• aquaculture sustainability
• fish nutrition
• fish welfare
• fishmeal replacement

As the world population grows, access to food becomes a priority. Aquaculture is growing rapidly as a food-producing sector and continues to flourish day by day. In this context, it is crucial to improve both the sustainability of the sector and the welfare of farmed fish. The present thesis explores innovative dietary strategies and aquaculture practices to enhance aquaculture sustainability, improve fish health and welfare, and reduce reliance on environmentally impactful feed ingredients. Using mainly zebrafish (Danio rerio) as a model species, several experiments were conducted to evaluate the effects of novel feed formulations, alternative protein sources, and welfare-enhancing treatments, with implications for broader aquaculture applications.
The introduction contextualises the study within the global aquaculture sector, highlighting its rapid growth, sustainability challenges, and the critical need for alternative feed ingredients to replace fishmeal and fish oil. Key alternatives, such as black soldier fly larvae meal (Hermetia illucens), plant-based proteins, and microbial biomass, are reviewed. Moreover, an analysis of the state-of-the-art in fish welfare is provided including stress responses, nociception, and ethical farming practices.
The first experimental study described focuses on replacing fishmeal with Black Soldier Fly Larvae meal (BSFLM) obtained on two different plant-based substrates. Growth performance, reproductive outcomes, gut morphology, and gene expression (nfkb1, gata4, slc15a2, slc15a4) were assessed. The results demonstrated improved growth rates and specific growth rates in diets containing BSFLM, with substrate-dependent differences in performance. Gut immunity was analyzed through macrophage and neutrophil density in gut tissue using confocal microscopy, revealing substrate-specific impacts on intestinal health.
Using alternative, sustainable fish diets and developing farming systems like the SIMTAP (self-sufficient integrated multi-trophic aquaponic) system are promising strategies to enhance aquaculture sustainability. In the second experimental study described the growth performances of Gilthead sea bream (Sparus aurata) fed three diets based on detritivores and filter-feeders organisms, have been tested and compared to a commercial feed. The alternative diets consisted of (on a dry matter basis) 50% mussels and 50% clams in Experiment 1 (initial body weight 6.78 g); 50% mussels, 30% clams, and 20% polychaetes in Experiment 2 (initial body weight 37.21 g); and 49% of mussels, 30% of clams, 20% of polychaetes, and 1% of mineral premix in Experiment 3 (initial body weight 249.17 g). In both Experiments 1 and 2, the group fed on commercial feed showed better growth performances, notably for final body weight and specific growth rate. On the contrary, in Experiment 3, the fish fed on the alternative diet showed better growth performances. In all the Experiments, the group fed on an alternative diet showed a better viscera-somatic index and hepatosomatic index. In conclusion, these results suggest that the experimental diets based on organisms candidate for production in a land-based IMTA system, such as the SIMTAP, did not negatively affect GSB performances when at least a mineral dietary integration is considered. Finally, to assess the improved sustainability of the experimental diets in comparison to the commercial feed, an accurate evaluation of their environmental and economic impact is necessary.
Then, there is described another study about the fishmeal replacement with Hermetia illucens meal using zebrafish as animal model. The present study investigated the effects of increasing the inclusion of Hermetia illucens meal (0%, 17%, 33% and 50% of the feed, equating to 0%, 34%, 66% and 100% fishmeal replacement) on fish mortality, growth performance, intestine morphology, and gene expression of intestinal carriers. The results showed no adverse effects on fish mortality, feed intake and body weight and a positive effect on feed conversion ratio. Body weight gain was higher when 17% and 50% of Black soldier fly meals’ feed included (34% and 100% fishmeal replacement, respectively). Gut morphology was not affected by the dietary treatments except for the area of PAS-positive goblet cells that was higher in the treatment fed 33% of insect meal. The mRNA expression of intestinal epithelium functionality-specific marker genes, such as slc15a1, gata4 and nfkb1b, confirmed that the insect meal-based diets might replace fishmeal-based diets without negative effects. Overall, the results of the present study suggest that using Hermetia illucens larvae meal as a replacement for fishmeal in aquafeeds might help to enhance sustainability while assuring favorable fish growth performance and gut health.
The following studies were conducted to promote fish health and welfare during farming practices. Fish sedation may help to secure fish health, welfare, preventing damages and negative side effects, when carrying out some farming procedures such as fish handling, as transportation, or vaccination. Consequently, the aim of the first study described was to investigate the potential sedative effects of an herbal and mineral blend on zebrafish swimming behaviour. The blend was mainly composed by Melissa officinalis extract and soluble magnesium. The study consisted of two trials, the first carried out on zebrafish larvae and using 5 different water concentration of the experimental blend (0-, 0.5-, 1.0-, 2.0-, and 4.0- ml L-1). Larvae locomotor performances were evaluated using DanioVision® at 5 days post fertilization (Noldus©, The Netherlands). Based on the observed results, the second trial was carried out on adult zebrafish. Four treatments (CTRL, CTRL-R, T1 and T2) and three different experimental blend water concentration (0-, 1.0- and 2.0- ml L-1) were used. On the 3rd, 6th, 9th day of treatment, fish behaviour was tested using the Novel Tank diving Test (NTT). Larvae locomotor performances showed sedative effect already at 1.0 ml L-1, with a dose-dependent effect. However, regarding adult locomotor performance, no sedative effects were detected until 9th day of treatment at 2.0 ml L-1 concentration. Fish exposed to 2.0 ml L-1 of the experimental blend showed more confidence to explore the new environment (p < 0.05). These results open up interesting applications in the aquaculture sector. To this regard, more data are still necessary to assess the proper concentration of Melissa officinalis and soluble magnesium to supply a slight sedation to fish. Despite that, it might be of great interest investigating about the possible administration of the sedative compounds through the inclusion in aquafeeds.
The second one described was about the use of a water and feed additives. Among mineral feed additives, MgCl2 is the most applied relaxant for research and commercial applications. MgCl2 sedative action is attributed to the magnesium ions (Mg2+), which serve as a calcium channel blocker, preventing calcium ions (Ca2+) from entering the cell. Aim of the study was to investigate the potential toxicity and sedative effects on larvae exposed to 6 different water concentration of AFI Calm™ (Mg rich mineral feed), on zebrafish eggs hatchability, post-hatching survival, larvae locomotion and mRNA gene expression. The results showed toxicity effect and reduced survival rate at 1 mg ml-1 and 5 mg ml-1 water concentrations. No differences between treatments were observed for eggs hatchability. Locomotor performances showed sedative effect already at the lowest concentration of AFI Calm™ (0.125 mg ml-1), with a dose-dependent effect. The qRT-PCR performed on stress-related genes showed that AFI Calm™ induced down-regulation of pomca, even at the lowest concentration. For a contrary, AFI Calm™ induced an over-expression of serta and hsp70 genes at the highest concentration (0.250 mg ml-1 and 0.5 mg ml-1, respectively). In conclusion, the water exposure of zebrafish larvae to low doses of AFI Calm™ showed no negative effects on eggs hatchability and embryo post-hatching survival and induced lower locomotor performances already at the lowest concentration. These findings support the hypothesis that magnesium might have sedative effects on fish and opening new opportunities for applications in the aquaculture sector.
The last study described in this thesis is about the evaluation of the physiological health of smuggled European glass eels (Anguilla anguilla). European eel is considered a “critically endangered” species due to its population decline (c.a. 98 %) in all European waters, primarily because human activities. The eel life cycle is very complex as it includes long migrations, and although aquaculture can help in repopulation, at this time artificially raised eel larvae have little survival. Therefore, to reverse this situation, in 2007 the EU adopted the Eel Regulation, starting a series of plans to improve the conservation and the recovery of the eel population, which included a ban on importing and exporting eels in all EU countries. However, an illegal global market and food fraud developed, turning Europe into the source of the international illegal eel trade. This study reports the evaluation of the welfare consequences of the illegal transport of European glass eels using their epithelial mucus, one of the most promising tools for studying endangered fish species. We also aimed to determine the recovery time required in controlled conditions before their release to the Ebro River. The results showed that high metabolite and cortisol levels were observed in mucus. Regarding the recovery time, we analysed the same parameters as for the initial transport point resulting in a stabilisation of stress parameters and an increased defensive capacity from day 3 to the final release, but the best results were observed after 2 months under optimal conditions. These findings suggest that transport stress is critical for the survival of glass eels, and the stress and defensive capacities analysis suggests the need for a recovery period prior to release. This approach represents a valuable contribution both for species subject to illegal trade and for the conservation of endangered species.
These findings highlight the potential of use of Hermetia illucens meal in aquafeed, welfare practices, and systems like SIMTAP to improve aquaculture sustainability and fish welfare, with implications for future environmental and economic impact assessments.