Vitrification of bovine oocytes impairs their reproductive capacity independently of maturation stage

Until the present literature has not achieved a consensus regarding the best maturation stage for oocyte to maintain their reproductive capacity after cryopreservation. The aim of this study was to determine, using an experimental bovine model, in which stage of development (VG stage, immature, or MII stage, post-maturation in vitro) the oocyte is less susceptible to damage during cryopreservation. Immature oocytes (VG) from the ovaries of slaughtered cows were selected for in vitro maturation or vitrification and divided into three groups. The first group (CONTROL) consisted of immature oocytes, matured in vitro without vitrification; the second group (CRYO-IVM) consisted of vitrified immature oocytes thawed and submitted to in vitro maturation; and the third group (IVM-CRYO) consisted of matured in vitro oocytes submitted to vitrification and thawing. The oocytes were evaluated for: nuclear maturation by acetic orcein staining; integrity of the zona pellucida using a polarized microscope; cell viability by the Dead-Live technique; and embryo development (cleavage, production and hatching rate) by in vitro fertilization and parthenogenetic activation. There was no difference in capacity of nuclear maturation between fresh and thawed oocytes (p=0.23). Regarding the zona pellucida (ZP), all oocytes (100%) of all three groups (control, CRYO-IVM and IVMCRYO) presented a positive ZP reading, with no correlation with later embryo evolution. DEAD-LIVE analysis of cell viability revealed reduction of viability in the IVM-CRYO group (27%) compared to control (84%) (p<0.0001) and to the CRYO-IVM group (56%) (p=0.017), with no difference between the last two groups (p=0.055). Analysis of the potential for embryo development by means of in vitro fertilization showed that the control group presented better cleavage and blastocyst formation rates than the CRYO-IVM (p<0.0001 and p<0.0001, respectively) and the IVM-CRYO (p<0.0001 and p=0.0004, respectively) groups. Analyzing the potential for embryo development the control group presented better cleavage by means of in vitro fertilization (80%) and parthenogenetic activation (58%) than the CRYOIVM (28%; p<0,0001; 28%; p=0,0002, respectively) and the IVM-CRYO groups (26%; p<0,0001; 22%, p<0,0001,respectively) Analysis of blastocyst formation rates and hatching after FIV and AT in CRYO-IVM and IVM-CRYO groups were null. Vitrification of bovine oocytes causes great impairment of their reproductive capacity regardless of the stage of maturation at the time of freezing. However the vitrified immature oocytes submitted to IVM maintained their capacity of nuclear maturation, as they achieved MII stage. This study was not able to determine which stage was better in reducing crio damage, as both stages (VG and MII) presented equally impaired by the process. (AU)