The aquaculture industry constantly seeks new raw materials to improve flexibility and sustainability, exploring alternatives such as soybean meal, feather meal, algae, yeasts, or poultry by-products. However, these alternatives often contain anti-nutritional factors that can negatively impact fish health and growth. Evaluating the suitability of these new ingredients generally requires in vivo feeding trials (with live animals), which are costly, time-consuming, and involve the use of a large number of animals.
Faced with this problem, a recent study published in Frontiers in Marine Science by scientists from the University of Milan, The National Centre for Mariculture, the Norwegian University of Life Sciences (NMBU), and Skretting Aquaculture Innovation presents the development and application of an in vitro platform using intestinal cell lines from rainbow trout (Oncorhynchus mykiss). The objective is to determine if this system can discriminate the functional differences of feed formulations with contrasting properties, offering a faster, more economical, and ethical way for selecting promising ingredients. This article delves into the findings of this research and its potential implications for aquaculture.
What is an in vitro intestinal platform and why is it necessary in aquaculture?
In vitro intestinal models are not new in science. In mammals, immortalized cell lines, such as Caco-2 (derived from human colon) or IPEC-J2 (from porcine intestine), have been used for years for toxicology and drug absorption studies, reducing the need for animal testing. These cells can proliferate indefinitely, allowing for repeatable experiments and long-term studies.
In fish, the development of stable intestinal cell lines is more recent. The first was derived from the distal intestine of rainbow trout a few years ago, and more recently, the same laboratory that conducted this study developed two cell lines from the proximal (RTpiMI) and distal (RTdiMI) intestine of the same species, which are those used in this research. These rainbow trout cell lines retain important characteristics of their organ of origin, such as notable cellular heterogeneity (including stem cells and cells in different states of differentiation) and a “memory” of their intestinal region, responding differently to the same stimuli.
The fundamental purpose of the study was to validate whether this in vitro platform, composed of the RTpiMI and RTdiMI cell lines, could distinguish the effects of different diets on intestinal function, providing a valuable tool for the preliminary selection of feed ingredients.
Testing the diets in the laboratory
To evaluate the discriminatory capacity of the in vitro platform, the researchers selected three diets with contrasting properties:
- Reference diet (FM): Rich in fish meal, known for its high digestibility and widespread use.
- Diet with high inclusion of soybean meal (SBM): Soybean meal is a common protein alternative, but known for its enteritis-inducing effects (intestinal inflammation) in salmonids at high concentrations.
- Diet with high inclusion of feather meal (FTHM): A by-product of poultry processing, characterized by its low digestibility if not properly processed.
How did the intestinal cells respond to the different diets?
The in vitro platform revealed distinct responses depending on the diet and the intestinal region of origin of the cells.
Intestinal barrier integrity (TEER)
The reference diet (FM) and the diet with feather meal (FTHM) caused an increase in TEER values in both cell lines (proximal and distal), suggesting a strengthening or thickening of the barrier. In the proximal intestine cells (RTpiMI), the increase with FM was significantly greater than with FTHM.
The diet with soybean meal (SBM) had opposite effects: in the proximal intestine cells (RTpiMI), TEER decreased drastically after only 3 days, indicating damage to the barrier. Surprisingly, in the distal intestine cells (RTdiMI), SBM induced a gradual increase in TEER, similar to FM and FTHM.
A crucial aspect was the recovery test performed on the RTpiMI cells damaged by SBM. When SBM was removed and replaced with normal culture medium, the cells managed to completely restore barrier integrity (TEER values) in the following 18 days.
Enzymatic activity (AAP) and cell differentiation
In general, all tested diets induced a significant increase in AAP activity compared to control cells (without exposure to diets).
In the proximal intestine cells (RTpiMI), the FTHM diet induced a greater increase in AAP than FM. Cells exposed to SBM showed a sharp initial decrease in AAP activity (coinciding with TEER damage), but after the recovery period, AAP activity reached levels similar to the control.
In the distal intestine cells (RTdiMI), all diets (FM, FTHM, and SBM) caused a similar and significant increase in AAP activity compared to the control.
Morphological changes and cell proliferation
Control cells (without diet) formed a compact monolayer.
Both the FM and FTHM diets induced cell proliferation, forming a stratified epithelium (multilayer) in both cell lines. There were subtle differences in the compaction of these layers between diets and cell lines, which correlated with TEER values.
Exposure to SBM for 3 days caused clear disruptions in the monolayer of RTpiMI (proximal) cells. However, after the recovery period, these cells were able to recreate a functional monolayer, and some areas even showed multiple layers. In contrast, continuous exposure of RTdiMI (distal) cells to SBM for 21 days resulted in the formation of a stratified epithelium.
Appearance of vacuolated cells (possible stress response)
The presence of vacuoles (round formations within the cells) was observed, especially in RTpiMI cells, and their quantity varied according to the diet. The SBM diet induced the largest number of vacuoles in just 3 days. Some of these vacuoles tested positive with PAS staining, indicating the presence of mucins, suggesting a possible differentiation towards mucus-secreting cells.
The researchers hypothesize that, in the absence of a protective mucus layer in vitro (present in vivo), the cells might be differentiating towards a secretory lineage as a protective response to diet-induced stress. This was more evident in the proximal cells, which in vivo have a higher number of goblet cells (mucus-producing cells).
Implications for rainbow trout aquaculture
The results of this study are promising and suggest that this in vitro platform has the potential to become a valuable tool for the aquaculture industry.
Differential sensitivity of intestinal regions
Cells derived from the proximal intestine (RTpiMI) proved to be more sensitive and capable of discriminating the effects of different diets. In contrast, cells from the distal intestine (RTdiMI) showed a more homogeneous response and a greater propensity for proliferation in response to all tested diets. This finding is interesting, as in vivo, soybean meal is usually observed to primarily affect the distal intestine in salmonids. The authors suggest that the greater sensitivity of proximal cells in vitro could be due to their more absorptive nature or a lower intrinsic proliferation rate, which would make them more vulnerable to damage before they can repair.
Cellular responses as indicators
The cell proliferation observed with the FM and FTHM diets, and even with SBM in distal cells, could be an adaptive or stress response. The absence of a protective mucus layer in the in vitro system could amplify these responses, as the cells are directly exposed to the diet components. This underscores the importance of interpreting in vitro results within the context of the model’s limitations.
Functional classification of diets
For the proximal intestine cells (RTpiMI), the platform allowed for a functional classification: the SBM diet resulted in the most severe effects (although reversible), FTHM induced mild stress responses, and FM was the least disruptive. Although it is complex to compare directly with in vivo results – where different parameters can yield different classifications – the reversibility of SBM-induced damage observed in vitro is consistent with in vivo studies showing that SBM-induced enteritis is reversible after removing the problematic diet.
Potential of the platform
This in vitro system offers several advantages:
- Rapid and economical screening: Allows for faster and cheaper evaluation of a larger number of ingredients or additives than in vivo trials.
- Reduction in animal use: Contributes to the 3Rs principles (Replacement, Reduction, and Refinement) in animal experimentation.
- Study of mechanisms: Facilitates research into the cellular and molecular mechanisms of how nutrients or anti-nutrients affect intestinal cells.
- Identification of solutions: Could be used to identify molecules or treatments that can mitigate the negative effects of certain ingredients, such as anti-nutritional factors in SBM.
Conclusion: towards a more precise and ethical feed evaluation
The study demonstrates that the in vitro intestinal platform developed with rainbow trout cell lines is capable of providing valuable and functional information on the effects of different diets. Proximal intestine cells, in particular, appear to be a good model for discriminating the effects of different formulations.
While no in vitro platform can fully replicate the complexity of a living organism, this system represents a significant advancement. It can serve as an efficient pre-screening tool, helping to identify the most promising or problematic ingredients before moving on to more costly and complex in vivo trials. Furthermore, the ability to observe the recovery of cellular damage opens doors to studying intestinal repair processes and developing strategies to improve fish gut health.
Reference (open access)
Camin, F., Verdile, N., Chacon, M. A., Løkka, G., Peggs, D., Tandler, A., Bitan, A., Kortner, T. M., Fontanillas, R., Brevini, T. A., & Gandolfi, F. (2025). Use of a rainbow trout (Oncorhynchus mykiss) intestinal in vitro platform to evaluate different diets. Frontiers in Marine Science, 12, 1576618. https://doi.org/10.3389/fmars.2025.1576618
Editor at the digital magazine AquaHoy. He holds a degree in Aquaculture Biology from the National University of Santa (UNS) and a Master’s degree in Science and Innovation Management from the Polytechnic University of Valencia, with postgraduate diplomas in Business Innovation and Innovation Management. He possesses extensive experience in the aquaculture and fisheries sector, having led the Fisheries Innovation Unit of the National Program for Innovation in Fisheries and Aquaculture (PNIPA). He has served as a senior consultant in technology watch, an innovation project formulator and advisor, and a lecturer at UNS. He is a member of the Peruvian College of Biologists and was recognized by the World Aquaculture Society (WAS) in 2016 for his contribution to aquaculture.
