The Promising Role of Synthetic Flavors in Advancing Fish Feeding Strategies: A Focus on Adult Female Zebrafish (Danio rerio) Growth, Welfare, Appetite, and Reproductive Performances

Abstract

The present study aimed to test over a six-month period different synthetic flavors in zebrafish (Danio rerio) as an experimental model. Specifically, two attractive and one repulsive synthetic flavors were added (1% w/w) to a specific zebrafish diet, which was administered to the fish during the whole life cycle (from larvae to adults), to evaluate their physiological responses, emphasizing fish welfare, feed intake, growth, reward mechanisms, and reproductive performances. Fish welfare was not affected by all tested flavors, while both attractive flavors promoted fish feed ingestion and growth. The results were supported by both molecular and immunohistochemical analyses on appetite-regulating neurohormonal signals, along with the influence of the feed hedonic properties induced by the brain reward sensation, as demonstrated by the dopamine receptor gene expression. Finally, the present study demonstrated that a higher feed intake also had positive implications on fish reproductive performances, suggesting a promising role of synthetic flavors for the aquaculture industry. In conclusion, the results highlighted the potential of synthetic flavors to improve fish feeding strategies by providing a consistent and effective alternative to traditional stimulants, thereby reducing dependence on natural sources.

Keywords: aquaculture; feed attractant; feed intake; histology; sustainable aquafeeds.

Figure 1

Final body weight (mg) (left) and survival rate (%) (right) of adult zebrafish fed the experimental diets. Boxplot shows minimum and maximum (whiskers), first quartile, median, and third quartile (box). ^a–d Different letters indicate statistically significant differences among the experimental groups (p < 0.05). Values are presented as mean ± SD (n = 3); ns, no significant differences.

Figure 2

Percentage of feed ingested by adult zebrafish, calculated 15 min post dietary administration. Results are expressed as mean + SD (n = 3). ^a,b Different letters indicate statistically significant differences among the experimental groups (p < 0.05).

Figure 3

Representative figures of intestine and liver parenchyma of adult zebrafish (a,b) from the present study: (a) details on mucosal folds (double-headed arrow: mucosal folds height); (b) liver parenchyma. Scale bars: 50 μm.

Figure 4

Relative mRNA abundance of genes involved in growth analyzed in liver samples from adult zebrafish. (a) igf1 and (b) mstnb. Results are expressed as mean + SD (n = 5). ^a,b Different letters denote statistically significant differences among the experimental groups; ns, no significant differences.

Figure 5

Relative mRNA abundance of genes involved in appetite regulation analyzed in brain (npy), intestine (ghrl), and liver (lepa) samples from adult zebrafish. (a) npy, (b) ghrl, and (c) lepa. Results are expressed as mean + SD (n = 5). ^a–c Different letters indicate statistically significant differences among the experimental groups.

Figure 6

Relative mRNA abundance of drd2a involved in brain reward system analyzed in brain samples from adult zebrafish. Results are expressed as mean + SD (n = 5). ^a,b Different letters denote statistically significant differences among the experimental groups.

Figure 7

Relative mRNA abundance of nr3c1 involved in stress response analyzed in liver samples from adult zebrafish. Results are expressed as mean + SD (n = 5); ns, no significant differences.

Figure 8

Neuropeptide Y (NPY) expression in the olfactory epithelium of zebrafish fed the different experimental diets. (a) Zebrafish olfactory organ, highlighting the morphology of epithelium arranged in lamellae. The central and medial region of each lamella includes a continuous sensory area, in which different olfactory sensory neurons (OSNs) are located, as well as a lateral non-sensory area (illustration by Conti F.); (b,c) DAPI (blue) correspond to nuclei while green (Alexa Fluor^® 488) correspond to NPY (CTRL group); (d) representative transverse section of zebrafish olfactory epithelium from CTRL group showing NPY-expressing cells (arrowheads); (e) representative transverse section of zebrafish olfactory epithelium from F35 (caramel) group showing NPY-expressing cells (arrowheads). Scale bar = 50 µm.

Figure 9

Oocyte developmental stages in zebrafish ovary. PreV, pre-vitellogenic stage; PostV, post-vitellogenic stage; N, nucleus. Scale bar: 200 µm.

Figure 10

Total number of spawned eggs per day and percentage of hatching rate observed in fish fed the different experimental diets. (a) Average daily number of eggs laid within a 10-day period. (b) Hatching rate expressed as percentage. Boxplots show minimum and maximum (whiskers), first quartile, median, and third quartile (boxes). ^a–c Different letters indicate statistically significant differences between experimental groups (p < 0.05). Values are presented as mean ± SD (n = 10 spawning days, n = 9 for hatching rate).

Figure 11

Relative mRNA abundance of genes involved in vitellogenin production analyzed in liver from adult female zebrafish. (a) vtg1, (b) vtg2, and (c) vtg3. Results are expressed as mean + SD (n = 5). ^a,b Different letters indicate statistically significant differences among the experimental groups.