The results from these experiments are presented in Table 4, where each

shade represents the appearance of the solution evidenced throughout the experiments. Crystallization of the solution (in light gray) was more frequently recorded when 0.25 ml plastic straw was used. Most of the solutions vitrified during cooling; however devitrification was frequently evidenced during warming (in dark gray). Among the 24 vitrification solutions, three Lumacaftor in vivo of them remained vitreous (Table 4, in black color) during both cooling and warming procedures. V2, V16 and V21 solutions were therefore selected for toxicity studies. The effect of toxicity of the vitrification solutions on membrane integrity of zebrafish ovarian follicles is shown in Fig. 1. When ovarian follicles were exposed to V21 solution the membrane integrity (77.9 ± 12.9%) did not differ (P > 0.05) from results obtained in the control group (91.0 ± 6.1%). Ovarian follicles exposed to V16 and V2 showed a decrease (P < 0.05) in membrane integrity compared to the control group. There was significant difference in membrane integrity of ovarian follicles between the room temperature control group and the vitrified groups (Fig. 2). Ovarian follicles showed membrane integrity of 59.9 ± 18.4% when fibreplug and V16 solution MAPK Inhibitor Library supplier were employed. When ovarian follicles were vitrified in V2 the membrane integrity decreased to 42.0 ± 21.0%,

using fibreplug as vitrification device (P < 0.05). After vitrification in V21 solution using plastic straw the largest decrease in membrane integrity was recorded, with a value of only 2.1 ± 3.6%.

Niclosamide Based on these results, V21 solution was not used for the subsequent experiments. The ATP concentration in the follicles declined significantly (P < 0.05) after vitrification. To make the comparisons clearer we normalised the data considering the ATP measured in the control group as 100% ( Fig. 3). Soon after warming, the ATP in the follicles vitrified in V2 declined to 22.0 ± 4.23%. Likewise, the ATP in ovarian follicles vitrified in V16 dropped to 6.9 ± 0.6% ( Fig. 3). Nevertheless, when measured 120 min post-warming the ATP in the ovarian follicles vitrified in V2 (15.1 ± 2.8%) did not differ (P > 0.05) to the ATP concentration recorded immediately after warming. In contrast, a decrease over time was observed in the follicles vitrified in V16 (3.5 ± 0.7%). The photomicrographs shown in Fig. 4 are representative examples of ovarian follicles obtained by confocal microscopy after exposure to JC-1 fluorescent probe. JC-1 was unable to penetrate deep inside the oocytes, therefore the fluorescence remained concentrated at the margins of the granulosa cells layer (Fig. 4AI and AII). Ovarian follicles from the control group displayed a contiguous peripheral aggregation of mitochondria in the granulosa cells that surround the oocytes, with a well-organized distributional arrangement and red fluorescence emission (Fig. 4AI and AII).