High-Yield Induced Ovulation in Adult Fat-Tailed Dunnarts by PMSG Treatment Combined with Estrus Synchronization

The fat-tailed dunnart, Sminthopsis crassicaudata , is an emerging laboratory-based marsupial model for research on comparative biology, reproduction and conservation. In females, the reproductive cycle lasts 31 days and approximately 10 oocytes are ovulated per cycle. Assisted reproductive technologies (ART) play a crucial role in marsupial conservation, but developing protocols to harvest large numbers of oocytes remains a key challenge. Specifically, producing sufficient mature metaphase II (MII) oocytes in dunnarts continues to be difficult. Ovarian follicle stimulation is common practice to achieve superovulation in many species and typically requires treatment of prepubertal female animals to avoid the impacts of endogenous hormone cycling. Alternatively, adult females can be stimulated during the intermediate or follicular phase. In this study, we aimed to develop a high-yield induced-ovulation protocol to collect higher number of MII oocytes from adult, cycling, female dunnarts. We first synchronized the female dunnart reproductive cycles using luteinizing hormone-releasing hormone (LHRH). The reproductive cycles were monitored by examining cytology of vaginal lavage samples. After administering four LHRH injections given at three-day intervals, 88.9% (n=36) of the adult female dunnarts responded to the treatment, with their estrous cycles synchronized at the diestrous stage. We then induced ovarian follicle development through two PMSG injections over 6 days, followed by hCG administration to trigger ovulation. By combining estrous cycle synchronization with PMSG stimulation, we achieved 77.8% (n=36) ovulation success and obtained an average of 20.1±9.1 (n=28) MII oocytes per adult dunnart. These data demonstrated that estrous cycle synchronization followed by the PMSG-hCG treatment yields consistent, highly efficient induced-ovulation in adult dunnarts. This approach of combining estrus synchronization and follicle stimulation to produce sufficient MII oocytes for ART purpose could be applied to other valuable marsupial species to support conservation efforts.