In the present study we aimed to analyse structural changes during in vitro maturation of the bovine zona pellucida (ZP) by scanning electron microscopy (SEM) ands zona pellucida birefringence (ZPB). Here we show that alterations during in vitro maturation invasively analysed by SEM are reflected in ZPB. In vivo-matured oocytes displayed significantly lower birefringence parameters and significantly higher blastocyst rates compared with in vitro-derived oocytes (39.1% vs 21.6%). The same was observed for in vitro-matured oocytes with cumulus-oocyte complex (COC) Quality 1 (Q1) compared with Q3-COCs with respect to zona birefringence and developmental capacity. Immature oocytes with Q1-COCs displayed higher ZPB values and a higher developmental capacity to the blastocyst stage (27.7% vs 16.9%) compared with immature Q3-COCs. Considering in vitro-matured oocytes, only those with Q1-COC showed a trend for ZPB similar to in vivo-matured oocytes. Therefore, a decreasing trend for ZPB during in vitro maturation seems to be typical for high-quality oocytes and successful cytoplasmic maturation. In accordance, fully-grown immature oocytes reached significantly higher blastocyst rates (32.0% vs 11.5%) and lower ZPB values compared with still-growing ones. In conclusion, we successfully evaluated the applicability of zona imaging to bovine oocytes: alterations during in vitro maturation invasively analysed by scanning electron microscopy were reflected in the birefringence of the zona pellucida of bovine oocytes affecting developmental capacity at the same value. Therefore ZPB measurement by live zona imaging has potential to become a new tool to assess correctness of in vitro maturation and to predict developmental competence.