Development of novel methods to evaluate availability of zinc, selenium and manganese in Atlantic salmon (Salmo salar)
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During the recent years, the composition of salmonids diets has changed from the use of mainly marine-based ingredients (e.g. fish meal and fish oil) to an increased use of plant-based ingredients (e.g. soybean meal and vegetable oil). These changes in diet composition have an impact on the mineral concentration and mineral availability. For instance, zinc (Zn) is naturally present in both fish meal and plant-based ingredients, but in different concentrations. The Zn concentration is usually higher in fish meal than in plant-based ingredients so, with the increased use of plant-based ingredients the Zn concentration tends to decrease in the basal mixes. In addition, compounds from plantbased ingredients can reduce mineral availability. For instance, phytic acid, which is typically found in plant-based ingredients, can decrease mineral availability due to its high binding affinity for metal ions. Therefore, minerals such as Zn, selenium (Se) and manganese (Mn) are supplemented to diets to cover the nutritional requirement of farmed fish. These minerals may be added as organic or inorganic forms. Taken all of this information into consideration, there is a need to study mineral availability in the current salmon feed composition which is formulated mainly using plant-based ingredients. Further knowledge in this area will provide a better understanding regarding mineral availability and necessary strategies to increase mineral availability in Atlantic salmon. Increasing mineral availability will promote fish health and robustness, but also decrease the environmental load via faeces.
Considering that several chemical species of minerals can be present in a fish feed, it was hypothesised that availability is affected by the chemical species. For that purpose, analytical methods were optimized for extraction, quantification and identification of Zn chemical species in fish feed. This included method optimization by fractional factorial design and evaluation of sample extracts by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry (SEC-ICP-MS) (Paper I). The impact of freshwater or seawater media ion composition and methionine chelation on Zn uptake was evaluated using a rainbow trout intestinal epithelial cell line (RTgutGC) (Paper II). This PhD work also compared the availability of Zn, Se and Mn from inorganic metal salts and their organic forms in Atlantic salmon diets. Sixteen experimental diets were prepared based on a two-level full factorial design for four factors. The tested factors were Zn additive source, Se additive source, Mn additive source and phytic acid level. The Zn, Se, Mn and yttrium concentration in diets and faeces were determined using inductively coupled plasma mass spectrometry (ICPMS) and the apparent availability of Zn, Se and Mn were estimated (Paper III). The availability of a nutrient depends on several factors, including solubility. An in vitro digestion method was developed to evaluate solubility of dietary Zn, Se and Mn in two diets for Atlantic salmon. The soluble fractions obtained were then evaluated as a measure to predict availability of Zn, Se and Mn (Paper IV). A summary of the work done in the PhD is described by the graphical abstract shown in Figure 1.
The procedure to extract the Zn species from the diet included extraction conditions to keep the Zn species intact. The highest recovery of Zn (9.9±0.2%) was obtained using 100 mM Tris-HCl, pH 8.5 at a temperature of 4°C for 24 h. The same soluble fraction was further evaluated for Zn species by SEC-ICP-MS. Four Zn containing peaks were found, each peak with different molecular weights: peak 1 (high molecular weight), peak 2 and peak 3 (medium molecular weight) were the least abundant peaks (1-6%), while peak 4 (low molecular weight) was the most abundant peaks (84-95%) (Paper I). In RTgutGC, Zn uptake was not different between freshwater and seawater media ion composition. Conversely, in the presence of methionine, Zn uptake in seawater media ion composition was lower compared to freshwater media ion composition, but only at high Zn concentrations (12 and 25 μM) (Paper II). The apparent availability of Zn was not affected by the Zn additive source. However, the Se and Mn additive sources affected their apparent availability. The apparent availability of Se was higher for selenomethionine than for selenite, and Mn sulphate was more available than Mn chelate of glycine. Several interactions between mineral additive sources and the phytic acid level affected the apparent availability of Zn, Se and Mn (Paper III). The solubility of Zn was similar in both diets tested. The amount of soluble Zn was low in the acidic hydrolysis (3-8%) and lower in the alkaline hydrolysis (0.4-2%). The solubility of Se was higher in the diet supplemented with organic mineral sources (7- 34%) when compared with diet supplemented with inorganic mineral sources (3-12%). Regarding Mn, during the acidic hydrolysis the solubility was higher in the diet supplemented with inorganic mineral sources (6-25%) than in the diet supplemented with organic mineral sources (4-17%) (Paper IV).
Several Zn species were found in the soluble fraction of the Atlantic salmon diet studied but further work is needed to evaluate the effect of the different Zn species on availability (Paper I). Zinc uptake in RTgutGC cell line was influenced by the ionic concentration in the media, indicating that the intestinal ionic composition in a freshwater or in a seawater environment can influence Zn availability (Paper II). Regarding the apparent availability of Zn, Se and Mn in Atlantic salmon, it was demonstrated that the availability of Zn, Se and Mn depended on both the chemical form of the mineral supplemented to diets and on several interactions between Zn, Se and Mn and phytic acid level (Paper III). The solubility of Zn, Se and Mn was influenced both by the mineral chemical form supplemented in diet and by the gastrointestinal environment (Paper IV). Moreover, solubility and apparent availability of Mn showed a strong positive correlation, but a week positive correlation was seen for Zn and Se (Paper IV). Consequently, more work needs to be done for improving the in vitro digestion method.