Introduction
The growth of the salmonid aquaculture industry has driven major changes in feed composition, leading to diets with higher lipid content and increased use of plant-based ingredients. As a result, these diets often contain reduced levels of the omega-3 fatty acids EPA and DHA. Additionally, due to environmental concerns and regulatory restrictions, zinc (Zn) content in salmon diets has also been reduced. The aim of this study was to investigate the interactions between dietary Zn and omega-3 fatty acids in relation to growth, body composition, skin health, and bone development in Atlantic salmon, using a combination of in vivo and in vitro approaches.
Material and Methods
Atlantic salmon parr (initial weight: 5 g) were fed six experimental diets for five weeks in triplicate tanks. These diets varied in Zn (120, 180, and 220 mg/kg, referred to as low [LZn], medium [MZn], and high [HZn], respectively) and EPA+DHA content (1% or 3%, referred to as low [LΩ] or high [HΩ], respectively). At the end of the feeding trial, skin and vertebra samples were collected from 10 fish per tank, flash-frozen in liquid nitrogen for chemical analyses, and fixed in 10% formalin for histological analysis. An additional 10 fish per tank were frozen at −20°C for whole-body fat analysis, fatty acid composition, mineral content, and vertebral X-ray and CT imaging.
To assess wound healing, 36 fish from each dietary group were transferred to a common tank and continued on their respective experimental diets for a 6-week wound healing trial. Two standardized skin wounds were inflicted: a superficial wound created by brushing five times with a toothbrush horizontally, and a deep wound involving a vertical incision approximately 2 mm long and 2 mm deep. Tissue samples from the wound areas were collected at 4 days and 6 weeks post-wounding for gene expression and histological analysis.
Three in vitro experiments were also conducted, using scales collected from six fish. Five scales per fish were placed in each well, totalling 30 scales per treatment, tested in triplicate. Four conditions were evaluated: control, 0.5 mg/L Zn, 10 µM DHA, and a combination of 0.5 mg/L Zn + 10 µM DHA. The effect on cell migration was measured as the ratio of scales with migrating keratocytes to those without.
Results and discussion
The experimental diets had no significant effects on growth performance, as measured by standard growth rate (SGR) and thermal growth coefficient (TGC). As expected, the dietary content of EPA+DHA was reflected in whole-body fatty acid composition, and Zn concentrations in the whole body, skin, and vertebrae increased proportionally with dietary Zn levels. Interestingly, a synergistic effect between Zn and EPA+DHA was observed, particularly at the highest inclusion levels (3% EPA+DHA and 220 mg/kg Zn), resulting in elevated tissue Zn concentrations. Fish receiving this diet also exhibited the highest bone density. Analysis of scale maturation in the skin showed that fish fed the lowest Zn levels (HΩ/LZn and LΩ/LZn) had the least mature scales, while those fed high Zn and high omega-3 (HΩ/HZn ) had the most mature scales by the end of the trial. Spinal deformities were observed in 6% of the fish, with twice as many deformities occurring in the LZn groups compared to those fed MZn or HZn, regardless of EPA+DHA content.
Wound healing assessments of mechanically induced injuries (deep cut, superficial wound, and surrounding skin) showed faster scale regeneration in fish fed diets containing the highest levels of both Zn and omega-3. Additionally, the in vitro model using salmon scales demonstrated that Zn and DHA both enhanced the migratory capacity of keratocytes. Since rapid keratocyte migration and scale regeneration are critical for wound healing and maintaining skin integrity, these findings underscore the role of these nutrients in fish health.
Conclusion
Overall, this study highlights the importance of balancing dietary Zn and omega-3 fatty acids to support optimal growth, skin and bone development, and overall welfare in Atlantic salmon.