Follicular fluid supports cumulus expansion in post-mortem recovered cumulus-oocyte complexes of Hippopotamus amphibius

Assisted reproduction is essential for the conservation of wildlife species. While obtaining oocytes from living individuals can be challenging, the retrieval of post-mortem ovaries provides a valuable alternative for developing reproductive protocols that could enhance preservation efforts. In this study, we aimed, to evaluate the impact of follicular fluid (FF) supplementation during in vitro maturation (IVM) on nuclear maturation and cumulus expansion of common hippo (Hippopotamus amphibius) oocytes. Ovaries were recovered during the post-mortem examination of a 33-year-old female hippo, and 44 oocytes were collected and randomly allocated into three groups for IVM. A control group matured in standard media, a Small-FF group with control standard supplemented with 10 % follicular fluid from small follicles, and a Large-FF group with standard media supplemented with 10 % follicular fluid from large follicles. Images were taken at 0, 16, and 24 h of IVM to evaluate cumulus expansion by measuring the diameter of each cumulus-oocyte complex (COC). Following 24 h of IVM, polar body (PB) extrusion was assessed. Results showed a significant increase in cumulus expansion in the FF-supplemented groups compared to the Control (16 h: Control: 13 ± 4 %, Small-FF: 43 ± 13 %, Large-FF: 44 ± 6 %; 24 h: Control: 27 ± 7 %, Small-FF: 71 ± 15 %, Large-FF: 66 ± 9 %). The Small-FF group (46.1 %) and Control group (40 %) exhibited similar percentages of PB extrusion, while PB could be observed in 6.3 % of the Large-FF group. These findings suggest that while FF supplementation promotes cumulus expansion, the size of the follicle from which the fluid is derived may significantly influence oocyte ability to achieve PB extrusion