Performance, immune response, disease resistance, and gut microbiota of rainbow trout, Oncorhynchus mykiss (Walbaum, 1792) juveniles fed ground leonardite with a high humic substance content

In the present study, the effects of feeding ground leonardite (LE), which contained 68% of humic substances, to juvenile Oncorhynchus mykiss were examined on overall growth and production performance, feed nutrient di-gestibility, gut microbiota, health condition, antioxidant status, immune response, and disease resistance. During two consecutive experiments, fish were fed four experimental diets with different LE supplementary levels: 0, 0.5, 1, and 3% of feed mass (LE0, LE0.5, LE1, and LE3, respectively). Firstly, the fish were tested in a growing experiment (GE, n = 300 fish/group, body weight 15.4 +/- 2.5 g) for 56 days. Thereafter, these fish (n = 75 fish/ group, body weight 51.9 +/- 8.7 g) were exposed to an intraperitoneal infection challenge experiment (IC) with Aeromonas salmonicida subsp. salmonicida for 28 days. In the GE, dietary LE did not affect the growth, production, or health performance of O. mykiss juveniles, although the fish more than tripled their initial body weight. The LE dose had an impact on the digestibility of feed nutrients (dry matter, gross energy, and ash), but not on the protein and lipid digestibility or solid waste outputs. The gut microbial communities were highly diverse across the experimental groups and remained distinct between the control and LE groups. The LE had a dose-dependent effect on the cell composition of the head kidney and peripheral blood leukocytes. The highest LE addition exerted the immune suppressive effect, and a decreased count of immune cells was observed in the LE3. The peritoneal cavity infection in the IC trial induced the rapid recruitment of myeloid cells in all groups shortly after the infection, except for the LE3, which had an apparent delayed response. The cumulative mortality and specific trout antibody levels against A. salmonicida did not significantly vary among tested groups, although their average initial level dropped by half at 3 days post-infection (dpi), and subsequently doubled at 28 dpi. The plasma biochemical parameters (TP, TAG, and CHOL) showed an overall decrease at 0-3 dpi, while increasing back to the initial levels at 3-6 dpi. Only LDH negatively correlated with LE inclusion, this can reflect the oxidative stress in the highest LE inclusion. Overall, our results support that there is a dose-dependent effect that should be optimised for ground LE di-etary use in aquaculture.