The Application of Synthetic Flavors in Zebrafish (Danio rerio) Rearing with Emphasis on Attractive Ones: Effects on Fish Development, Welfare, and Appetite

Abstract

The aim of the present study was to test synthetic flavors as potential feed attractants in zebrafish (Danio rerio) during early development. Six experimental groups were set up in triplicate: (i) a CTRL group fed a zebrafish commercial diet; (ii) a PG group fed a control diet added with Propylene Glycol (PG); (iii) A1⁺ and A2⁺ groups fed a control diet added with 1% of the two attractive flavors (A1⁺ cheese odor made by mixing Propylene Glycol (PG) with the aromatic chemicals trimethyamine, 2-acetylpyrazine, 2-acetylpyridine, and dimethyl sulfide; and A2⁺ caramel odor, made of PG mixed with the aromatic chemicals vanillin, maltol, cyclotene, acetoin, butyric acid, and capric acid with traces of both gamma-octalactone and gamma-esalactone) or the repulsive flavor (A^- coconut odor, made by mixing PG with the aromatic chemicals gamma-eptalactone, gamma-nonalactone, delta-esalactone, and vanillin with trace of both delta-octalactone and maltol), respectively; (iv) an ROT group fed the two attractive diets, each administered singularly in a weekly rotation scheme. All the tested synthetic flavors did not affect the overall health of larval and juvenile fish and promoted growth. Due to the longer exposure time, results obtained from the juvenile stage provided a clearer picture of the fish responses: zebrafish fed both attractive diets showed higher appetite stimulus, feed ingestion, and growth, while the brain dopaminergic activity suggested the A2⁺ diet as the most valuable solution for its long-lasting effect over the whole experiment (60-day feeding trial, from larvae to adults). The present study provided important results about the possible use of attractive synthetic flavors for aquafeed production, opening new sustainable and more economically valuable opportunities for the aquaculture sector.

Keywords: feed attractant; feed intake; growth factors; histology; zebrafish development.

Figure 1

Specific growth rate (% day ⁻¹) of zebrafish (a) larvae and (b) juveniles fed the experimental diets. Boxplots show minimum and maximum (whiskers), first quartile, median, and third quartile (box). ^a–e Different letters indicate statistically significant differences among the experimental groups (p < 0.05).

Figure 2

Percentage of feed ingested after 15 min administration of experimental diets in zebrafish juveniles. Results are expressed as mean + SD (n = 3). ^a–c Different letters indicate statistically significant differences among the experimental groups (p < 0.05).

Figure 3

Example of histomorphology of intestine and liver parenchyma of zebrafish (a–c) larvae and (d–f) juveniles from the present study. (a) Representative section of liver (*) and intestine (arrowhead) of a whole-embedded zebrafish larva fed CTRL diet; (b) details of mucosal folds of intestine from a zebrafish larva fed PG diet (double-headed arrow indicate mucosal folds height); (c) liver parenchyma from a zebrafish larva fed A1⁺ diet; (d) section of intestine (arrowhead) from a zebrafish juvenile fed A2⁺ diet (# indicate perivisceral adipose tissue); (e) details of mucosal folds of intestine from a zebrafish juvenile fed ROT diet (double-headed arrow indicate mucosal folds height); (f) liver parenchyma from a zebrafish juvenile fed A⁻ diet. Scale bars: a = 200 μm; b,c,e,f = 20 μm; d = 100 μm.

Figure 4

Relative mRNA abundance of genes involved in growth analyzed in whole larvae or in liver samples from juveniles. (a) igf1 and (b) mstnb in larvae; (c) igf1 and (d) mstnb in juveniles. 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 whole larvae or in intestine (ghrl), brain (npy), and liver (lepa) samples from juveniles. (a) ghrl, (b) npy, and (c) lepa in larvae; (d) ghrl, (e) npy, and (f) lepa in juveniles. Results are expressed as mean + SD (n = 5). ^a–d Different letters denote statistically significant differences among the experimental groups; ns, no significant differences.

Figure 6

Relative mRNA abundance of genes involved in reward system analyzed in whole larvae or in brain samples from juveniles. (a) drd1b, (b) drd2a, and (c) drd3 in larvae; (d) drd1b, (e) drd2a, and (f) drd3 in juveniles. Results are expressed as mean + SD (n = 5). ^a–e Different letters denote statistically significant differences among the experimental groups; ns, no significant differences.

Figure 7

Relative mRNA abundance of genes involved in immune and stress response analyzed in whole larvae or intestine (immune response: il1b, il10, and litaf) and liver (stress response: nr3c1) samples from juveniles. (a) il1b, (b) il10, (c) litaf, and (d) nr3c1 in larvae; (e) il1b, (f) il10, (g) litaf, and (h) nr3c1 in juveniles. Results are expressed as mean + SD (n = 5). ns, no significant differences.

Figure 8

Total lipid content (g/100 g) of zebrafish juveniles. ^a–f Different letters show statistically significant differences among experimental groups (p < 0.05). Values are reported as mean + SD (n = 3).