46 SPINDLE CONFIGURATION OF IN VITRO-MATURED BOVINE OOCYTES EXPOSED TO SODIUM CHLORIDE OR SUCROSE PRIOR TO CRYOTOP VITRIFICATION

Abstract

It has been previously described that a simple treatment with medium containing elevated NaCl or sucrose concentrations increases the cryotolerance and developmental competence of in vitro-matured porcine oocytes after vitrification and parthenogenetic activation (Lin et al. 2009 Reprod. Fertil. Dev. 21, 338–344). This work was designed to study whether the exposure to increased concentrations of NaCl or sucrose before vitrification improves cryotolerance of in vitro-matured bovine oocytes. In Experiment 1, in vitro-matured oocytes were exposed to different NaCl and sucrose concentrations (from 375 to 808 mOsm) for 1 h. In Experiment 2, and according to the results obtained in the first experiment, oocytes were exposed to 375 mOsm NaCl or sucrose solution, vitrified, and warmed. Nontreated oocytes were used as controls. In both experiments, oocytes were fixed after treatment and microtubule, and chromosome distribution was analysed by immunocitochemistry. All statistical analyses were conducted with the IBM SPSS 19 for Windows (IBM corp., Chicago, IL). ANOVA was performed to analyse differences in meiotic spindle. Statistical significance was set at P < 0.05. After exposure to 375 mOsm of NaCl or sucrose, similar percentages of oocytes showing normal chromosome distribution were obtained compared to the control group (83.4, 71.8, and 85.0%, respectively). Groups treated with higher concentrations (443 to 808 mOsm) triggered significantly lower proportions of normal spindles. After vitrification/warming, no significant differences were observed between nonvitrified oocytes (71.3%) and those treated with NaCl before vitrification/warming procedure (41.9%) when normal chromosome organisation was analysed. Significantly higher percentages of normal chromosome configuration were observed when oocytes were exposed to sucrose before vitrification (34.2%) compared with control-vitrified oocytes (23.3%). However, pretreatment with NaCl or sucrose before vitrification did not trigger significant differences in terms of percentages of normal microtubule configuration (41.9 and 32.9%, respectively) compared with control-vitrified oocytes (40.2 and 24.4%, respectively), although both treatments differed significantly from control (79.1 and 81.7%, respectively). In conclusion, this study showed that a 375-mOsm NaCl or sucrose pretreatment of bovine oocytes before vitrification did not have a deleterious effect on the organisation of the meiotic spindle of vitrified/warmed bovine oocytes. Further experiments are required to investigate whether in vitro-matured oocytes subjected to this osmotic treatment could improve their development competence after being vitrified/warmed.