Prosjektnummer
910440
Optimizing Zinc Supplementation in Salmon Feed for Improved Bioavailability, Fish Health, and Reduced Environmental Impacts (Zinc-SAFE)
Background
Zinc (Zn) is an essential trace mineral for Atlantic salmon (Salmo salar), acting as a structural element of enzymes and a co-factor in numerous biological processes. Deficiency can lead to poor growth, cataracts, impaired skin and immune function, and higher mortality. Zn bioavailability in modern aquafeeds is low due to the increased use of plant ingredients containing ligands such as phytic acid that reduce Zn uptake. As a result, much dietary Zn is lost to the environment, raising concerns for ecosystem health. Current EU limits (150–180 mg Zn kg⁻¹) are below estimated salmon requirements (181–218 mg Zn kg⁻¹), especially in the seawater phase, making it urgent to improve Zn utilization in diets.
Intestinal uptake is complex, involving active transporters but largely dominated by less predictable passive mechanisms in salmon feeds. Absorption is influenced by Zn source (inorganic versus organic), luminal conditions (pH, salts, temperature), feed composition, and interactions with other minerals. While organic Zn and additives such as phytase may improve uptake, results remain inconsistent. The role of the gut microbiota in competing for Zn is also poorly understood in fish.
Due to these complexities, conventional in vivo trials are costly and limited in identifying causal effects. In contrast, 3R (Replace, Reduce, Refine) methods provide ethical, efficient, and controlled approaches to disentangle these processes and identify key factors. For example, in vitro digestibility assays have great potential in assessing Zn bioaccessibility during digestion. Gut sac models have proven useful for measuring mineral fluxes, providing insight into absorption. Cell cultures reveal molecular and tissue-specific responses that can be used to assess the health implications of different Zn dietary strategies. Together, these complementary models enable precise evaluation of Zn bioavailability, supporting improved feed formulations, fish health, and reduced environmental losses.
Zinc (Zn) is an essential trace mineral for Atlantic salmon (Salmo salar), acting as a structural element of enzymes and a co-factor in numerous biological processes. Deficiency can lead to poor growth, cataracts, impaired skin and immune function, and higher mortality. Zn bioavailability in modern aquafeeds is low due to the increased use of plant ingredients containing ligands such as phytic acid that reduce Zn uptake. As a result, much dietary Zn is lost to the environment, raising concerns for ecosystem health. Current EU limits (150–180 mg Zn kg⁻¹) are below estimated salmon requirements (181–218 mg Zn kg⁻¹), especially in the seawater phase, making it urgent to improve Zn utilization in diets.
Intestinal uptake is complex, involving active transporters but largely dominated by less predictable passive mechanisms in salmon feeds. Absorption is influenced by Zn source (inorganic versus organic), luminal conditions (pH, salts, temperature), feed composition, and interactions with other minerals. While organic Zn and additives such as phytase may improve uptake, results remain inconsistent. The role of the gut microbiota in competing for Zn is also poorly understood in fish.
Due to these complexities, conventional in vivo trials are costly and limited in identifying causal effects. In contrast, 3R (Replace, Reduce, Refine) methods provide ethical, efficient, and controlled approaches to disentangle these processes and identify key factors. For example, in vitro digestibility assays have great potential in assessing Zn bioaccessibility during digestion. Gut sac models have proven useful for measuring mineral fluxes, providing insight into absorption. Cell cultures reveal molecular and tissue-specific responses that can be used to assess the health implications of different Zn dietary strategies. Together, these complementary models enable precise evaluation of Zn bioavailability, supporting improved feed formulations, fish health, and reduced environmental losses.
Objectives
Main objective
To optimise the use of Zn in feed for salmon farming that ensures good fish health under relevant farming conditions, with minimal environmental impact.
Sub-objectives
Main objective
To optimise the use of Zn in feed for salmon farming that ensures good fish health under relevant farming conditions, with minimal environmental impact.
Sub-objectives
1. To determine the effect of internal and external parameters on bioaccessibility and uptake of dietary Zn, using in vitro digestibility and gut sac models.
2. To determine the effect of dietary Zn on gut transcriptome as the main site of uptake using salmon gut cell lines, to improve insight into transport mechanisms and impacts on barrier function and health.
3. To determine the effect of dietary Zn on transcriptome of liver as the main site of Zn storage, detoxification, and metabolism, using salmonid liver cell line, to provide novel insight into the regulation of Zn and associated health impacts.
4. To identify molecular indicators of Zn deficiency in salmon using cell culture data.
5. To recommend optimum Zn forms, concentrations, and dietary conditions to maximize Zn bioavailability in the diet and manage good health in salmon.
2. To determine the effect of dietary Zn on gut transcriptome as the main site of uptake using salmon gut cell lines, to improve insight into transport mechanisms and impacts on barrier function and health.
3. To determine the effect of dietary Zn on transcriptome of liver as the main site of Zn storage, detoxification, and metabolism, using salmonid liver cell line, to provide novel insight into the regulation of Zn and associated health impacts.
4. To identify molecular indicators of Zn deficiency in salmon using cell culture data.
5. To recommend optimum Zn forms, concentrations, and dietary conditions to maximize Zn bioavailability in the diet and manage good health in salmon.
Expected project impact
This project has been conceptualized in response to the challenges faced by the aquaculture industry in providing adequately bioavailable dietary levels of Zn while still following increasingly strict regulatory guidelines. The project draws on our expertise in fish nutrition, physiology, and health, and is shaped through continuous engagement with industry stakeholders and leverages alternative 3R methodologies to develop cost-effective, ethically sound models that support the principles of Replacement and Reduction in animal use.
The project aims to develop and validate a set of models to offer a comprehensive pipeline to assess the bioavailability and health impacts of dietary Zn in an aquaculture setting. Once established, this pipeline can be adapted for other trace elements, significantly reducing industry time and costs associated with in vivo trials, as well as the number of live fish used in feed testing. This will improve animal welfare, align with ethical and regulatory expectations in aquaculture research, and further minimize costs for the industry.
Moreover, by uncovering the mechanistic pathways and factors influencing Zn uptake and metabolism, these 3R models will help identify key contributors and gaps where further research or product innovation is needed. These insights will support the industry in optimising feeding strategy for Zn, increasing fish performance and welfare while mitigating environmental impacts. Beyond generating results and recommendations that directly benefit feed companies in terms of zinc concentration, form, and supplementation strategies, the pipeline itself can be routinely applied in feed formulation practices. In parallel, the developed markers of zinc deficiency can be exploited by fish farmers as an early diagnostic tool, enabling adjustment of feed additives before severe disorders such as cataracts and other related conditions appear and cause major losses.
This project has been conceptualized in response to the challenges faced by the aquaculture industry in providing adequately bioavailable dietary levels of Zn while still following increasingly strict regulatory guidelines. The project draws on our expertise in fish nutrition, physiology, and health, and is shaped through continuous engagement with industry stakeholders and leverages alternative 3R methodologies to develop cost-effective, ethically sound models that support the principles of Replacement and Reduction in animal use.
The project aims to develop and validate a set of models to offer a comprehensive pipeline to assess the bioavailability and health impacts of dietary Zn in an aquaculture setting. Once established, this pipeline can be adapted for other trace elements, significantly reducing industry time and costs associated with in vivo trials, as well as the number of live fish used in feed testing. This will improve animal welfare, align with ethical and regulatory expectations in aquaculture research, and further minimize costs for the industry.
Moreover, by uncovering the mechanistic pathways and factors influencing Zn uptake and metabolism, these 3R models will help identify key contributors and gaps where further research or product innovation is needed. These insights will support the industry in optimising feeding strategy for Zn, increasing fish performance and welfare while mitigating environmental impacts. Beyond generating results and recommendations that directly benefit feed companies in terms of zinc concentration, form, and supplementation strategies, the pipeline itself can be routinely applied in feed formulation practices. In parallel, the developed markers of zinc deficiency can be exploited by fish farmers as an early diagnostic tool, enabling adjustment of feed additives before severe disorders such as cataracts and other related conditions appear and cause major losses.
Project design and implementation
The project consists of the following work packages (WPs):
WP1: Feeds preparations and in vivo material and data collection
Leader: Naouel Gharbi (NORCE)
Activities
This work package (WP) provides the experimental feeds and biological material required to conduct and/or validate the 3R models developed in WPs 2–4. Given the complex nature of Zn bioavailability and the conflicting evidence on the optimal form and concentration of dietary Zn for salmon at different life stages, we will test two Zn sources (inorganic and organic) at three dietary inclusion levels (deficient, adequate, and supra-optimal) in parr (freshwater) and post-smolt (seawater) stages. This design will enable the generation of robust dose–response curves for key biological functions assessed in the subsequent WPs.
Experimental feeds will be formulated to resemble commercial diets and produced at a small scale for use in the 3R pipeline developed in WPs 2–4. Water, feed, and fish samples will be collected from one or two commercial production sites (FishBase).
The collected material will serve two purposes: (1) to provide enzyme extracts for in vitro digestibility assays (WP2) and intact gut tissues for the gut sac model (WP3); and (2) to generate in vivo data for validating Zn solubilization, uptake, and retention predicted by the models in WPs 2–4. Short-term Zn status will be evaluated in faeces, intestine, and blood, while long-term status will be assessed in liver and whole-body samples.
WP2: In vitro digestibility assays to simulate zinc bioaccessibility
Leaders: Neda Gilannejad (NORCE) and Francisca P. Martínez Antequera University of Almeria (UAL)
Activities
In vitro digestibility assays will be developed to assess Zn bioaccessibility in Atlantic salmon. This approach will allow us to evaluate the influence of multiple factors on Zn potential availability, including feed-related parameters such as Zn form and concentration or feed composition (e.g., additives like phytase or acidifiers), as well as physiological and environmental conditions such as gastrointestinal pH and water salinity, which affect Zn solubilization.
For this purpose, enzyme extracts from salmon stomach and intestine will be used to simulate the digestive process in semi-permeable membrane bioreactors, and the soluble Zn fraction will be quantified by ICP-MS. Key factors will be identified, and through factorial trials and statistical modeling, predictive tools will be generated and subsequently validated using in vivo data from WP1.
WP3: Gut explants and microbiome data for zinc absorption
Leader: Simon Menanteau-Ledouble (NORCE)
Activities
The gut sac method will be used to model Zn intestinal absorption under different dietary conditions. For this, the intact gut lumen will be excised from the anterior intestine of salmon and sealed. Zn-containing digesta samples produced in WP2 will then be introduced into the sacs, and Zn flux (both active and passive pathways) will be assessed by measuring zinc concentrations across five compartments: gut lumen, mucus layer, mucosal epithelium, muscle layer, and serosal saline. This approach allows the study of the three main steps of intestinal transport: mucus binding, accumulation in the mucosal epithelium, and transfer to the blood side. The obtained models will be validated using data from WP1.
In addition, we will investigate Zn sequestration by intestinal microbial communities as a potential cause of Zn loss in the gut. A previously assembled metagenomic and metatranscriptomic database from Norwegian salmon will be interrogated, and bioinformatic platforms will be used to identify microbial strains or metabolic pathways with an above-average abundance of Zn-dependent metalloenzymatic genes, suggesting high Zn requirements. These microbial fingerprints will then be correlated with environmental data and aquaculture practices associated with increased Zn sequestration by the microbiota.
WP4: Cell culture models for zinc uptake and health impacts
Leaders: Patrik Tang and Simon Menanteau-Ledouble (NORCE)
Activities
Using a combination of gut and liver cell cultures with transcriptomic analysis will provide comprehensive insight into the uptake, regulation, and metabolism of Zn, helping to optimize its supplementation in salmon feeds to support fish health and reduce environmental runoff.
RNA-sequencing of standard salmonid intestinal cell lines exposed to the dietary conditions tested in WP2 will be performed to identify differences in the activation of pathways regulating Zn bioavailability, including both active uptake and non-specific routes. These results will be complemented with barrier flux data from ex vivo gut models in WP3, enabling a holistic evaluation of Zn transport across the gut barrier under different environmental and biological conditions simulated in WP2.
Salmonid liver cell lines will be used to assess the effects of different Zn concentrations in a dose-dependent manner on metallothionein production, antioxidant activity, inflammation, apoptosis, and general liver function. This will provide insight into the benefits and the potential toxicity associated with varying plasma Zn gradients.
To validate the cell culture models, selected pathways will be targeted using qPCR (quantitative polymerase chain reaction) technique in the corresponding fish tissue samples from WP1.
The project consists of the following work packages (WPs):
WP1: Feeds preparations and in vivo material and data collection
Leader: Naouel Gharbi (NORCE)
Activities
This work package (WP) provides the experimental feeds and biological material required to conduct and/or validate the 3R models developed in WPs 2–4. Given the complex nature of Zn bioavailability and the conflicting evidence on the optimal form and concentration of dietary Zn for salmon at different life stages, we will test two Zn sources (inorganic and organic) at three dietary inclusion levels (deficient, adequate, and supra-optimal) in parr (freshwater) and post-smolt (seawater) stages. This design will enable the generation of robust dose–response curves for key biological functions assessed in the subsequent WPs.
Experimental feeds will be formulated to resemble commercial diets and produced at a small scale for use in the 3R pipeline developed in WPs 2–4. Water, feed, and fish samples will be collected from one or two commercial production sites (FishBase).
The collected material will serve two purposes: (1) to provide enzyme extracts for in vitro digestibility assays (WP2) and intact gut tissues for the gut sac model (WP3); and (2) to generate in vivo data for validating Zn solubilization, uptake, and retention predicted by the models in WPs 2–4. Short-term Zn status will be evaluated in faeces, intestine, and blood, while long-term status will be assessed in liver and whole-body samples.
WP2: In vitro digestibility assays to simulate zinc bioaccessibility
Leaders: Neda Gilannejad (NORCE) and Francisca P. Martínez Antequera University of Almeria (UAL)
Activities
In vitro digestibility assays will be developed to assess Zn bioaccessibility in Atlantic salmon. This approach will allow us to evaluate the influence of multiple factors on Zn potential availability, including feed-related parameters such as Zn form and concentration or feed composition (e.g., additives like phytase or acidifiers), as well as physiological and environmental conditions such as gastrointestinal pH and water salinity, which affect Zn solubilization.
For this purpose, enzyme extracts from salmon stomach and intestine will be used to simulate the digestive process in semi-permeable membrane bioreactors, and the soluble Zn fraction will be quantified by ICP-MS. Key factors will be identified, and through factorial trials and statistical modeling, predictive tools will be generated and subsequently validated using in vivo data from WP1.
WP3: Gut explants and microbiome data for zinc absorption
Leader: Simon Menanteau-Ledouble (NORCE)
Activities
The gut sac method will be used to model Zn intestinal absorption under different dietary conditions. For this, the intact gut lumen will be excised from the anterior intestine of salmon and sealed. Zn-containing digesta samples produced in WP2 will then be introduced into the sacs, and Zn flux (both active and passive pathways) will be assessed by measuring zinc concentrations across five compartments: gut lumen, mucus layer, mucosal epithelium, muscle layer, and serosal saline. This approach allows the study of the three main steps of intestinal transport: mucus binding, accumulation in the mucosal epithelium, and transfer to the blood side. The obtained models will be validated using data from WP1.
In addition, we will investigate Zn sequestration by intestinal microbial communities as a potential cause of Zn loss in the gut. A previously assembled metagenomic and metatranscriptomic database from Norwegian salmon will be interrogated, and bioinformatic platforms will be used to identify microbial strains or metabolic pathways with an above-average abundance of Zn-dependent metalloenzymatic genes, suggesting high Zn requirements. These microbial fingerprints will then be correlated with environmental data and aquaculture practices associated with increased Zn sequestration by the microbiota.
WP4: Cell culture models for zinc uptake and health impacts
Leaders: Patrik Tang and Simon Menanteau-Ledouble (NORCE)
Activities
Using a combination of gut and liver cell cultures with transcriptomic analysis will provide comprehensive insight into the uptake, regulation, and metabolism of Zn, helping to optimize its supplementation in salmon feeds to support fish health and reduce environmental runoff.
RNA-sequencing of standard salmonid intestinal cell lines exposed to the dietary conditions tested in WP2 will be performed to identify differences in the activation of pathways regulating Zn bioavailability, including both active uptake and non-specific routes. These results will be complemented with barrier flux data from ex vivo gut models in WP3, enabling a holistic evaluation of Zn transport across the gut barrier under different environmental and biological conditions simulated in WP2.
Salmonid liver cell lines will be used to assess the effects of different Zn concentrations in a dose-dependent manner on metallothionein production, antioxidant activity, inflammation, apoptosis, and general liver function. This will provide insight into the benefits and the potential toxicity associated with varying plasma Zn gradients.
To validate the cell culture models, selected pathways will be targeted using qPCR (quantitative polymerase chain reaction) technique in the corresponding fish tissue samples from WP1.
Dissemination of project results
Dr. Gharbi and Prof. Ebbesson (NORCE) will take the lead on stakeholder communication, leveraging their strong networks in both the scientific and industrial sectors. Their efforts will be supported by active involvement from all project partners—particularly Fishbase, whose industry presence will be key in facilitating dialogue and outreach.
The project aims to generate at least three peer-reviewed publications, and findings will also be shared at relevant scientific and industry conferences.
Dissemination will be a central focus, with results communicated through established networks, meetings, conferences, scientific journals, and platforms. Other dissemination measures include publication industry industry-relevant journals, as well as participation in aquaculture-related podcasts. In addition, a summary report will be prepared for FHF and shared with relevant stakeholders in the aquaculture sector.
Dr. Gharbi and Prof. Ebbesson (NORCE) will take the lead on stakeholder communication, leveraging their strong networks in both the scientific and industrial sectors. Their efforts will be supported by active involvement from all project partners—particularly Fishbase, whose industry presence will be key in facilitating dialogue and outreach.
The project aims to generate at least three peer-reviewed publications, and findings will also be shared at relevant scientific and industry conferences.
Dissemination will be a central focus, with results communicated through established networks, meetings, conferences, scientific journals, and platforms. Other dissemination measures include publication industry industry-relevant journals, as well as participation in aquaculture-related podcasts. In addition, a summary report will be prepared for FHF and shared with relevant stakeholders in the aquaculture sector.