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In mammalian research, the implementation of the 3Rs is ubiquitous. However, the adaptation of procedures for experimental work on fish seems less extensive in comparison, even though fish model organisms are common in a broad range of research fields already. To strengthen animal welfare in experimental research, we provide an overview of current research results, including studies on the nociception of fishes. Furthermore, we describe the potential of implementing the 3Rs in fish experimental research. In the context of "Reduction", we show alternative research methods to lethal sampling. Considering "Refinement", we point out possibilities to improve fish handling and indicate that adaptations to the individual species ecology are necessary. Under the aspect of "Replacement", we describe the high potential of cell cultures that can be obtained from fish tissue and give an overview of the already extensive use in ecotoxicology and virology. In addition, we illustrate that cell cultures could also be increasingly used for basic research. Graphical abstract

Pikeperch (Sander lucioperca) is a freshwater fish that has become increasingly popular as a food fish. Within this study, the influence of the spawning process on meat quality was investigated. For this purpose, adult pikeperch was examined directly before and after spawning, and compared regarding physical meat parameters and lipid composition. The results indicate that after spawning, the values of the pH, the electrical conductivity and the yellowness value of fillet were significantly higher than those of the animals sampled before spawning. Analysis of the sum of the total lipid content indicates no differences before and after closed season, but differences in the fatty acid profile were present. Despite significant lower MUFA concentrations, the EPA and DHA showed unaffected high contents. Therefore, the fish muscle indicated an equivalent meat quality. Nevertheless, the significant changes of some physical meat quality parameters after spawning season could have a particular impact on the shelf life and storage of the pikeperch fillet, highlighting the need for further research.

In this study, a cell line of the fish species Coregonus maraena was produced for the first time. C. maraena is an endangered species, and studies indicate that this fish species will be affected by further population declines due to climate change. This cell line, designated CMAfin1, has been maintained in Leibovitz L-15 supplemented with 10% fetal bovine serum over 3 years. Both subculturing and storage (short-term storage at -80°C and long-term storage in liquid nitrogen) was successful. Cell morphology and growth rate were consistent from passage 10 onwards. Immunocytochemical examination of cellular proteins and matrix components confirmed the mechanical stability of the cells. Actin, fibronectin, vinculin, vimentin, and tubulin are present in the cells and form a network. In addition, the transport of molecules is ensured by the necessary proteins. Gene expression analysis showed a shift in the expressions of stem cell markers between younger and higher passages. While SOX2 and IGF1 were more highly expressed in the seventh passage, SOX9 and IGF2 expressions were significantly increased in higher passages. Therefore, the stable cell culture CMAfin1 can be used for applied analysis to further understand the cell physiology of C. maranea.

Exposure to elevated temperatures during embryogenesis can influence the plasticity of tissues in later-life. Despite these long-term changes in plasticity, few differentially expressed genes are ever identified, suggesting that the developmental programming of later-life plasticity may occur through the modulation of other aspects of the transcriptomic architecture, such as gene network function. Here, we use network modelling approaches to demonstrate that warm temperatures during embryonic development (developmental warming) have consistent effects in later-life on the organisation of transcriptomic networks across four diverse species of fishes:Scyliorhinus canicula, Danio rerio, Dicentrarchus labrax, andGasterosteus aculeatus. The transcriptomes of developmentally warmed fishes are characterised by an increased entropy of their pairwise gene interaction networks, implying a less structured, more ‘random’ set of gene interactions. We also show that, in zebrafish subject to developmental warming, the entropy of an individual gene within a network is associated with that gene’s probability of expression change during temperature acclimation in later-life. However, this association is absent in animals reared under ‘control’ conditions. Thus, the thermal environment experienced during embryogenesis can alter transcriptomic organisation in later-life, and these changes may influence an individual’s responsiveness to future temperature challenges.

Salmon pancreas disease virus (SPDV) has been affecting the salmon farming industry for over 30 years, but despite the substantial amount of studies, there are still a number of recognized knowledge gaps, for example in the transmission of the virus. In this work, an ultrastructural morphological approach was used to describe observations after infection by SPDV of an ex vivo cardiac model generated from Atlantic salmon embryos. The observations in this study and those available on previous ultrastructural work on SPDV are compared and contrasted with the current knowledge on terrestrial mammalian and insect alphaviral replication cycles, which is deeper than that of SPDV both morphologically and mechanistically. Despite their limitations, morphological descriptions remain an excellent way to generate novel hypotheses, and this has been the aim of this work. This study has used a target host, ex vivo model and resulted in some previously undescribed features, including filopodial membrane projections, cytoplasmic stress granules or putative intracytoplasmic budding. The latter suggests a new hypothesis that warrants further mechanistic research: SPDV in salmon may have retained the capacity for non-cytolytic (persistent) infections by intracellular budding, similar to that noted in arthropod vectors of other alphaviruses. In the notable absence of a known intermediate host for SPDV, the presence of this pattern suggests that both cytopathic and persistent infections may coexist in the same host. It is our hope that the ultrastructural comparison presented here stimulates new research that brings the knowledge on SPDV replication cycle up to a similar level to that of terrestrial alphaviruses.

Sander lucioperca is an organism of growing importance for the aquaculture industry. Nonetheless, the rearing of S. lucioperca larvae is proving to be a difficult task as it is facing a high mortality rate during hatching and the change to exogenous feeding. To gain insight into growth patterns during this period, the authors analysed pikeperch embryos and larvae from 9 days before hatching to 17 days after hatch. Hereby they were able to describe a natural development by using close to natural conditions based on using a direct flow-through supply of lake fresh water on specimens from a local wild population. The results show that between the early embryonic stages a steady growth was visible. Nonetheless, in between hatching and the start of exogenous feeding, a phase of growth stagnation took place. In the following larval stages, an increased growth with large size variations between individual specimens appeared. Both factors are conspicuous as they can indicate a starting point for cannibalism. With this analysis, the authors can provide a fundament to support the upcoming research on S. lucioperca and aid to optimize size-sorting procedures for a higher survival of pikeperch stock in aquaculture.

The zebrafish (ZF) has become an essential model for biomedical, pharmacological and eco-toxicological heart research. Despite the anatomical differences between fish and human hearts, similarities in cellular structure and conservation of genes as well as pathways across vertebrates have led to an increase in the popularity of ZF as a model for human cardiac research. ZF research benefits from an entirely sequenced genome, which allows us to establish and study cardiovascular mutants to better understand cardiovascular diseases. In this review, we will discuss the importance of in vitro model systems for cardiac research and summarise results of in vitro 3D heart-like cell aggregates, consisting of myocardial tissue formed spontaneously from enzymatically digested whole embryonic ZF larvae (Zebrafish Heart Aggregate - ZFHA). We will give an overview of the similarities and differences of ZF versus human hearts and highlight why ZF complement established mammalian models (i.e. murine and large animal models) for cardiac research. At this stage, the ZFHA model system is being refined into a high-throughput (more ZFHA generated than larvae prepared) and stable in vitro test system to accomplish the same longevity of previously successful salmonid models. ZFHA have potential for the use of high-throughput-screenings of different factors like small molecules, nucleic acids, proteins and lipids which is difficult to achieve in the zebrafish in vivo screening models with lethal mutations as well as to explore ion channel disorders and to find appropriate drugs for safety screening.

The renin‐angiotensin system promotes oxidative stress, apoptosis, necrosis, fibrosis, and thus heart failure. Secretory renin plays a central role in these processes, initiating the generation of angiotensins. Nevertheless, alternative renin transcripts exist, which code for a cytosolically localized renin isoform (cyto‐renin) that is cardioprotective. We tested the hypothesis that the protective effects are associated with a beneficial switch of metabolic and mitochondrial functions. To assess H9c2 cell mitochondrial parameters, we used the Seahorse XF analyser. Cardiac H9c2 cells overexpressing cyto‐renin exhibited enhanced nonmitochondrial oxygen consumption, lactate accumulation, and LDH activity, reflecting a switch to more aerobic glycolysis known as Warburg effect. Additionally, mitochondrial spare capacity and cell respiratory control ratio were enhanced, indicating an increased potential to tolerate stress conditions. Renin knockdown induced opposite effects on mitochondrial functions without influencing metabolic parameters. Thus, the protective effects of cyto‐renin are associated with an altered bioenergetic profile and an enhanced stress tolerance, which are favourable under ischaemic conditions. Therefore, cyto‐renin is a promising new target for the prevention of ischaemia‐induced myocardial damage.

This study compares the trunk skeletal muscle anatomy in 870- and 2900-degree-day-old lesser-spotted dogfish larvae (Scyliorhinus canicula) via haematoxylin/eosin staining as well as immunohistochemistry and Western blot analysis. The results showed poorly differentiated muscle formation in the trunk segments in the younger larvae and fully developed skeletal muscle with a division of red and white cells in the older larvae. The stem cell marker PAX7, which is present in all developmental stages of teleost fish, is only expressed in the younger dogfish. The results show the necessity of examining the skeletal muscle development in sharks to understand the evolutional changes from cartilaginous fishes to teleosts.

Elasmobranchs display various reproductive modes, which have been key to their evolutionary success. In recent decades there has been a rise in the number of reported cases of foetal abnormalities including fertilised, double-embryos held within one egg capsule, hereafter referred to as twins. Previously, the occurrences of twin egg cases have been reported in two batoid and one shark species. We report the first cases of twins in three species of oviparous elasmobranchs: the undulate ray (Raja undulata), the nursehound (Scyliorhinus stellaris), and the small-spotted catshark (Scyliorhinus canicula). We investigated the genetic relationships between the twins in S. stellaris, and S. canicula using microsatellite markers. Whilst the S. stellaris twins displayed the same genotypes, we found that the S. canicula twin individuals arose through heteropaternal superfecundation. This is the first reported incidence of such a paternity in elasmobranchs. The relationship between environmental change and reproductive strategy in elasmobranchs is unclear and further research is needed to determine its effect on the prevalence and mechanisms of formation of elasmobranch twins.

Background/Aims: Renin is known as a secretory glycoprotein that ultimately leads to angiotensin II generation. In this way renin exerts pro-inflammatory effects and promotes cardiac injury. Additional transcripts have been identified encoding for a cytosolic renin isoform that - in contrast to secretory renin - exhibits cardioprotective effects under ischemic conditions. The promoter of these transcripts is unknown. Methods: Using qRT-PCR and dual-luciferase reporter assay we examined the expression and promotor activity of cytosolic renin as well as the regulation by glucose starvation in H9c2 cardiomyoblasts. Results: We identified a promoter in intron1 of the rat renin gene with two glucose starvation-sensitive regions. One region contains a binding motif for serum response factor (SRF). Under glucose depletion expression of SRF increased prior to cytosolic renin. SRF knock down selectively decreased cytosolic renin expression and attenuated the increase of cytosolic renin expression under glucose depletion. Conclusions: Transcripts encoding for secretory and cytosolic renin are differentially expressed. The low basal expression of cytosolic renin as well as its induction under ischemia-related conditions represents an efficient system regulated in accordance with its previously identified unfavorable effects under control situations but protective effects seen after myocardial infarction or glucose depletion.

Background/Aims: Cellular models are an interesting tool to study human heart diseases. To date, research groups mainly focus on mouse models, but important murine physiology is different from human characteristics. Recently, scientists found that the electrophysiology of fish cardiomyocytes largely resembles that of humans. So far, cardiomyocyte models were generated using differentiation medium, were stimulated electrically or, when contracting spontaneously, only did so over a short time period. We established an in vitro spontaneously, long-term beating heart model generated from rainbow trout, with the potential to be used as a new human heart model system because of its electrophysiology. Methods: Spontaneously contracting 3D cell layers from rainbow trout were generated in vitro and analyzed using PCR and immunochemistry. Further, electrophysiology was measured via intra – and extracellular recordings. Results: Contracting cardiomyogenic aggregates were generated without differentiation medium and were beating autonomously for more than one month. Electrophysiological measurements exhibit that the action potential properties of fish cardiomyocytes in part resemble the characteristics of human cardiomyocytes. The sensitivity of the beating cell aggregates to drugs could also be confirmed. Conclusion: Spontaneously contracting cardiomyogenic cell aggregates from rainbow trout generated in vitro are suitable for human heart research and pharmacology.

We describe here a novel, fast and inexpensive method for producing a 3D ‘heart’ structure that forms spontaneously, in vitro, from larval zebrafish (ZF). We have named these 3D ‘heart’ structures ‘zebrafish heart aggregate(s)’ (ZFHAs) and have characterised their basic morphology and structural composition using histology, immunohistochemistry, electron microscopy and mass spectrometry. After 2 days in culture the ZFHA spontaneously form and become a stable contractile syncytium consisting of cardiac tissue derived by in vitro maturation, which beats rhythmically and consistently for more than 8 days. We propose this model as a platform technology, which can be developed further to study in vitro cardiac maturation, regeneration, tissue engineering and safety pharmacological/toxicology testing.

Cellular models are an interesting tool to study human heart diseases. To date, research groups mainly focus on mouse models, but important murine physiology is different from human characteristics. Recently, scientists found that the electrophysiology of fish cardiomyocytes largely resembles that of humans. So far, cardiomyocyte models were generated using differentiation medium, were stimulated electrically or, when contracting spontaneously, only did so over a short time period. We established an in vitro spontaneously, long-term beating heart model generated from rainbow trout, with the potential to be used as a new human heart model system because of its electrophysiology.Spontaneously contracting 3D cell layers from rainbow trout were generated in vitro and analyzed using PCR and immunochemistry. Further, electrophysiology was measured via intra - and extracellular recordings.Contracting cardiomyogenic aggregates were generated without differentiation medium and were beating autonomously for more than one month. Electrophysiological measurements exhibit that the action potential properties of fish cardiomyocytes in part resemble the characteristics of human cardiomyocytes. The sensitivity of the beating cell aggregates to drugs could also be confirmed.Spontaneously contracting cardiomyogenic cell aggregates from rainbow trout generated in vitro are suitable for human heart research and pharmacology.

In primary cell preparations from larvae of rainbow trout Oncorhynchus mykiss, the formation of autonomously contracting cell aggregates was observed after 7 days. These contracting elements could be propagated and some aggregates were maintained over a period of 35 days. Electron microscopical and immunocytochemical examination revealed the presence of cardiomyocytes.