Intravaginal Progesterone Application as an Efficient and Reproducible Tool for Synchronizing the Mouse Estrous Cycle

ABSTRACT Purpose The synchronization of the estrous cycle is crucial for optimizing reproductive procedures and improving experimental consistency in female mice. Traditional progesterone (P4) administration methods, such as oral or subcutaneous routes, have limitations including invasiveness and variable efficacy. We aimed to develop intravaginal P4 application as a new method for synchronizing the mouse estrous cycle. Methods A c ustom‐prepared P4 cream was applied to the vagina and washed out the following day. A vaginal smear test was conducted for 4–5 consecutive days. A combination of an anti‐inhibin antibody (AIMA) and P4 was used to improve reproductive performance. The P4‐treated, synchronized female mice were tested to determine whether they could serve as pseudopregnant recipients for embryo transfer (ET). Results Intravaginal P4 application effectively synchronized the estrous cycle and did not adversely affect pregnancy rate or litter size. The combined use of AIMA and P4 significantly increased the number of offspring compared to natural mating ( p = 0.006). Successful offspring production was achieved with improved procedural consistency when this protocol was used to produce pseudopregnant females for ET. Conclusions Our method reduces animal discomfort and the need for vasectomized males, offering a practical approach for enhancing reproductive efficiency and animal welfare in research settings.