FIGURE 4.

Glycine or glycine plus melatonin supplementation during vitrification, thawing and in vitro maturation protected against apoptosis in the subsequent oocytes. (A): Representative images of early apoptosis labeled with Annexin-V in each examined group. Green, Annexin-V staining; PI (propidium iodide), DNA; DIC, differential interference contrast image; Merge, overlapping of green, DNA and DIC. Scale bar: 40 μm. (B): The relative fluorescent intensity of Annexin-V staining in oocytes of each experimental group. In each set of experiments, the fluorescence intensity of Annexin-V staining was normalized to the value of oocytes in the control group. All experiments were performed in triplicate. Data are shown as mean ± SEM. Different lowercase letters above columns indicate statistical difference at p < 0.05. The same lowercase letters above columns denote that the data are not significantly different at p > 0.05. Control: oocytes were neither vitrified/thawed nor supplemented with glycine or melatonin to assist their maturation; Vitrified: oocytes were sequentially vitrified, thawed and cultured for maturation without glycine or melatonin supplementation; Vitrified + Gly: oocytes were sequentially vitrified, thawed and cultured for maturation with glycine supplementation; Vitrified + Gly + MT: oocytes were sequentially vitrified, thawed and cultured for maturation with glycine plus melatonin supplementation.