6th RESAMA Days

Cephalopods are currently the only invertebrate organisms to be included in the European directive (2010/63/EU) regulating the use of animals for scientific purposes. Between 2013 and 2017, the COST action FA1301 CephsInAction attempted to respond to these new legislative concerns by mobilizing the cephalopod research community. Five years after the program ended, what emerged was an interdisciplinary network of professionals and institutions, several publications that primarily synthesize available knowledge on cephalopod care and procedures, and many unanswered questions. Considerable work has been initiated to develop guidelines for the care and welfare of cephalopods, but recommendations remain largely general and vague. Further efforts are needed to achieve consensus and standardization. Some researchers have pointed out that the lack of scientific data makes it difficult to define categorical recommendations, which are therefore based on prolonged practical experience in successful cephalopod breeding or on "grey literature".

Studies of cephalopods did not begin until the advent of marine stations in the second half of the 19th century. During the 20th century, numerous works demonstrated the astonishing cognitive abilities of cephalopods. There is no longer any doubt that squid, octopuses and cuttlefish are capable of learning and memorizing information or extracting complex sets of information from their environment. They can also demonstrate causal reasoning and are capable of making decisions. In certain areas, this “mollusk intelligence” is comparable to that of vertebrates (birds and mammals). This presentation aims to review the significant results of recent years in terms of intelligence and sentience in cephalopods.

Cephalopods are the only invertebrates subject to animal experimentation at the moment; despite numerous ethological studies, there is no clear benchmark to quantify the state of well-being of these animals in experimentation.

Research carried out within the Marine Biological Resources Center of the Roscoff Biological Station, in association with the BOREA laboratory of the Natural History Museum of Paris, allows the development of tools for assessing the well-being of the common cuttlefish (Sepia officinalis).

Development and validation of a white muscle biopsy method to monitor fatty acid composition in European sea bass. This non-lethal method is part of a reduction context according to the 3R rule. Biopsy could be applied in several areas of research in order to combine reduction and sampling quality.

Since the first demonstration of the capacity for regeneration following an amputation of the apex of the cardiac ventricle in 2002, and although widely used by the scientific community interested in this phenomenon, the classically implemented apex resection protocol in zebrafish has evolved very little. At the same time, over the last 20 years, knowledge of the perception of pain in teleosts, as well as ethical awareness of the need to address animal suffering, have greatly progressed.

It now appears essential to provide a robust protocol, ethically compatible with regulatory obligations (directive 2010/63/EC, decree 2013-118), involving real pain management during surgical interventions via the systematic use of analgesics, but also by providing an environment and control of effective post-operative parameters, promoting optimal recovery of zebrafish.

One of the major challenges of implementing this protocol, notably the use of analgesic molecules, must however confirm the absence of interaction with the complex mechanisms of cardiac regeneration already demonstrated.

Summary not communicated.

The presentation retraces the entire journey we have taken to set up a cichlid farm (Astatoreochromis alluaudi species). This is feedback, step by step, from the theoretical design of the animal facility to the reproduction of the species raised. It obviously addresses the problems encountered specific to cichlids but also the more general obstacles that must be overcome when one wishes to set up a new breeding with a new species. This presentation may be relevant to anyone doing aquatic model breeding, from project designers to animal technicians.

Killifish or Notobranchius furzeri is the vertebrate animal model with the shortest lifespan that has been described to date, i.e. 6 months. This model is particularly interesting for working on questions related to aging, however the breeding of such special fish presents many challenges compared to the breeding of the fish most used in research, the zebrafish. During my presentation, I will address the monitoring of the longevity and well-being of animals, the particularities associated with their diet, reproduction and breeding conditions.

Summary not communicated.

The African Turquoise Killifish is a new model for studying aging since it has a compressed lifespan of a few months and exhibits all the characteristics of vertebrate aging. Since 2020, the Turquoise Killifish has been implemented at the RESTORE institute in Toulouse where numerous projects aimed at better understanding the reasons for its accelerated aging are under development.

The animal experiment presented aims to evaluate the impact of a mixture of pesticides on flounder, a flatfish commonly found in estuaries.

The fish were exposed for a month to sublethal doses of pesticides reflecting the concentrations typically found in an estuary whose watershed is subject to strong agricultural pressure.

The objective is to identify the metabolic modifications of flounder in response to this exposure to pesticides. The results obtained will make it possible to validate biomarkers whose deregulation, in fish taken from the wild, is a sign of the presence of pesticides.

This project will therefore make it possible, in the long term, to improve the procedures for assessing the quality of estuarine waters.

The porbeagle shark, Lamna nasus, is a mobile species found in cold waters. Since 2012, reported sightings of this species have been increasing, during the summer period, near the coasts in Trégor-Goëlo (22). This increase in observations led to the creation of a project combining research, management and awareness-raising approaches. The aim of the research was to better understand the spatio-temporal dynamics of the species using porbeagle shark individuals. Access to individuals of porbeagle shark in the natural environment is restricted, therefore the biological samples taken are refined in order to optimize the acquisition of data on a little-known species. In order to respond to the challenges of knowledge on this species, analyzes of stable isotopes of carbon and nitrogen were carried out on the basis of muscle biopsies. Biopsy techniques in a natural environment depend on the method of restraining individuals as well as the ease of operation in varied weather conditions. Thus, the biopsy "punch" conventionally used and machined for this species have been replaced by biopsy guns in order to ensure the success and speed of samples whatever the conditions while reducing the impact on the animals.

Development of a non-lethal detection protocol for the parasite and its bryozoan host in rivers, and for parasitic infection in fish.

Proliferative kidney disease (PKD), caused by Tetracapsuloides bryosalmonae, is responsible for significant declines in salmonids. Understanding the distribution of the parasite in the environment and in its hosts therefore seems essential for the development of management plans. I present to you a case study in a natural environment using new molecular tools.

With a view to controlling the health status of zebrafish in order to standardize infectious disease protocols, the INRAe-IERP unit is engaged in the development of a project for the production of zebrafish with controlled flora. These poisons will be a major asset for the study of host-microbiota interactions in infectious or non-infectious contexts.

The Brain Institute (ICM) holds Xenopus for the needs of a research team. The captivity of these animals requires veterinary involvement, particularly for the implementation of the health surveillance program and the management of sick animals.

This review focuses on 6 articles published between 1999 and 2014 which demonstrate the feasibility and interest of developing lab-made (DIY) and low-cost breeding systems. Some technical points of these installations are detailed and the costs are updated by integrating the exchange rate of the time and inflation until 2023. This is to better compare them with each other and with the costs of stand-alone systems that professional manufacturers generally offer for our aquatic species. The discussion attempts to identify the advantages and disadvantages of this type of structure for housing model or wild aquatic animals. A final article on a lab-made food distribution system is associated with this review. The issues of breeding systems and housing environmental parameters with respect to 3R approaches are addressed and discussed within the framework of animal welfare and different scientific fields.

The Flavobacteriaceae family is one of the major problems in fish farming, due to the importance of the problems generated but also due to the ubiquity of these pathogens. This family includes both species of the genus flavobacterium which are particularly problematic in fresh water but also bacteria of the genus tenacibaculum which are problematic in saline environments. Despite the great diversity of hosts, bacterial species and environments, the pathogenicity of this family is ultimately quite homogeneous.

The diagnosis of diseases in fish requires several combined techniques including histopathology. This technique allows you to have an overview and direct you towards other complementary examinations. Some cases encountered in zebrafish, xenopus, etc., but not only that, are presented here.

The National Reference Laboratory (NRL) for regulated fish diseases carries out fish virology analyses. It intervenes in the context of confirmations of positive analyzes for regulated viruses of Viral Hemorrhagic Septicemia (vSHV) or Infectious Hematopoietic Necrosis (vHIN), the first-line analyzes being carried out by one of the 7 approved laboratories of the network. The NRL has at its disposal numerous tools, based on cellular or molecular virology techniques, which allow it to target a wide range of fish viruses, regulated viruses but also emerging viruses potentially problematic for professional sectors or for the natural environment.

Our project aims to improve knowledge on spring viremia in carp. In the context of the 3Rs, we have developed non-invasive in vivo imaging techniques (refinement) for the study of host-pathogen interactions in zebrafish, an alternative model to animal experimentation (reduction and replacement).

Aquaculture is the animal production sector that has developed most rapidly in recent decades (more farmed fish are now raised in volume than cattle), while the issue of animal welfare appears to be an increasingly present concern in our societies. The use of enrichments is certainly a beneficial element in the practice of fish farming, such as rainbow trout, and the experiments carried out by the IBECA platform, in addition to studying the benefits of enrichments for fish, aim to propose and validate innovative and minimally restrictive solutions which can be easily implemented on the production site.

Description of the fish experimental facilities at Ifremer Brest. Presentation of research themes and marine species used as well as the refinement applied in the structures.

Aquaculture is the animal production sector that has developed most rapidly in recent decades (more farmed fish are now raised in volume than cattle), while the issue of animal welfare appears to be an increasingly present concern in our societies. The use of enrichments is certainly a beneficial element in the practice of fish farming, such as rainbow trout, and the experiments carried out by the IBECA platform, in addition to studying the benefits of enrichments for fish, aim to propose and validate innovative and minimally restrictive solutions which can be easily implemented on the production site.

At the University of Perpignan (UPVD), several laboratories require the creation of an “establishment using animals for scientific purposes”.

In this context, the UPVD made available a 100m² premises and €150k for its development.

The presentation briefly describes this aquatic animal facility and emphasizes the measures intended to reduce its environmental impact.

In particular the installation of a double flow VMC to reduce energy consumption and the use of peracetic acid as a disinfectant in order to minimize polluting discharges.

Presentation of the Aquatic Animal Center platform of the Vision Institute (Paris): equipment, accommodation and breeding conditions, services offered to users

The Coastal Environment Research Center is home to small flying foxes Scyliorhinus canicula. In order to improve breeding conditions and comply with regulations, various actions have recently been carried out: 1) drafting of a species sheet for users; 2) validation of a method for photo-identification of individuals. This information could be used to carry out concrete action to improve breeding conditions with behavioral monitoring carried out before and after a restructuring of the groups, carried out in order to limit agonistic interactions between individuals.

Design and construction of a low-cost breeding structure intended for the early life stages of Arctic char (Salvelinus alpinus). This structure was initially designed for ecotoxicology research but remains adaptable to many research questions and can accommodate various organisms.

Currently, the European sardine (Sardina pilchardus) is a species little used in the laboratory even though it is a pelagic fish with major ecological, economic and public health roles. Indeed, it occupies a central place in marine food webs, represents a significant part of global fisheries, and constitutes an important source of long-chain omega 3 for humans. So, could this species become a new model organism in the coming years? This presentation will address different zootechnical aspects (fishing of wild sardines, acclimatization to experimental structures, long-term conditioning, etc.) and biological aspects specific to sardines.

In 2020, Aquaprimeur created the first frog hatchery in Europe. The species bred is the laughing frog P ridibundus, selected by INRA in 1992 and included on the list of domestic animals in 2006: RIVAN92. Agnès JOLY presents the breeding process of this species from reproduction to the froglet just weaned after metamorphosis. The rest of the cycle is carried out by growers, who supply starred restaurants, until now obliged to serve imported frog legs. Then the zootechnical difficulties are mentioned, in particular reproduction, a real obstacle to date: too few fertilized eggs per female.

A large part of aquatic animal experimentation is carried out on zooplankton, either explicitly (as model organisms) or implicitly (as fish prey for example). Using various illustrations in ecology, parasitology and ecotoxicology, we will discuss how we should and can consider the notions of animal welfare and apply the 3Cs. Without giving an answer, the objective will rather be to question the implementation of animal experimentation on zooplankton.