Fig. 1
Optimization of cryopreservation conditions for Tokyo bitterling testes. A-C Haematoxylin‒eosin image of a fully immature (A) slightly mature (B) and mature testis (C) of Tokyo bitterling. A’-C’. The external morphology of Tokyo bitterling, which retains fully immature (A’), slightly mature (B’), and mature testes (C’). The black pattern on the outer edge of the anal fin and the white pattern on the outer tip of the dorsal fin (sign of nuptial colouration), which are indicators of donor fish selection, are shown in the insets. (D) Trypan blue-stained image of testicular cells showing live cells (black arrowhead) and dead cells (yellow arrowhead). (E) Recovery rates of testicular cells cryopreserved in a cryomedium containing 1.3 M DMSO, PG, GLYC, EG, and MeOH. (F) Recovery rates of testicular cells cryopreserved in cryomedium containing 1.0, 1.3, or 1.6 M DMSO. (G) Recovery rates of testicular cells cryopreserved in cryomedium containing 1.3 M DMSO and 0.1 or 0.2 M glucose or 0.05 or 0.1 M trehalose or sucrose. (H) Recovery rates of cryopreserved testicular cells after equilibration in a cryomedium containing 1.3 M DMSO and 0.1 M trehalose for 15, 30 and 45 min. (I) Fluorescence images of testicular cells after cryopreservation obtained by immunostaining with the Vasa antibody. DAPI staining was used to visualize the nucleus. Bars: 20 µm.
