Their results showed that the Tg of the solutions rose as the proportions of these sugars in the vitrification solution increased. The results from

the present study showed that solutions were better vitrified using fibreplug when compared to 0.25 ml plastic straws. It has been shown in the literature that the most effective way for increasing cooling rates is to use the smallest possible volume of cryoprotectant solution in order to establish a direct contact (without any thermal insulating layer) between the solution and the liquid nitrogen [42]. A smaller volume may also offer a special advantage: it prevents heterogeneous ice formation. In zebrafish, it has been shown that methanol and propylene glycol are less toxic to stage III oocytes than other cryoprotectants, such as ethylene glycol and Me2SO [24] and [31]. This explains the higher membrane integrity of ovarian follicles after exposure to V16 solution (1.5 M methanol + 4.5 M propylene Z-VAD-FMK clinical trial glycol) when

compared to the results recorded for the follicles exposed to V2 (1.5 M methanol + 5.5 M Me2SO). Me2SO at 5.5 M became toxic to stage III zebrafish Epacadostat solubility dmso ovarian follicles. Although ethylene glycol is considered to be the most toxic among the CPAs used in this experiment [43], ovarian follicles exposed to V21 (1.5 M methanol + 6.0 M ethylene glycol + 0.5 M sucrose) displayed the highest membrane integrity of all treated groups. The presence of sucrose may have lowered the toxicity of ethylene glycol and worked as an osmotic buffer

stabilizing the follicles membrane and consequently preserved its integrity. Studies have shown that the use of sucrose as non-permeating CPA provides additional protection to membranes from the consequences of dehydration in fish embryos and optimizes the performance of permeable CPAs when used Tolmetin in combination [1], [11], [15], [23] and [36]. The present study showed that the membrane integrity of ovarian follicles after vitrification, assessed by TB staining, was not preserved when using plastic straws. This result suggests that intracellular ice crystal formation may have taken place during vitrification process. No changes were observed in solution appearance in the straws during both cooling and warming procedures; however, even transparent solutions may contain countless ice nuclei and ice crystals, because the ice crystals only are detectable optically once they become larger than the wavelength of light [33]. The volume of the vitrification solution was minimized when fibreplug was used, increasing the probability of vitrification, which may have contributed to the higher membrane integrity of the ovarian follicles vitrified in V16 and V2. Guan et al. [12] reported a slightly higher membrane integrity after vitrification of isolated stage III zebrafish ovarian follicles than the results obtained here using ovarian fragments, when assessed by TB staining.