Shilpa Dange ovariectomized them under anesthesia. Then after one to two week period of recovery ; subcutaneously inject estradiol benzoate (5μg/rat) and progesterone (500 μg/rat) 48 h and 4–7 h, respectively.

Is your research definitely affected by reproductive cycle? In many treatment efficacy or toxicity models, we do not attempt to alter the cycle but include reproductive tissue histopathology so we could determine cycle stage if needed (you can also use vaginal cytology for this staging purpose).