Animal ethics and experimentation design

The experimental animal research was approved by the ethics committee of Nanjing Medical University. Unless otherwise stated, all chemicals used in this study were purchased from Sigma Aldrich Chemical Company (St. Louis, Missouri, USA).

Female ICR mice aged 3–4 weeks and males aged 9–10 weeks were separately placed in indoor ventilated cages with a 12 h light/12 h dark cycle at a constant temperature of 21–22 °C. The animals were provided sufficient drinking water and feed. There were three main experimental schemes: Experiment 1: To study the effective concentration of NAM in the oocytes during in vitro maturation (IVM). Cumulus expansion, mother cell maturation rate, and fertilization rate were evaluated after co-culture with immature oocytes in IVM medium with the addition of 0.01, 0.1, 1, 5 or 10 mM NAM for 16–18 h [26–29], in com-parison to a control. Experiment 2: To establish a murine oxidative stress model. Oocytes were pretreated with different concentrations of hydrogen peroxide (H₂O₂; 0, 20, 50, or 100 μM) for 1 h to evaluate the maturation rate and fertilization rate of the oocytes. Experiment 3: To study whether NAM was able to enhance the quality of oocytes when subjected to oxidative stress. Based on the results of the previous two experiments, IVM was conducted in culture medium supplemented with 0 mM or 5 mM NAM after pretreatment of the oocytes for 1 h with 100 μM H₂O₂. The same experimental conditions described above were studied, with the levels of ROS in the oocytes, and the ratio of abnormal spindle analyzed.