Introduction
One of the primary goals of modern aquaculture is to develop sustainable aquafeeds, largely focusing on alternative protein sources to replace traditional marine-derived ingredients. Meals derived from plant, livestock, insects and microbial sources, are the main alternatives currently employed, that however often result in lower diet’s palatability. In fact, the off-flavour substances present in these ingredients often affect aquafeeds attractiveness, depressing fish feed intake, eventually having negative consequences for growth and welfare. Additionally, the resulting feed wastage derived from uneaten feed has side effects on both the farm’s economy and the environment. In fact, not ingested feed remains in wastewater outflows and thus can contribute to the eutrophication of aquatic ecosystems, representing a direct financial loss as well.
To improve aquafeed palatability, marine-derived feed attractants are regularly included in aquafeeds. Particularly, meals derived from shrimp, anchovy, and squid are well-recognized feed attractants, but due to their natural origin they present both unsustainability issues and highly variable attractiveness. In fact, their attractive effect depends on their harvesting location, the raw material quality and composition, the freshness, and processing methods, posing sustainability concerns and fluctuations in the product availability. The current alternative solution is represented by a limited number of molecules, such as mixture of free amino acids, nucleosides, and nucleotides, or substances such as betaine and taurine, that can be used as feed attractants. Particularly, among these substances, amino acids have been shown the strong attractive effects, although the limitations and disadvantages of their use as feed attractants are well-known. In this context, synthetic flavours are emerging as a promising and sustainable alternative to conventional feed attractants, obtained through standardized processes. Preliminary studies conducted throughout the zebrafish life cycle (Danio rerio) demonstrated that the dietary supplementation of attractive synthetic flavours had positive effects on appetite stimulus, feed ingestion, reward pathways and, consequently, on the growth of fish. Zebrafish is widely recognized as an optimal model for finfish nutrition research, and preliminary findings in this species can serve as a foundation for further studies in farmed fish.
In this regard, the aim of the present study was to test the effectiveness same synthetic flavours previously tested in zebrafish, on European seabass (Dicentrarchus labrax), during a 90-day feeding trial, evaluating the fish physiological responses. The European seabass is a strictly carnivorous species with a high market value and of particular importance in the aquaculture sector of the Mediterranean region. The application of synthetic flavours as feed attractants can help reduce feed wastage and improve farm’s productivity. This approach also supports the shift toward more sustainable and eco-friendly practices in aquaculture.
Materials and Methods
Experimental diets. Two attractive (F1, cheese; F2, caramel) and one repulsive (F3, coconut) flavours, previously tested throughout zebrafish life cycle, were produced by To Be Pharma S.r.l. (S. Egidio alla Vibrata, Teramo, Italy). The selected flavours were then included in a commercial diet for European seabass (Ecoprime, Veronesi, VR, Italy), rich in land animal proteins (LAPs). Then, four experimental diets were prepared starting from the commercial CTRL diet by adding Propylene Glycol (basic solvent used for flavours’ production), F1, F2, and F3 flavours (named PG, F1, F2, or F3 diets, respectively). The PG and all the flavours were added at 1% (w/w) to the CTRL diet.
Experimental design. Five hundred and forty European seabass juveniles (initial body weight: 72.48 ± 8.04 g) were initially acclimated for two weeks. At the end of acclimation period, fish were randomly divided into six experimental groups (in triplicate) as follows: (i) CTRL group, fish fed the CTRL diet; (ii) PG group, fish fed PG diet; (iii) F1 group, fish fed F1 diet; (iv) F2 group, fish fed F2 diet; (v) F3 group, fish fed F3 diet; (vi) ROT group, fish fed the two attractive diets (F1 and F2), each administered singularly in a weekly rotation scheme, to avoid a possible olfactory receptors adaptation in response to a long-term stimulation. The duration of the feeding trial was 90 days during which fish were hand-fed the experimental diets at 1.5% of body weight, daily monitoring the feed consumption. At the end of the feeding trial, all the fish were euthanized and then individually weighted.
Analyses. Biometric data were used to calculate the zootechnical parameters, and the daily feed intake was evaluated. In addition, samples from brain, liver, and distal intestine, were collected for: i) histological analyses in liver to determine the hepatic lipid and glycogen deposition and in distal intestine to verify eventual alterations or signs of inflammation; ii) molecular analyses to evaluate the gene expression of markers involved in fish brain reward system (drd3), serotonergic system (5ht1a), and immune response (il1b, il10, tnfa).
Results and Discussion
The PG and all the tested synthetic flavours had no adverse effects on liver and distal intestine, as widely demonstrated by the histological analyses. However, fish fed caramel and coconut diets showed an upregulation of immune-related markers in distal intestine, despite the clear absence of inflammatory process evidenced by the histological analyses. However, the various roles of interleukins may explain result inconsistencies, highlighting the need for more research on how synthetic flavours affect cytokine gene expression. Despite this, fish fed F2 (caramel) diet showed a higher feed intake, confirming, even in this species, the attractive effectiveness of the caramel flavour, as already demonstrated in zebrafish. This led to direct consequences on the amount of energy available for fish to support growth performance and storage of nutrients. In fact, caramel group showed the highest weight gain and growth rates and exhibited a significantly higher fat percentage in the liver. Finally, the positive modulation of monoaminergic pathways in F2 group may contribute to the hedonic value of the diet and the desire to consume feed, driven by reward sensation.
Overall, the caramel flavour notably improved feed palatability, enhancing its intake and fish growth, highlighting the role of sensory cues in feeding motivation. This study confirmed the potential of synthetic flavours to enhance aquaculture practices in a sustainable and cost-effective approach for the sector.