The use of fish in scientific research is increasing worldwide, due to both the rapid expansion of the fish farming industry and growing awareness of questions concerning the humane use of mammalian models in basic research and chemical testing. As fish are lower on the evolutionary scale than mammals, they are considered to be less sentient. Fish models are providing researchers, and those concerned with animal welfare, with opportunities for adhering to the Three Rs principles of refinement, reduction and replacement. However, it should be kept in mind that fish should also be covered by the principles of the Three Rs. Indeed, various studies have shown that fish are capable of nociception, and of experiencing pain in a manner analogous to that in mammals. Thus, emphasis needs to be placed on the development of alternatives that replace, as much as possible, the use of all living vertebrate animals, including fish. This review gives the first comprehensive and critical overview of the existing alternatives for live fish experimental studies. The alternative methods described range from cell and tissue cultures, organ and perfusion models, and embryonic models, to in silico computer and mathematical models. This article aspires to guide scientists in the adoption of the correct alternative methods in their research, and, whenever possible, to reduce the use of live fish.

A thorough understanding of host−microbe interactions is crucial for more efficient disease management in the marine larviculture industry. As demonstrated in terrestrial animal research, gnotobiotic systems (involving animals cultured in germ-free conditions or inoculated with known microorganisms) are excellent tools to extend our understanding of the mechanisms involved in host−microbe interactions and allow the evaluation of new treatments for diseases. In this study, we introduce a germ-free European sea bass Dicentrarchus labrax larval model, independent of the continuous addition of antimicrobial agents. This model has an experimental set-up that allows addition of live feed to the larvae without compromising the germ-free status. This model will facilitate and render aquaculture research more effective in terms of mitigation fish larval diseases.KEY WORDS: Aquaculture · Gnotobiotic · Pathogen · Larvae Resale or republication not permitted without written consent of the publisherDis Aquat Org 117: [177][178][179][180][181][182][183][184][185] 2016 towards more strict regulations on the use of antibiotics in the aquaculture sector (Suba singhe et al. 2001, Romero et al. 2012, hence calling for alternative, sustainable methods to which the aquaculturist can resort for preventing and controlling disease outbreaks. Several environmentally friendly prophylactic disease treatments have been the focus of recent research, i.e. probiotics, prebiotics, vaccines, immunostimulants or antimicrobial peptides (Merrifield & Ringø 2014).A judicious and scientifically supported application of the abovementioned alternatives warrants a thorough testing of their efficacy and safety under standardized and controlled experimental conditions (Smith et al. 2003). However, the stochastic colonization of larvae by microorganisms may hinder the establishment of a reproducible experimental set-up by generating high inter-individual and inter-batch variability in the composition of the standing microbial community. Hence, from a microbiological point of view, iterating experimental conditions using conventional animals is almost impossible (Fjellheim et al. 2012). The development of test systems in which the researcher has complete control over the microbial community structure, by adopting a germ-free or gnotobiotic model, was revolutionary in this respect. As already demonstrated in multiple terrestrial animal studies, gnotobiotic models are an excellent tool to extend our understanding of (1) the nutritional requirements of host organisms, (2) host−microbe interactions and (3) host metabolic functions (Gordon & Pesti 1971, Wostmann 1996, Marques et al. 2005. Despite the significant increase in the use of fish as experimental animals during the last decades (Marques et al. 2005, Schaeck et al. 2013) and the stressed importance of raising aquatic organisms gnotobiotically (Bates et al. 2006), the current know-how of rearing gnotobiotic aquatic organisms is much more limited compared to the more traditional mammalian l...

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