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.

Incorporation and proliferation of fresh and cryopreserved spermatogonia. The incorporation and proliferation of fresh (A) and cryopreserved (B,C) vasa::egfp spermatogonia within the testes (A,B) and ovaries (C) of non-sterilized recipients. Testes (arrows) of the control MO-sterilized recipients (D) appear undeveloped under the stereomicroscope (D1), while the histological analysis (D2) displayed a lack of developing germ cells. Recipients of actb:yfp spermatogonia displayed either one (E) or both (F) developed testes (arrows). Developed testes displayed strong green fluorescence originating from donor actb:yfp spermatogonia (F1), while histological analyses displayed clear differentiation of germ cells into spermatozoa (F2). (G) Incorporation of fresh and cryopreserved spermatogonia into non-sterilized (NST) or MO-sterilized (ST) recipients. Values are presented as mean ± SD. Scale bars: (A, B, C) 500 µm; (top panels) 100 µm; (D, E, F) 1 mm; (D1, F1) 200 µm; (D2, F2) 20 µm.

Production of donor-derived spermatozoa and progeny from MO-sterilized recipients. Milt (A) and individual spermatozoa (A’) stripped from recipients demonstrating yfp fluorescence. (B) RT-PCR amplification of yfp in milt obtained from wild-type AB (AB), β-actin transgenic (pku341Tg; Actb) and recipient (REC) zebrafish. Milt volume (C) and sperm count (D) of milt obtained from wild-type AB (AB), β-actin transgenic fish (pku341Tg; Actb) and recipients of fresh (REC_F), frozen (REC_SR) and vitrified (REC_V) spermatogonia. (E) Fertilization rates after spawning control fish as well as recipient males and wild-type AB females. (F) Three dpf larvae obtained from crossing control fish and recipient males with wild-type AB females under fluorescent stereomicroscope. (G) PCR amplification of yfp in offspring obtained from control crossings, as well as from crossing recipient males with wild-type AB females. (H) F1 individuals developed normally and donor-derived individuals displayed fluorescent signal compared to the control crossing six months after fertilization. Values in C–E are presented as mean ± SD. Lack of different letters above SD lines indicate the lack of statistical significance (Tukey’s HSD, p > 0.05). Scale bars: (A) 100 µm; (A’) 20 µm; (F) 1 mm; (H) 2.5 mm.