28 Successful production of kangaroo ICSI embryos

Although Australia is home to the greatest diversity of marsupial fauna on the planet, it also has the distinction of possessing the highest mammal extinction rate. Assisted reproductive technologies have offered significant benefits for reproductive and genetic management in eutherian mammals. However, progress in marsupials has been limited. The overabundant Eastern-Grey Kangaroo (EGK) offers a great experimental model for later translation to other threatened marsupials. Thus, this work aimed to assess EGK oocyte recovery and morphometrics, IVM rates, and ovarian follicular growth in medium developed for bovine. In addition, we explored in vitro embryo development, cell number, and DNA fragmentation levels after intracytoplasmic sperm injection (ICSI). Ovaries were collected from euthanized EGK females, transported to the laboratory at room temperature, and punctured using 27 G needles to recover oocytes and follicles. Oocyte diameter and zona pellucida (ZP) thickness were assessed using the Research Instruments Pte Ltd., Viewer Imaging Software. IVM medium was prepared as reported for bovine (Ynsaurralde et al. 2020 Theriogenology 148, 140–148). Oocytes were matured at 35.5°C, with evaluation of the first polar body extrusion at 24 and 48 h of IVM. Primary and secondary follicles were cultured in 10-µL drops of IVM medium for 8 days and the diameter was measured daily. For ICSI, mature and immature oocytes were injected with a single EGK refrigerated epididymal spermatozoa from two bucks. Injected oocytes were cultured in vitro in a commercial bovine embryo medium (VitroCleave, ART Lab Solutions). Cleavage was assessed at 48 h after injection, and embryos were allowed to grow for up to 6 days. Then, embryos were fixed, permeabilized, and stained with yH2AX Alexa 647 and DAPI for nuclear staining. Cell number and the percentage of cells with DNA double-strand breaks (+yH2AX) were analyzed using confocal microscopy. A total of 68 oocytes were recovered from five females. Morphometrics (mean ± SEM) revealed an oocyte diameter of 181.8 ± 2.02 µm and a ZP thickness of 7.4 ± 0.17 µm. At 24 h, none of the oocytes exhibited the first polar body; however, after 48 h of IVM, 27% (10/37) showed the presence of a polar body. A total of 32 ovarian follicles were cultured from one female; 78% exhibited some degree of growth, and 12% of these doubled in size. ICSI resulted in 83% (5/6) of cleaved embryos. Out of the five cleaved embryos, 40% arrested at the 4- to 8-cell stage and 60% reached the 10- to 14-cell stage. DNA fragmentation levels varied from 0% to 38%. Immature injected oocytes (3/3) did not cleave. Our findings report for the first time oocyte collection, morphometrics, and IVM after 48 h of EGK oocytes together with in vitro follicular development. Additionally, our study showed that ICSI can successfully induce early embryonic development using medium designed for bovine, marking the first reported case of in vitro embryo production in this species. Although further investigation is required to optimize in vitro culture conditions, our results represent a significant advancement toward in vitro embryo production in marsupials.

This study was funded by Hidden Vale Research Support Grant 701460.