Introduction
One of the main goals of modern aquaculture is the formulation of sustainable aquafeeds, mainly based on alternative protein sources respect to the conventional marine-derived ones. The first widely used alternative was represented by vegetable ingredients which, however, have led to negative side-effects on fish growth and welfare, especially considering many carnivorous species. However, the improvement of processing methods for vegetable meals production as well as the introduction of new and more sustainable alternatives (e.g., processed animal proteins, insect meal, and single-cell proteins derived from cultures of different microbial sources), have partially solved these drawbacks. Nevertheless, the use of alternative ingredients often results in lower diet palatability that can consequently impair fish zootechnical performance, water quality, and the farm’s economy. In fact, not ingested feed remains in wastewater outflows and thus can contribute to the eutrophication of aquatic ecosystems, affecting the company’s economy 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 their use on large-scale is still limited by different factors. In fact, they still rely on fisheries activity that poses sustainability concerns and fluctuations in the product availability. Furthermore, their attractive effect is highly variable depending on raw material composition, freshness, and processing methods. The current alternative solution is represented by a limited number of molecules, such as a mixture of free amino acids, nucleosides, and nucleotides, or substances such as betaine and taurine, that can be used as feed attractants. Particularly, amino acids have been widely studied for their attractive properties, although the limitations and disadvantages of their use as feed attractants are well-known. Therefore, a novel, standardized, and sustainable alternative to marine-derived feed attractants is represented by synthetic ones, obtained through standardized processes. Existing literature indicates that few specific studies exist on this topic, which instead represents a great opportunity for the aquaculture sector.
The aim of this study was to identify new different synthetic feed additives, widely used in other industrial sectors, testing them on zebrafish (Danio rerio; ZF) to assess their potential role as feed attractants. ZF represents a well-validated model for aquaculture nutritional studies and allows to investigate possible dietary effects on all its life cycle stages, in a relatively short time.
Materials and Methods
A behavioural screening test was conducted on ZF larvae to select three positive and a negative commercial feed additives (To Be Pharma S.r.l., Stant’Egidio alla Vibrata, Teramo (TE), Italy) within a plethora of 40 ones. The selected feed attractants (positive: B, C, D; negative: E) were then included in a commercial diet for ZF (Zebrafeed, Sparos ltd, Portugal) to be tested from the larval to the juvenile stage. Particularly, the commercially available standard diet for ZF was used as Control, while five experimental diets were obtained by adding the previously selected feed additives to the Control diet (diets B, C, D, and E). All feed additives were added at 1% (w/w) concentration using a micropipette, coating the pellet by mixing homogeneously. ZF larvae obtained from broodstock AB strain were divided to set up seven experimental groups: Control, group A (Control diet + feed additive basic solvent), group B (Control diet + feed additive B), group C (Control diet + feed additive C), group D (Control diet + feed additive D), group E (Control diet + feed additive E), and group F. This last group was fed the three attractive diets (B, C, and D), each administered singularly in a weekly rotation scheme since it has been demonstrated that teleost, in response to a repeated stimulation, develop olfactory receptors habituation to the same stimulus. ZF were daily fed the experimental diets (3% of the body weight) from 5 to 60 days post fertilization (dpf). The required number of fish per tank were sampled at 21 dpf to evaluate dietary effect of feed additives administration during larval development. The remaining fish from each tank were gently transferred to bigger tanks, fed on the same diets and, at 60 dpf, were euthanized to evaluate the long-term dietary effect of feed additive administration on juvenile stage. For both larvae and juveniles the following analysis were performed: (i) growth performances calculating the specific growth rate (SGR%); (ii) histological analyses to evaluate eventual alterations or the occurrence of inflammatory conditions in both the intestinal tract and the hepatic parenchyma; (iii) molecular analyses to evaluate the relative expression of genes involved in fish growth (igf1, mstnb), appetite regulation (ghrl, npy, lepa), stress response (gr), immune response (il1b, il10, tnfa), and brain reward system (drd1b, drd2a, drd3).
Results and Discussion
Fish welfare was not affected by all the feed additives tested, while considering SGR%, significant differences (p < 0.05) were detected among groups, supporting the fact that the positive feed additives here tested stimulated fish feed intake and thus growth. All these results are well supported by both histological and molecular analysis on fish welfare (immune-response, stress response, gut/liver anatomical architecture) and appetite regulation and showed some interesting analogies or differences between the larval and juvenile stages which will be presented during the research discussion. As regards the analysis on gene expression involved in reward analysed in brain, results evidenced a different activity among groups of the dopamine receptors, the main neurotransmitter involved in the pleasure sensation, suggesting a pivotal role of the tested feed additives in their activation/inactivation. In conclusion, the positive feed additives administration promoted higher feed intake, acting on the fish brain reward system without impairing welfare and represents a very promising result for the aquaculture industry since a faster and complete feed intake by the fish has both ecological and economic benefits for the sector.