In vitro Selection of Synbiotics and in vivo Investigation of Growth Indices, Reproduction Performance, Survival, and Ovarian Cyp19α Gene Expression in Zebrafish Danio rerio

Abstract

In this study, we tested the compatibility of two extracts from the plant Jerusalem artichokes and button mushrooms with two different Lactobacillus probiotics (Lactobacillus acidophilus; La and Lactobacillus delbrueckii subsp. Bulgaricus; Lb) to develop a synbiotic formulation to improve the growth, survival, and reproductive performances of farmed fishes. Initially, we employed in vitro approach to monitor the growth of the probiotic lactobacilli in the presence of the different doses of the plant-based prebiotics, with the aim of selecting interesting combination(s) for further verification under in vivo conditions using zebrafish as a model. Results from the in vitro screening assay in the broth showed that both the probiotic species showed a preference for 50% mushroom extract as a source of prebiotic. A synbiotic formulation, developed with the selected combination of L. acidophilus, L. bulgaricus, and 50% mushroom extract, showed a positive influence on the growth and reproductive performances of the zebrafish. Our findings also imply that the improvement in the reproductive indices was associated with the upregulation of a cyp19a gene. Overall results suggest that a combination of L. acidophilus, L. bulgaricus, and mushroom extract can be considered as a potential synbiotic for the successful production of aquaculture species.

Keywords: artichoke; cyp19a gene; mushroom; prebiotics; probiotics; reproduction; zebrafish.

Figure 1

Growth of Lactobacillus acidophilus in De Man Rogosa Sharpe (MRS) broth medium with and without Jerusalem artichoke extract added at an increasing concentration of 2, 25, 50, 75, or 100%. Lactobacillus acidophilus grown in MRS broth medium with a standard amount of glucose served as a positive control. The glucose-free MRS medium with 100% of artichoke extract and without L. acidophilus served as negative control 1. The MRS medium free from both glucose and extract but with L. acidophilus served as negative control 2. The data points are the mean values of three replicates.

Figure 2

Growth of L. acidophilus in MRS broth medium with and without mushroom extract added at an increasing concentration of 2, 25, 50, 75, or 100%. Lactobacillus acidophilus grown in MRS broth medium with a standard amount of glucose served as a positive control. The glucose-free MRS medium with 100% of mushroom extract and without L. acidophilus served as negative control 1. The MRS medium free from both glucose and extract but with L. acidophilus served as negative control 2. The data points are the mean values of three replicates.

Figure 3

Growth of Lactobacillus delbrueckii subsp. bulgaricus in MRS broth medium with and without artichoke extract added at an increasing concentration of 2, 25, 50, 75, or 100%. Lactobacillus acidophilus grown in MRS broth medium with a standard amount of glucose served as a positive control. The glucose-free MRS medium with 100% of artichoke extract and without L. acidophilus served as negative control 1. The MRS medium free from both glucose and extract but with L. acidophilus served as negative control 2. The data points are the mean values of three replicates.

Figure 4

Growth of L. delbrueckii subsp. bulgaricus in MRS broth medium with and without mushroom extract added at an increasing concentration of 2, 25, 50, 75, or 100%. Lactobacillus acidophilus grown in MRS broth medium with a standard amount of glucose served as a positive control. The glucose-free MRS medium with 100% of mushroom extract and without L. acidophilus served as negative control 1. The MRS medium free from both glucose and extract but with L. acidophilus served as negative control 2. The data points are the mean values of three replicates.

Figure 5

Comparison of the (A) specific growth rate (μmax; h⁻¹), (B) doubling time (T_d; h), and (C) pH of the medium of L. acidophillus with that of L. delbrueckii subsp. bulgaricus grown in MRS medium containing 50% mushroom or 100% artichoke extract. Bars with different letters indicate a significant difference (p<0.05). Data are presented as mean±SE.

Figure 6

Effects of feeding various experimental diets for 120days on the relative expression of cyp19a gene in the ovary of zebrafish, normalized against β-actin factor as reference gene. Bars with different letters indicate significant differences (p<0.05). Data are presented as mean±SE of three replicates. Diet 1 (control diet), Diet 2 (supplemented with 1% mushroom extract prepared from 50% concentrated extract), Diet 3 (supplemented with La at 10⁷CFUg⁻¹ of diet), Diet 4 (supplemented with Lb at 10⁷CFUg⁻¹ of diet), Diet 5 (supplemented with La at 10⁷CFUg⁻¹ of diet +1% mushroom extract prepared from 50% concentrated extract), Diet 6 (supplemented with Lb at 10⁷CFUg⁻¹ of diet +1% mushroom extract prepared from 50% concentrated extract), and Diet 7(supplemented with La and Lb, each at 10⁷CFUg⁻¹ of diet +1% mushroom extract prepared from 50% concentrated extract).