Evaluation of different factors affecting the efficiency of oocytes cryopreservation in the bovine model

Interest in oocyte cryopreservation has recently increased. Cattle oocytes are sensitive to low temperatures, and despite the efforts of numerous research groups cryopreservation of oocytes remains a difficult task. This problem may be in part due to the large size of bovine oocytes, which consequently have a low surface to volume ratio, making it more difficult for water and cryoprotectants (CP) to move across the cell plasma membranes. Several attempts to improve the survival rate of oocytes pioneered the establishment of different cryopreservation techniques. Recently, vitrification (based on direct plugging of samples into liquid nitrogen) has been used for the cryopreservation of oocytes as an alternative technique over traditional slow-rate freezing method. One of the most successful ultrarapid vitrification techniques is the Cryotop vitrification (CTV) that has resulted in excellent survival and developmental rates with human and bovine Metaphase II (MII) oocytes. However, despite the improvement in survival and cleavage rates, development into blastocysts is still poor, probably because ultrastructural and physiological changes that occur as a consequence of vitrification. For this reason, the aim of this study was the evaluation of different factors affecting the efficiency of oocytes cryopreservation in the bovine model. It is known that vitrification causes several ultrastructural and structural alterations, including damages of meiotic spindle apparatus in oocyte of several species. So, the starting point of this work was the evaluation of structural changes of in vitro matured bovine oocytes following cryopreservation and simple exposure to CPs (toxicity test). The first conclusion of this experiment was that the simple exposure of oocytes to CPs causes damages to the meiotic spindle and chromosome organization similar to those induced by the whole vitrification protocol. The second conclusion, according to our opinion, was even the most interesting: we observed the occurrence of spontaneous ...