Apparent availability and requirements of microminerals in salmon (APREMIA)

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present project aimed to describe a systematic approach combining different methodologies to assess the availability and requirement of zinc (Zn), manganese (Mn) and selenium (Se) in Atlantic salmon (Salmo salar). Considering that several chemical species of these metals can be present in an Atlantic salmon feed, it was hypothesised that micro-mineral availability was influenced by the chemical species present in the feed. Thus, in this project, the first step was to develop a method to extract the different chemical species (Zn) from the feed. Subsequently, in vitro methods were developed to evaluate the solubility of dietary Zn, Se and Mn in Atlantic salmon feeds. Further, an in vitro gut cell model (RTgutGC) was used to study the impact of changing chemical species composition on the intestinal uptake (Zn and Mn). Together, the findings from the in vitro methods were compared with in vivo studies examining the apparent availability and requirement of Zn, Se and Mn to Atlantic salmon from inorganic and organic sources. The results showed that several chemical species can be found in feeds and the efficiency of an organic source depends very much on the amino acid ligand used to chelate. The findings of the in vitro methods corresponded well with that of the in vivo studies for a qualitative evaluation but not on quantitative terms. Nevertheless, in vitro protocols described in this article provided crucial information regarding micro-mineral availability and its assessment in fish feeds. Moreover, the estimates of dietary levels required to meet the requirements of Zn, Se and Mn in fish feeds were derived from specific feeding trials. The mean estimate of dietary Mn required to maintain tissue Mn saturation ranged between 26 and 34 mg Mn kg?1 diet; the corresponding Mn supplementation (as MnSO4) ranged from 14 to 22 mg Mn kg?1 diet. The supplementation level can be reduced to 4.9 to 5.7 mg Mn kg?1 diet by using Mn-gly as Mn source without compromising growth or Mn status of Atlantic salmon. Dietary Se inclusion level required to the basal diet containing 0.24 mg Se kg-1 was 0.4 mg (as SS, total 0.65 mg kg-1) or 0.15 mg (as SM, total 0.4 mg kg-1), based on body Se homeostasis or tissue Se status. SM inclusion at 0.4 mg kg-1 diet (total, 0.65 mg kg-1) was able to enhance innate immunity and attenuate bacterial or viral induced pro-inflammatory responses in vitro. Overall, dietary Se level required to maintain body Se homeostasis and improved health status of Atlantic salmon fed plant-based diets exceed the existing EU maximum limit of 0.5 mg Se kg-1 diet. The apparent availability of Zn, Zn status in the whole body, plasma and vertebrae were significantly influenced by dietary Zn levels; and were significantly lower in seawater phase compared to freshwater phase. The organic source Zn-glycine used did not have an improved effect on any of the analysed parameters. The final whole-body Zn concentration of 33 mg kg-1 wet weight achieved with the highest Zn level (280 mg kg-1 diet) corresponds to the recent data reported by Åsgård et al. (2018), however, is lower than the level reported by Shearer et al. (1994). Considering the existing EU legislation on maximum permitted total Zn level of 180 mg kg-1 feed for salmonids (EC, 2016) and an EFSA (2017) scientific opinion suggesting a further decrease to 150 mg kg-1 feed, the present results are of high significance. From the perspective of the environment, the load of Mn and Se from Norwegian salmon farming was estimated to be 62 and 0.8 tonnes per year at present, which can be reduced by 30 to 60% (for Mn) and by 50 to75% (for Se) without compromising the growth and health of Atlantic salmon.

The major outcomes are, (i) capacity development with one PhD and one early stage researcher with two mobility arrangements each strengthening international collaboration. (ii) Seven published peer-reviewed open-access research articles and 17 presentations. The achieved outcomes will impact (i) better production performance of farmed salmon by meeting their requirements for Zn, Mn and Se for uncompromised welfare, health and growth; (ii) quantitative information on dietary mineral levels required in plant-based feeds and the efficacy of different sources for the feed industry; (iii) regulation of trace mineral levels in salmon feeds by Mattilsynet and EFSA; (iv) the in vitro methods will strengthen 3Rs in aquaculture research; (v) reduced emissions through improved availability and retention will reduce environmental impact; (vi) the society will benefit from the improved sustainability of Atlantic salmon farming (iv) equal opportunity and gender balance in capacity development.

Due to increasing inclusion of vegetable ingredients in salmon feeds, new challenges have arisen with regards to minerals. Substitution of marine feed ingredients alters the level and total composition of minerals. Substitution of marine feed ingredients with plant ingredients will also lower the bioavailability of already supplemented minerals due to the presence of plant fibers and phytate. The lower mineral bioavailability in novel aquafeeds will both affect essential elements required for fish health and robustness, but also increase the environmental load by increased loss via the faeces.High supplementation of several minerals to fish feeds today, merely based on precautions, has further challenges in that the minerals added to the feed may interact with each other both on intestinal uptake and biological function. Furthermore, the chemical form (e.g. inorganic or organic forms)of supplemented minerals has been suggested to alter the bioavailability. The route of uptake and tissue distribution for chemical forms has, however not been demonstrated in fish. Challenges to assess mineral requirements in the Atlantic salmon relates to chemical forms in feed ingredients and complete diets, interacting factors that negatively influence digestibility, uptake, tissue distribution and functional status of the individual minerals, as well as farming conditions that may elevate the requirements. The activity in the project is divided into three work packages: 1) we aim to develop a chemical speciation method of selected minerals to predict mineral digestibility depending on mineral chemical species. 2)We will apply rapid in situ and in vitro methods to assess intestinal mineral uptake with combined factors that can affect bioavailability. 3) Based on the digestibility and availability studies, we want to establish safe lower requirement levels of Zn,Mn and Se to support growth performance and robustness in farmed Atlantic salmon fed future environmentally friendly feeds.