Perspective Chapter: <em>In vitro</em> Contracting Cardiomyogenic Models from Whole Fish Embryos and Larvae – Method, Properties, and Applications

Heart diseases remain a leading cause of mortality worldwide. The development of effective treatments and interventions relies on a deep understanding of cardiac biology. Traditional two-dimensional (2D) cell cultures and animal models fall short in replicating crucial physiological and pathological features of cardiac tissue. In response, 3D cardiac models have emerged, offering a more faithful replication of the native heart tissue’s architecture and functionality in a controlled environment. Although technical hurdles limit the widespread adoption of in vitro 3D models, they hold promise for advancing cardiovascular research. This chapter provides a description of the development of 3D spontaneously contracting cardiac primary cultures derived from fish embryos and larvae, presenting an easily accessible model for diverse applications, including the investigation of viral heart infections, as well as biomedical, pharmacological, and cardiology research. In this chapter, we will highlight the importance of in vitro model systems for modern cardiac research. Additionally, we will provide an overview of the protocol and results concerning the creation of in vitro 3D heart-like cell aggregates using enzymatically digested whole fish embryos/larvae. These aggregates exhibit long-term stability and spontaneous contractions, making them promising candidates for high-throughput screenin

Atlantic salmon cardiac primary cultures:An in vitro model to study viral host pathogen interactions and pathogenesis

Development of Salmon Cardiac Primary Cultures (SCPCs) from Atlantic salmon pre-hatch embryos and their application as in vitro model for cardiotropic viral infection research are described. Producing SCPCs requires plating of trypsin dissociated embryos with subsequent targeted harvest from 24h up to 3 weeks, of relevant tissues after visual identification. SCPCs are then transferred individually to chambered wells for culture in isolation, with incubation at 15-22°. SCPCs production efficiency was not influenced by embryo's origin (0.75/ farmed or wild embryo), but mildly influenced by embryonic developmental stage (0.3 decline between 380 and 445 accumulated thermal units), and strongly influenced by time of harvest post-plating (0.6 decline if harvested after 72 hours). Beating rate was not significantly influenced by temperature (15-22°) or age (2-4 weeks), but was significantly lower on SCPCs originated from farmed embryos with a disease resistant genotype (F = 5.3, p<0.05). Two distinct morphologies suggestive of an ex vivo embryonic heart and a de novo formation were observed sub-grossly, histologically, ultra-structurally and with confocal microscopy. Both types contained cells consistent with cardiomyocytes, endothelium, and fibroblasts. Ageing of SCPCs in culture was observed with increased auto fluorescence in live imaging, and as myelin figures and cellular degeneration ultra-structurally. The SCPCs model was challenged with cardiotropic viruses and both the viral load and the mx gene expression were measurable along time by qPCR. In summary, SCPCs represent a step forward in salmon cardiac disease research as an in vitro model that partially incorporates the functional complexity of the fish heart

Pre-Hatching Ontogenetic Changes of Morphological Characters of Small-Spotted Catshark (<i>Scyliorhinus canicula</i>)

The small-spotted catshark, Scyliorhinus canicula, provides an optimal model organism to include chondrichthyans in studies comparing morphology or physiology through vertebrate evolution. In particular, for investigations with ontogenetic aspects, there are only a limited number of alternative taxa. Therefore, a detailed staging system is a prerequisite to allowing comparison between different studies. This study supplements information on the latest stages of the established system by Ballard and colleagues in 1993 and complements the respective staging system by including the latest pre-hatching stages. During this phase, some significant ontogenetic shifts happen, e.g., reduction of external gill filament length and complete flattening of rostral angle until Size Class 6, change in the ratio of pre- to post-vent length, and establishment of body pigmentation in Size Classes 7 and 8. All these shifts finally transform the embryo into a hatchling prepared for living outside the eggshell. This study provides a framework allowing comparison of investigations on pre-hatchings of the small-spotted catshark

Pre-Hatching Ontogenetic Changes of Morphological Characters of Small-Spotted Catshark (Scyliorhinus canicula)

The small-spotted catshark, Scyliorhinus canicula, provides an optimal model organism to include chondrichthyans in studies comparing morphology or physiology through vertebrate evolution. In particular, for investigations with ontogenetic aspects, there are only a limited number of alternative taxa. Therefore, a detailed staging system is a prerequisite to allowing comparison between different studies. This study supplements information on the latest stages of the established system by Ballard and colleagues in 1993 and complements the respective staging system by including the latest pre-hatching stages. During this phase, some significant ontogenetic shifts happen, e.g., reduction of external gill filament length and complete flattening of rostral angle until Size Class 6, change in the ratio of pre- to post-vent length, and establishment of body pigmentation in Size Classes 7 and 8. All these shifts finally transform the embryo into a hatchling prepared for living outside the eggshell. This study provides a framework allowing comparison of investigations on pre-hatchings of the small-spotted catshark

In Vitro Fish Models for the Analysis of Ecotoxins and Temperature Increase in the Context of Global Warming

Rising temperatures can affect fish survival, especially from shallower waters, as temperatures increase faster and more intensively in these areas; thus, species-specific temperature tolerance can be exceeded. Additionally, the amounts of anthropogenic pollutants are higher in coastal waters. Although increasing metabolic activity at higher temperatures could lead to stronger effects of toxins, there are hardly any studies on this topic. Subsequently, the aim was to investigate the response of fish cells upon exposure to industrial solvents (ethanol, isopropanol, dimethyl sulfoxide (DMSO)) in relation to a temperature increase (20 °C and 25 °C). Concerning the 3Rs (the replacement, reduction and refinement of animal experiments), in vitro tests were used for two threatened, vulnerable fish species: maraena whitefish (Coregonus maraena) and Atlantic sturgeon (Acipenser oxyrinchus). Both cell lines exhibited higher proliferation at 25 °C. However, ecotoxicological results indicated significant differences regarding the cell line, toxin, temperature and exposure time. The evolutionarily older fish lineage, Atlantic sturgeon, demonstrated lower mortality rates in the presence of isopropanol and recovered better during long-term ethanol exposure than the maraena whitefish. Atlantic sturgeon cells have higher adaptation potential for these alcohols. In summary, fish species respond very specifically to toxins and changes in temperature, and new ecotoxicological questions arise with increasing water temperatures

Fatty Acid Composition in Blubber, Liver, and Muscle of Marine Mammals in the Southern Baltic Sea

Marine mammals play an important role in marine ecosystems. However, as they are less accessible for research, relatively little is known about their physiology compared to terrestrial mammals. The stranding scheme of the Deutsches Meeresmuseum (Stralsund, Germany) continuously collects strandings and by-catches of marine mammals in the Baltic Sea in Mecklenburg-Western Pomerania. In this project, the fatty acid composition of the liver, skeletal muscles, and blubber of harbour porpoises and grey seals from the southern Baltic Sea was investigated for the first time. In the liver and blubber tissue, the values and concentrations measured for both species are consistent with studies on other marine mammals. In a direct comparison of the focus species, the skeletal muscles of harbour porpoises exhibit higher concentrations of fatty acids than those of grey seals. In the future, these studies will be extended to the entire Baltic Sea, as we suspect that fatty acid composition can be used to determine the nutritional status of the animals and thus will allow for an objective assessment of the body condition.Peer Reviewe

Observations of growth changes during the embryonic‐larval‐transition of pikeperch (Sander lucioperca) under near‐natural conditions

Abstract 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

In Vitro Fish Models for the Analysis of Ecotoxins and Temperature Increase in the Context of Global Warming

Rising temperatures can affect fish survival, especially from shallower waters, as temperatures increase faster and more intensively in these areas; thus, species-specific temperature tolerance can be exceeded. Additionally, the amounts of anthropogenic pollutants are higher in coastal waters. Although increasing metabolic activity at higher temperatures could lead to stronger effects of toxins, there are hardly any studies on this topic. Subsequently, the aim was to investigate the response of fish cells upon exposure to industrial solvents (ethanol, isopropanol, dimethyl sulfoxide (DMSO)) in relation to a temperature increase (20 °C and 25 °C). Concerning the 3Rs (the replacement, reduction and refinement of animal experiments), in vitro tests were used for two threatened, vulnerable fish species: maraena whitefish (Coregonus maraena) and Atlantic sturgeon (Acipenser oxyrinchus). Both cell lines exhibited higher proliferation at 25 °C. However, ecotoxicological results indicated significant differences regarding the cell line, toxin, temperature and exposure time. The evolutionarily older fish lineage, Atlantic sturgeon, demonstrated lower mortality rates in the presence of isopropanol and recovered better during long-term ethanol exposure than the maraena whitefish. Atlantic sturgeon cells have higher adaptation potential for these alcohols. In summary, fish species respond very specifically to toxins and changes in temperature, and new ecotoxicological questions arise with increasing water temperatures