Protective effects of Bifidobacterium longum subsp. longum BL21 against cyclophosphamide-induced reproductive dysfunction in zebrafish

Abstract

This study aimed to investigate the effects of probiotic Bifidobacterium longum subsp. longum BL21 on reproductive health in zebrafish (Danio rerio), focusing on hormonal modulation, sperm vitality, and overall reproductive function improvement. Adult male zebrafish of the wild-type AB strain were divided into four groups: control (CTL), model control with cyclophosphamide-induced oligospermia (CS), probiotic-treated (BL21), and a positive control treated with clomiphene citrate (CC). All groups, except CTL, were exposed to cyclophosphamide from day 1 to day 9 to induce reproductive dysfunction, with subsequent monitoring until day 16. Key metrics assessed included body and testicular weight, sperm vitality, male courtship behavior, hormone levels, sperm DNA fragmentation, and gene expression of PCNA, NANOG, ZBTB16, mTOR, DDX4, CYP26A1, and ALDH1A2. In this study, BL21 demonstrated significant therapeutic potential in treating reproductive dysfunction in zebrafish. Metabolomic analysis revealed that BL21 influenced crucial pathways, notably upregulating mTOR signaling and isoflavone biosynthesis, while downregulating the TCA cycle and pyruvate metabolism, suggesting a broad biochemical impact. The probiotic treatment notably improved testicular weight (p < 0.01) and sperm count and vitality (p < 0.001) compared to the cyclophosphamide-induced model control, underscoring its efficacy in enhancing reproductive parameters. Additionally, BL21 intervention led to a marked increase in chasing behavior and sex hormone levels, surpassing those of the positive control. The treatment also significantly reduced sperm DNA fragmentation (p < 0.001) and increased the expression of genes crucial for spermatogenesis and testicular function (p < 0.05), confirming its potential to restore reproductive health at multiple biological levels. These results highlight the promise of BL21 as a multi-faceted agent for improving reproductive health, warranting further investigation in clinical settings. Probiotic BL21 enhances reproductive parameters in zebrafish by modulating hormone levels, improving sperm quality, and positively affecting reproductive behavior. These findings suggest potential therapeutic applications of probiotics in managing reproductive health issues.

Keywords: Hormonal modulation; Male infertility; Probiotic; Sperm vitality; Zebrafish (Danio rerio).

Fig. 1

Metabolomic profiling of Bifidobacterium longum subsp. longum BL21. (A) Pathway analysis displays pathways significantly influenced by the fermentation product. “Up_hits” are pathways with increased activity (blue), “Down_hits” denote pathways with decreased activity (yellow), and “Hits” are pathways with detected activity that has not significantly changed. (B) Amino acid and (C) organic acid levels show the concentrations of various amino acids and organic acids contained in the inactivated bacterial mass.

Fig. 2

Experimental design to evaluate the impact of Bifidobacterium longum subsp. longum BL21 on cyclophosphamide-induced reproductive dysfunction in zebrafish. (A) Experimental flowchart detailing the procedural steps, (B) Body weight comparisons, (C) Testicular weight measurements across different treatment groups: CTL (normal control), CS (cyclophosphamide-only), BL21 (BL21 treatment), and CC (clomiphene citrate as positive control). NS indicates no significant difference. ** p < 0.01.

Fig. 3

Impact of Bifidobacterium longum subsp. longum BL21 on sperm parameters in zebrafish experiencing cyclophosphamide-induced reproductive dysfunction. (A) Sperm count, (B) Viability, (C,D) DNA fragmentation. The groups are as follows: CTL (normal control), CS (cyclophosphamide-only), BL21 (BL21 treatment), and CC (positive control with clomiphene citrate). ***p < 0.001. FITC-H denotes fluorescence intensity of stained DNA, reflecting DNA fragmentation levels, and SSC-H represents side scatter height, assessing cellular complexity.

Fig. 4

Effects of Bifidobacterium longum subsp. longum BL21 on reproductive behaviors and hormonal responses in zebrafish. (A) Diagram of the experimental tank setup, divided into a starting zone ('1') and a chasing zone ('2') to assess zebrafish mating behaviors. (B) Table presenting the frequency of male zebrafish chasing behaviors per minute across four groups: Control (CTL), Cyclophosphamide-only (CS), treated with BL21 (BL21), and a Clomiphene Citrate positive control (CC). (C) Movement trajectory plots for male (blue) and female (pink) zebrafish in the CS group showing baseline activities. (D) Enhanced trajectories in the BL21 group illustrate more frequent and interactive chasing, indicating improved reproductive behaviors after treatment. (E) Testosterone levels graph comparing responses across all groups, highlighting hormonal adjustments following treatments. (F) Follicle-stimulating hormone (FSH) levels graph, demonstrating gonadotropic responses to the treatments. (G) Luteinizing hormone (LH) levels graph, detailing endocrine responses across the treatment groups. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 5

Impact of Bifidobacterium longum subsp. longum BL21 on gene expression related to spermatogenesis and testicular development in zebrafish. (A) PCNA, (B) NANOG, (C) ZBTB16, (D) mTOR, (E) DDX4, (F) CYP26A1, (G) ALDH1A2 gene expression. Groups are control (CTL), cyclophosphamide only (CS), BL21 treated (BL21), and clomiphene citrate positive control (CC). *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 6

Effects of Bifidobacterium longum subsp. longum BL21 on reproductive recovery in cyclophosphamide-treated zebrafish.