Figure 1

Figure 1

Optimization of the slow-rate freezing (A–D) and vitrification (F) protocols. (A) Viability of spermatogonia after freezing with 1.3 M dimethyl sulfoxide (Me₂SO), ethylene glycol (EG), propylene glycol (PG) and glycerol (Gly) (N = 3). (B) Viability of spermatogonia after slow-rate freezing with either 1.0, 1.3 or 1.6 M of Me₂SO (N = 3). The effects of sugar (C) and protein (D) supplementation of spermatogonia viability (N = 3). (E) Testes (arrows) pinned on an acupuncture needle for the needle-immersed vitrification (NIV) method. (F) The effects of different equilibration (ES) and vitrification (VS) solutions on spermatogonia viability after NIV (N = 3). Reproducibility of the developed freezing (G) and vitrification protocols (H) demonstrated on AB wild-type (AB), vasa (ddx4^{sa6158/sa6158}; VASA), Wilms tumor (wt1b; WT), leopard (gja5b^t1; LEO), casper (mitfa^w2/w2; mpv17^a9/a9; CASP) and β-actin (pku341Tg; ACTB) zebrafish lines. (I) Testicular cell suspensions prior to, and after cryopreservation. All values are presented as mean ± SD. Different letters above the SD bars indicate statistical significance (Tukey’s HSD, p < 0.05). Scale bars: (I) 20 µm.