Abstract
BACKGROUND: Recirculating aquaculture systems can cause chronic stress in fish when stocking density is too high. However, this study tested whether low stocking density can cause fish stress. Adult rainbow trout, with an average weight of 1.517 kg (± 0.39), were subjected to low (12 kg/m3 ± 0.94) and moderate (43 kg/m3 ± 2.03) stocking densities for 24 days in a recirculating system maintained at 15 °C. At the end of the experiment, fecal microbiome analysis was carried out using a 16S rRNA amplicon sequencing. Additionally, an untargeted plasma metabolomics analysis was conducted. RESULTS: The moderate stocking density group harboured greater numbers of commensals, such as C. somerae, R. lituseburensis, and L. plantarum. In contrast, detrimental species such as S. putrifacens and P. putida were abundant in the low-stocking density fish. Functional microbiome profiling revealed vitamin B12 salvage and synthesis in moderate stocking densities, which may support intestinal tight junction function. Additionally, vitamin B1 biosynthesis pathways were more abundant in the moderate stocking density group, which may function towards oxidative energy metabolism and protect against oxidative stress. A complementary plasma metabolomics study, although done at slightly different stocking densities and duration, confirmed the presence of blood metabolic stress markers. Elevated levels of L-lactic acid and L-Norvaline, L-Valine, and L-glutamine, indicate low stocking density fish were under stress. Furthermore, a P4HA2 stress gene biomarker confirmed the occurrence of stress in low-density fish. This study suggests that low stocking density can induce stress in fish. Moreover, moderate stocking density leads to a distinct and beneficial fecal microbiome profile. CONCLUSION: Our study highlights the potential benefits of optimizing the stocking density of fish in recirculating aquaculture systems. This can improve fish health and welfare, promoting a more resilient fecal microbiome.