Introduction
Convenient sampling methods for non-invasive stress monitoring are needed to support welfare assessment in modern salmon aquaculture. Fecal samples have been validated and used for measuring stress levels of Atlantic salmon (Salmo salar). The fecal sampling method in the field is usually conducted by stripping fish or collected fecal material after dissecting fish in commercial sea cages(Cao et al., 2017). In the present study, we developed and validated a fecal swab method for quantifying cortisol in Atlantic salmon under commercial farming conditions. FLOQSwabs�� Minitip were evaluated across six validation components: swab material uptake, field and laboratory comparability, biological validation, analytical validation, and storage stability.
Materials and Methods
Atlantic salmon were sampled from commercial sea-cage sites in Norway. All sampling was performed during routine operational procedures, including sea-lice counting and regular health check. All procedures involving fish handling and sampling followed Directive 2010/63/EU and complied with Norwegian regulations governing the use of animals in scientific research.
Blood samples were collected from the caudal vein using 4���mL heparinized BD Vacutainer�� tubes (Becton, Dickinson and Co., NJ, USA). Fecal samples were firstly collected by inserting a sterile Mini Copan FLOQSwabs�� (Cambio, Cambridge, UK), through the anus until the breakpoint of the swabs, and gently rotating the swab five times along the mucosal surface. This approach ensures sampling of both feces and gut mucosa. According to Ruiz et al., (2024), this method does not elicit abnormal behavior or visible signs of stress or injury in Atlantic salmon. Additional fecal material was collected by gently applying pressure along the abdomen to expel feces through the vent. The expelled fecal matter was transferred into sterile 2 mL tubes, homogenized thoroughly and stored at ���80���°C before further analysis.
Plasma and fecal cortisol concentrations were determined using a commercial Neogen cortisol ELISA kit, following extraction and analysis procedures adapted from Ding et al., (2024)
Results
Swab could sample enough material for fecal cortisol analysis. The weight of material collection by one swab was consistent across three different fecal textures (mean 0.118 g ± 0.01 g; CV = 8.23%; p = 0.609). Field swabs produced cortisol concentrations comparable to laboratory resampling (paired p > 0.05) with a strong correlation (r = 0.94). Biological validation across three different commercial sits with four net pens in Norway demonstrated significant associations between plasma and fecal cortisol among sample groups (mixed-effects model: p = 0.044; Spearman's �� = 0.67, p < 0.001). Significant increased cortisol levels were detected by fecal swabs after fish were posted to acute handling stress. In addition, baseline fecal cortisol concentrations differed among net pens, indicating that the method is sensitive enough to detect variation associated with different farming environments. Spike-and-recovery tests showed an overall recovery of 90.5% (range 62.9–127.5%). Repeatability testing of five homogenised fecal samples yielded a mean technical CV of 10.23%. Storage stability analysis indicated rapid degradation at room temperature, stable cortisol profiles at 4 °C for up to 14 days, and high stability at ���20 °C for at least 60 days.
Summary
Overall, the fecal swab method provides a practical, non-invasive, and robust tool for stress hormone monitoring in farmed salmon. This approach offers strong potential for integration into routine welfare assessments and operational decision support in commercial aquaculture.
References
Cao, Y., Tveten, A.-K., & Stene, A. (2017). Establishment of a non-invasive method for stress evaluation in farmed salmon based on direct fecal corticoid metabolites measurement. Fish & shellfish immunology, 66, 317–324 https://doi.org/10.1016/j.fsi.2017.04.012.
Ding, J., Gansel, L. C., Finstad, B., Stene, A., Bakke, S., Aas, G. K. F. H., & Cao, Y. (2024). Investigating the biological relevance of measuring gastrointestinal cortisol metabolite levels to assess stress responses in Atlantic salmon (Salmo salar L.) after an acute stress. Fish & shellfish immunology, 109729 https://doi.org/10.1016/j.fsi.2024.109729.
Ruiz, A., Torrecillas, S., Kashinskaya, E., Andree, K. B., Solovyev, M., & Gisbert, E. (2024). Comparative study of the gut microbial communities collected by scraping and swabbing in a fish model: a comprehensive guide to promote non-lethal procedures for gut microbial studies. Frontiers in Veterinary Science, 11, 1374803. https://doi.org/https://doi.org/10.3389/fvets.2024.1374803