Date Published: July 20, 2017
Publisher: Public Library of Science
Author(s): Patricia A. Noguera, Bianka Grunow, Matthias Klinger, Katherine Lester, Bertrand Collet, Jorge del-Pozo, Lloyd Vaughan.
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.
Farmed Atlantic salmon (Salmo salar) is one of the most important cold water diadromous species produced by aquaculture worldwide, exceeding 2.3 million tonnes in 2014 . In the UK, Scotland’s record production in 2014 was associated with revenues of over £1.8 billion . As with most species under intensive farming, disease management is one of the major challenges faced by the industry. Consequently, fish diseases and host-pathogen interaction remains among the top priorities for aquaculture research in Europe  and the UK .
The reduced availability of wild-capture fish in the context of increased global demand for seafood, has resulted in continuous growth of the aquaculture industry worldwide . In this context, the study of diseases affecting salmon, one of the most important species in cold water aquaculture, requires methodologies in agreement with bioethical principles seeking to investigate, facilitate and promote the advancement of alternative approaches . The SCPCs model presented here is in line with the aim of this Directive to minimise the number of animals used in research while providing reliable results and allowing for optimal extrapolation into target species. Furthermore, non-hatched fish embryos are not classified as protected animals under UK regulations  and therefore, the approach poses no ethical conflict with regulations under the Animals (Scientific Procedures) Act 1986 (ASPA). On our work the SCPCs production efficiency per embryonated egg varied but on average, was not higher than 1. This compares favourably yet with egg to smolt survival of 70–80% under the greatly improved farmed conditions and only 0.15% to 3.2% among wild salmon [38–41]. Moreover, the cost of embryonated eggs compared to that of fry/parr or smolts, also contributes to a significant economical saving of the SCPCs approach.