5A). These vesicles were not seen in fresh or frozen morulaes of same quality. Cytoplasm discontinuities, as well as organelle-free

areas were common after vitrification (Fig. 5B), as in frozen embryos. Vitrified grade III and also frozen embryos had heterogeneous cytoplasm in addition to mitochondrial LGK-974 cell line and SER swelling (Fig. 5C). Large vesicles occupying great areas of the cytoplasm (Fig. 5D) and degenerated cells among viable embryonic cells (not shown) were characteristics found only in the vitrified group. In this study, fresh embryos revealed intense mitochondrial activity. Active mitochondria were distributed throughout the cytoplasm, regardless of the embryonic developmental stage. However, no mitochondrial activity could be observed in cryopreserved embryos, either frozen or vitrified. The evaluation of mitochondrial activity after cryopreservation are unpublished in this species and it is known that mitochondrial dysfunctions check details or abnormalities may compromise developmental processes by inducing chromosomal segregation disorders, maturation and fertilization failures or even embryo fragmentation [4]. Sohn et al. [35] studied the effect of two frequencies of liquid N2 infusion on the cryopreservation of mice two-cell embryos on the mitochondrial activity and actin filaments distribution using

fluorescent markers similar to those used on the present work. Very similar to what this study revealed, those authors [35] showed that the number of mitochondria with high membrane potential decreased on cryopreserved embryos, and described gaps or discontinuities in the peripheral actin fibers (those in close association with the cell membrane), especially on the low frequency N2 infusion treatment. Disturbances in function and distribution of mitochondria, as well as changes in the organization of cytoskeleton related to insufficient culture conditions or cryopreservation are expected to occur and may reduce developmental capacity [12] and [15]. Previous studies have demonstrated succesfull cryopreservation

of mitochondria isolated from rat liver [17], muscles [25] and brain [29]. In brain tissue, mitochondria showed a reduction in respiratory activity after cryopreservation. However, this effect was not due to mitochondrial membranes rupture [29]. Penetration of the fluorochrome used in this experiment is proportional to the inner mitochondrial Thymidine kinase membrane activity and equilibrium [28], which was surely altered. However, in the present work no rupture of mitochondrial membranes was seen on the ultrastructural analysis. Nukala et al. [29] also found that freezing mitochondria without any cryoprotective agent destroyed their structural integrity and functional viability, and that the use of a cryopretective agent prevents most but not all damages. Moreover, the ability to restore a satisfactory metabolic activity or regenerate damaged structures after exposure to low temperatures requires time. For example, Leoni et al.