FIGURE SUMMARY
Title

Slow freezing versus vitrification for the cryopreservation of zebrafish (Danio rerio) ovarian tissue

Authors
Marques, L.S., Fossati, A.A.N., Rodrigues, R.B., Da Rosa, H.T., Izaguirry, A.P., Ramalho, J.B., Moreira, J.C.F., Santos, F.W., Zhang, T., Streit, D.P.
Source
Full text @ Sci. Rep.
Figure 1

Membrane integrity after vitrification. V1 (1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose); V2 (1.5 M methanol + 5.5 M Me2SO + 0.5 M sucrose + 10% egg yolk solution); V3 (1.5 M methanol + 5.5 M Me2SO + 0.5 M trehalose); SV4 (1.5 M methanol + 5.5 M Me2SO + 0.5 M trehalose + 10% egg yolk solution). Control = fresh ovarian tissue fragments. Mean ± SE followed by different letters differ by Tukey’s test (P = 0.0004).
Figure 2

Membrane integrity after slow freezing. SF1 (2 M methanol + 0.1 M sucrose); SF2 (2 M methanol + 0.1 M sucrose + 10% egg yolk solution); SF3 (2 M methanol + 0.1 M trehalose); SF4 (2 M methanol + 0.1 M trehalose + 10% egg yolk solution); SF5 (2 M Me2SO + 0.1 M sucrose); SF6 (2 M Me2SO + 0.1 M sucrose + 10% egg yolk solution); SF7 (2 M Me2SO + 0.1 M trehalose); SF8 (2 M Me2SO + 0.1 M trehalose + 10% egg yolk solution). Control = fresh ovarian tissue fragments. Means ± SE followed by different letters differ by Tukey’s test (P < 0.0001).
Figure 3

Histological analysis after slow freezing and vitrification. Not significant (P > 0.05). Cell integrity (%): P = 0.3452; Nuclear damage (%): P = 0.4627; Membrane damage (%): P = 0.9517.
Figure 4

Histology of fresh (A), vitrified (B) and frozen (C,D) primary growth (PG) oocytes of zebrafish (Danio rerio) after thawing.In A, fresh PG oocyte with intact nucleus and cell membrane.In B,vitrified PG oocytes with condensed chromatin (arrow) and oocytes with intact nucleus (asterisk).In C, frozen PG oocyte with follicular membrane rupture (arrow). In D, three frozen PG oocytes with nucleus damage.Light microscope 40x. Stain: HE. Bar = 10 µm.
Figure 5

Transmission electron microscopy (TEM) of fresh (control), vitrified and frozen primary growth (PG) oocytes of zebrafish (Danio rerio). (A–C) Fresh PG oocytes with normal cell organelles and intact plasma and nuclear membranes. (B,C) Presence of large number of mitochondria in the cytoplasm. (D–F) Vitrified PG oocyte showing an intact plasma membrane, chromatin condensation (arrow), signs of a ruptured nuclear membrane and a lower number of mitochondria. (G,H) Frozen PG oocyte showing a ruptured plasma membrane and a loss of intracellular contents (arrow). (I) Cytoplasm of a frozen PG oocyte with swollen mitochondria (arrow). PG = primary growth, Mt = mitochondria.
Figure 6

(A) Reactive oxygen species (ROS) by DCFH, and (B) total antioxidant capacity by FRAP (Ferric reducing/antioxidant power) after slow freezing and vitrification. Means ± SE followed by different letters differ by Student’s t test. ROS: P < 0.0001; FRAP: P < 0.0001.
Figure 7

Mitochondrial activity by thiazolyl blue tetrazolium bromide (MTT). Means ± SE followed by different letters differ by Student’s t test (P = 0.0081).
Figure 8

DNA damage (%) by comet assay of fresh (control), frozen and vitrified primary growth (PG) oocytes of zebrafish (Danio rerio). Means ± SE followed by different letters differ by the analysis of Kruskal-Wallis, followed by Dunn’s test (P = 0.0013).
Acknowledgments
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