Generation and assessment of high-quality fat-tailed dunnart oocytes following superovulation in prepubertal animals†.

The fat-tailed dunnart, Sminthopsis crassicaudata, is a mouse-sized, polyovular, solitary dasyurid marsupial found in central and southern Australia. With the establishment of a chromosome-scale genome assembly, induced pluripotent stem cells, and targeted genetic editing, the dunnart is emerging as the laboratory marsupial model for comparative developmental, reproductive and conservation biology. The development of assisted reproductive technologies (ART) are critical to achieving these goals in this species. ART requires a large number of mature oocytes which are typically collected through stimulated and synchronised female reproductive cycles. While protocols for induced-ovulation or superovulation are standard in many placental mammals, there are no methods to date designed for marsupials. In the present study, prepubertal dunnarts were stimulated with pregnant mare serum gonadotrophin and human chronic gonadotrophin across 6 different treatment regimens. Our best regimen resulted in over 70% of prepubertal dunnarts ovulating with 82% normal oocytes. When the primed females were mated with stud males, 4-cell stage embryos were collected 48 h post-hCG administration. At around 96 h post-hCG, 50% (n = 8) and 78% (n = 9) of the embryos developed to blastocysts. Our results demonstrated successful stimulation of ovulation and mature oocyte collection in prepubertal dunnarts. Furthermore, we confirmed developmental competence of the induced ovulated oocytes through to at least the blastocyst stage. These findings represent the first robust hormonal regimen for predictable oocyte generation in any marsupial and will significantly contribute to the use of the dunnart in developmental and conservation biology.