This project aims to develop a protocol for oocyte freezing Prochilodus lineatus. To this solution will be evaluated three extensor (Ringer's solution, Hank's solution, and phosphate buffer) and three cryoprotectant substances (Dimethyl sulfoxide, propylene glycol and methanol) at a concentration of 0.2M to 4M linked glucose. The oocytes are packaged in straws of 0.5 ml and exposed to a cooling curve 2 ° C / min to -12.5 ° C (for 5 minutes), 0.3 ° C / min to -40 ° C, 10 ° C / min to -80 ° C and 50 ° C / min to -160 ° C, and then immersed in liquid nitrogen (LN2) and stored for 24 hours. The samples are defrosted in a "bath" to 27 ° C for 10s, and rehydrated by soaking in solutions of decreasing osmolarity of cryoprotectants (2M, 1M, 0.5M). The subsequent membrane integrity is assessed by using trypan blue dye, and the viability of the oocytes fertilization. The experiment will include samples consisting of 100 oocytes and 5 replicates in each treatment and their data were assessed for normality (Shapiro-Wilk) and homogeneity (Bartlett) and means were compared using an analysis of variance (ANOVA) and Tukey (± = 0 , 05). Morphologically, the embryos will undergo optical microscopy (stained with PAS, HE and toluidine blue), scanning electron microscopy and transmission electron microscopy.
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