Confirmation of pancreatic β-cell ablation and onset of diabetes in zebrafish males. (A) Graphic representation of the construct inserted in the Tg(ins:nfsb-mCherry) genome. (B) Observation of pancreas with lack of fluorescence in Tg(ins:nfsb-mCherry) diabetic males, with evident pancreatic β-cell ablation and onset of diabetes. (C) Observation of pancreatic fluorescence in Tg(ins:nfsb-mCherry) control males after dissection, with the presence of live β cells (white area showing magnification of the pancreas).

Gene expression of insulin metabolism markers in zebrafish sperm pools: (A) expression of insa in spermatozoa pools from AB males in control (n = 4) and metronidazole (Met) treatment (n = 4) groups and Tg(ins:nfsb-mCherry) in control (n = 5) and Met treatment (n = 7) groups; (B) expression of insra in spermatozoa pools from AB males in control (n = 4) and Met treatment (n = 4) groups and Tg(ins:nfsb-mCherry) in control (n = 7) and Met treatment (n = 7) groups; (C) expression of slc2a2 in spermatozoa pools from AB males in control (n = 4) and Met treatment (n = 4) groups and Tg(ins:nfsb-mCherry) in control (n = 5) and Met treatment (n = 6) groups. The plotted values represent means ± SD. Different letters represent statistical differences (one-way ANOVA, post hoc SNK, p < 0.05).

Spermatozoa plasma membrane viability of fresh and cryopreserved sperm from untreated Tg(ins:nfsb-mCherry) males (n = 5) and metronidazole (Met)-treated males (n = 6). The plotted values represent means ± SD. Different superscript letters represent statistical differences (independent samples t-test, p < 0.05).

Sperm motility parameters of fresh sperm obtained from untreated Tg(ins:nfsb-mCherry) males (n = 5) and metronidazole (Met)-treated males (n = 8) and of cryopreserved sperm from untreated (n = 5) and Met-treated males (n = 10). Sperm was activated, and motility parameters were recorded every 10 s for 1 min in terms of: (A) total motility (%); (B) progressive motility (%); (C) curvilinear velocity (μm/s); (D) straight-line velocity (μm/s), and (E) linearity (%). The plotted values represent means. The dashed lines represent fresh sperm and the continuous lines represent cryopreserved sperm. Sperm from untreated males is represented with a white circle and sperm from Met-treated males is represented with a dark circle.

DNA integrity in zebrafish sperm: (A) comet with high DNA fragmentation; (B) comet with low DNA fragmentation; (C) DNA fragmentation of fresh sperm obtained from untreated Tg(ins:nfsb-mCherry) males (n = 4) and metronidazole (Met)-treated males (n = 8) and of cryopreserved sperm from untreated males (n = 5) and Met-treated males (n = 6). The plotted values represent means ± SD. Different letters represent statistical differences between treatments and the asterisk represents a statistical difference between fresh and cryopreserved sperm (independent samples t-test, p < 0.05).

Hypothetic model of putative effects of diabetes on sperm quality in type I diabetes zebrafish strain [Tg(ins:nfsb-mCherry)].