Periodic Reporting for period 3 - Fish-AI (Developing an Artificial Intestine for the sustainable farming of healthy fish)
Okres sprawozdawczy: 2022-10-01 do 2024-12-31
• What is the problem/issue being addressed?
A healthy, balanced diet has a fundamental role in preventing a wide range of chronic diseases. Aquaculture plays a substantial role in this perspective because fish is an important source of well-balanced proteins. However, aquaculture’s sustainability has been criticized. Indeed, the industry is trying to address these concerns by actively implementing the substitution of marine-derived protein and lipid sources by using plant-based feeds from sustainable agriculture. At the same time insects and microorganisms are being explored. However, alternative feedstuffs often have characteristics that make them less suitable for use in aquafeeds compared to fishmeal, for example by not supporting optimal fish health. The search for viable and sustainable feed ingredients for aquafeeds requires a continuous and extensive raw material evaluation program.
• Why is it important for society?
This project has developed a reliable in vitro platform that can provide relevant information on the biological properties of fish feeds that will enable a faster progression in feed formulations by reducing the number of animals required.
• What are the overall objectives?
The overall objective is the development of an in vitro platform able to screen efficiently and reliably the health and nutritional value of sustainable fish feeds while reducing the need for experiments with fish.
A healthy, balanced diet has a fundamental role in preventing a wide range of chronic diseases. Aquaculture plays a substantial role in this perspective because fish is an important source of well-balanced proteins. However, aquaculture’s sustainability has been criticized. Indeed, the industry is trying to address these concerns by actively implementing the substitution of marine-derived protein and lipid sources by using plant-based feeds from sustainable agriculture. At the same time insects and microorganisms are being explored. However, alternative feedstuffs often have characteristics that make them less suitable for use in aquafeeds compared to fishmeal, for example by not supporting optimal fish health. The search for viable and sustainable feed ingredients for aquafeeds requires a continuous and extensive raw material evaluation program.
• Why is it important for society?
This project has developed a reliable in vitro platform that can provide relevant information on the biological properties of fish feeds that will enable a faster progression in feed formulations by reducing the number of animals required.
• What are the overall objectives?
The overall objective is the development of an in vitro platform able to screen efficiently and reliably the health and nutritional value of sustainable fish feeds while reducing the need for experiments with fish.
In the first part of the project the consortium derived two new rainbow trout intestinal cell lines that are stable and represent both the proximal and the distal portion of the organ. At the same time an efficient and reliable method for extracting the bioactive fraction of the feed pellets was developed. This enables the pellets to be administered to the cells. Finally, an extensive range of Hydrogel -based scaffolds and culture devices have been developed and tested, but any of the resulting combinations proved to be adequate to the scope of the project.
During the last 27 months the consortium developed and tested two functional prototypes based on commercially available cell supports. Both are based on a bicameral system where the upper chamber mimics the intestinal lumen, and the lower chamber is the functional equivalent of the blood vessels. One includes only epithelial cells, while the other includes also a tri-dimensional scaffold that hosts fibroblasts and extracellular matrix. After a thorough comparison the conclusion was that the simpler prototype is more reliable and enables a wider range of analytical tests.
In the final part of the project we tested with a traditional nutritional trial in vivo 5 diets (see Deliverable 6.1) containing protein sources known to vary in nutritional value and health value, i.e. fish meal, soybean meal, feather meal, as well as one single cell protein at two inclusion levels. At the same time, we used the protocol previously developed to extract the bioactive fractions of same diets and added them to the selected in vitro platform prototype. Finally, the two sets of results were compared to determine the degree of correspondence between the two and, therefore, establish Fish-AI predictive power.
The Fish-AI prototype function and predictive ability can be summarized as follows:
• The final Fish-AI prototype can provide a high-resolution ranking of different diets.
• Fish-AI provides repeatable results within the same laboratory.
• Fish-AI provides different ranking depending on the analysis performed with it.
• Good correlation with in vivo results was achieved.
• Since in vivo trials measure parameters very different from in vitro tests, additional work is required to further improve how to correlate these two heterogenous sets of results
During the last 27 months the consortium developed and tested two functional prototypes based on commercially available cell supports. Both are based on a bicameral system where the upper chamber mimics the intestinal lumen, and the lower chamber is the functional equivalent of the blood vessels. One includes only epithelial cells, while the other includes also a tri-dimensional scaffold that hosts fibroblasts and extracellular matrix. After a thorough comparison the conclusion was that the simpler prototype is more reliable and enables a wider range of analytical tests.
In the final part of the project we tested with a traditional nutritional trial in vivo 5 diets (see Deliverable 6.1) containing protein sources known to vary in nutritional value and health value, i.e. fish meal, soybean meal, feather meal, as well as one single cell protein at two inclusion levels. At the same time, we used the protocol previously developed to extract the bioactive fractions of same diets and added them to the selected in vitro platform prototype. Finally, the two sets of results were compared to determine the degree of correspondence between the two and, therefore, establish Fish-AI predictive power.
The Fish-AI prototype function and predictive ability can be summarized as follows:
• The final Fish-AI prototype can provide a high-resolution ranking of different diets.
• Fish-AI provides repeatable results within the same laboratory.
• Fish-AI provides different ranking depending on the analysis performed with it.
• Good correlation with in vivo results was achieved.
• Since in vivo trials measure parameters very different from in vitro tests, additional work is required to further improve how to correlate these two heterogenous sets of results
At the end of the project, given the results obtained and the milestones achieved, we confirm many of the expected impacts. Moreover, following feedback received from stakeholders and potential users of the platform, the Fish-AI impacts were expanded and integrated and are summarized below:
Scientific and technological contributions to the foundation of a new future technology
• The possibility of offering a reliable in vitro technology as a relevant alternative to the use of animal models
• The possibility of shortening the time required for new raw material development and screening. According to our results, preliminary data can be available within 24/48 hours of exposure.
• The possibility of using the artificial intestine as a reliable model for neonatological application and for premature infant nutrition. During the project we also investigated the possibility of adapting this technology to other animals’ species (e.g swine, model of choice when studying infant gastro-intestinal pathologies such as short-bowel diseases)
• The possibility of using the platform to validate and verify in vivo experimental hypothesis by examining the molecular mechanisms at play at cellular level
• The possibility of using the platform for eco-toxicological studies and specifically to evaluate the uptake microplastics mechanisms at intestinal cellular level. In fact, during the project, we also explored the versatility and the flexibility of the platform for parallel applications involving the gut as a target organ
• the possibility of using the platform as an efficient screening system to define the power of analysis and estimate the sufficient and required number of animals to obtain significant results before conducting an in vivo feeding trial
• The generation of new IPs and POC initiatives aimed at sustaining the efforts for improving and refining the platforms and the know-how developed so far as a legacy of the project.
Potential for future social or economic impact or market creation
• The possibility of addressing climate change and environmental issues by promoting the identification of alternative sources and compounds for feed, while preserving their nutritional quality
• the possibility of improving aquaculture practices and enhancing worldwide food production in terms of quantity and quality. As mentioned above, using the Fish-AI platform the feed industries will be able to substantially speed up the procedures for the identification of irritating and/or inflammatory substances as well as bioactive compounds acting on intestinal health
volving the gut as a target organ
• the possibility of using the platform as an efficient screening system to define the power of analysis and estimate the sufficient and required number of animals to obtain significant results before conducting an in vivo feeding trial
• The generation of new IPs and POC initiatives aimed at sustaining the efforts for improving and refining the platforms and the know-how developed so far as a legacy of the project.
Potential for future social or economic impact or market creation
• The possibility of addressing climate change and environmental issues by promoting the identification of alternative sources and compounds for feed, while preserving their nutritional quality
• the possibility of improving aquaculture practices and enhancing worldwide food production in terms of quantity and quality. As mentioned above, using the Fish-AI platform the feed industries will be able to substantial speed up the procedures for the identification of irritating and/or inflammatory substances as well as bioactive compounds acting on intestinal health
Scientific and technological contributions to the foundation of a new future technology
• The possibility of offering a reliable in vitro technology as a relevant alternative to the use of animal models
• The possibility of shortening the time required for new raw material development and screening. According to our results, preliminary data can be available within 24/48 hours of exposure.
• The possibility of using the artificial intestine as a reliable model for neonatological application and for premature infant nutrition. During the project we also investigated the possibility of adapting this technology to other animals’ species (e.g swine, model of choice when studying infant gastro-intestinal pathologies such as short-bowel diseases)
• The possibility of using the platform to validate and verify in vivo experimental hypothesis by examining the molecular mechanisms at play at cellular level
• The possibility of using the platform for eco-toxicological studies and specifically to evaluate the uptake microplastics mechanisms at intestinal cellular level. In fact, during the project, we also explored the versatility and the flexibility of the platform for parallel applications involving the gut as a target organ
• the possibility of using the platform as an efficient screening system to define the power of analysis and estimate the sufficient and required number of animals to obtain significant results before conducting an in vivo feeding trial
• The generation of new IPs and POC initiatives aimed at sustaining the efforts for improving and refining the platforms and the know-how developed so far as a legacy of the project.
Potential for future social or economic impact or market creation
• The possibility of addressing climate change and environmental issues by promoting the identification of alternative sources and compounds for feed, while preserving their nutritional quality
• the possibility of improving aquaculture practices and enhancing worldwide food production in terms of quantity and quality. As mentioned above, using the Fish-AI platform the feed industries will be able to substantially speed up the procedures for the identification of irritating and/or inflammatory substances as well as bioactive compounds acting on intestinal health
volving the gut as a target organ
• the possibility of using the platform as an efficient screening system to define the power of analysis and estimate the sufficient and required number of animals to obtain significant results before conducting an in vivo feeding trial
• The generation of new IPs and POC initiatives aimed at sustaining the efforts for improving and refining the platforms and the know-how developed so far as a legacy of the project.
Potential for future social or economic impact or market creation
• The possibility of addressing climate change and environmental issues by promoting the identification of alternative sources and compounds for feed, while preserving their nutritional quality
• the possibility of improving aquaculture practices and enhancing worldwide food production in terms of quantity and quality. As mentioned above, using the Fish-AI platform the feed industries will be able to substantial speed up the procedures for the identification of irritating and/or inflammatory substances as well as bioactive compounds acting on intestinal health