspects and the challenges of international scientific collaboration with regard to animal ethics.