35 Koala sperm induces bovine oocyte activation after intracytoplasmic sperm injection

Assisted reproductive technologies have become vital in conserving endangered species and as tools for studying their fundamental reproductive biology. Intracytoplasmic sperm injection (ICSI) is a powerful technique for investigating sperm-related events, including DNA repair. Due to the difficulty in obtaining koala oocytes, this study examined whether oocytes from more accessible eutherian species might provide an alternative for the assessment of sperm fertilization ability and as a means of investigating their potential to induce early embryo events. This work aimed to assess oocyte activation rates through evaluating pronuclear formation and DNA fragmentation levels of bovine oocytes after injection of a koala sperm cell with or without assisted chemical activation. Semen collection and cryopreservation of koala sperm was performed as described by (Johnston et al. 2012 J. Androl. 33, 1007–1015). Cumulus–oocyte complexes were collected from slaughterhouse cattle ovaries and matured in vitro for 24 h as described by (Cabeza et al. 2022 Animals 3560, 1–12). For ICSI, cryopreserved sperm cells that were motile and had normal morphology after thawing were used. Half of the injected oocytes were incubated 4 min in 5 mM ionomycin (ICSI+IONO). As control group, noninjected matured oocytes were parthenogenetically activated (PA) by incubation for 4 min in 5 mM ionomycin + 4 h incubation in 1.9 mM 6-DMAP. Half of the PA-embryos were cultured for 7 days. Presumptive zygotes were fixed in 4% formaldehyde solution 16–18 h after injection or activation, permeabilized and incubated with γH2AX (a sensitive molecular marker of DNA damage and repair) Alexa 647 antibody (1:100). After confocal microscope analysis, zygotes were classified according to the presence of pronuclei (PN) into those with two PN (2-PN), one PN with the presence of a semi-condensed or condensed sperm (1-PN) and semi-condensed or condensed sperm with no evidence of PN (no activation). An area of interest was delineated around each PN based on 4′,6-diamidino-2-phenylindole staining in a single mid plane, and ImageJ image processing software was utilised to calculate the average pixel intensity of γH2AX staining. Data were analysed by Fisher’s exact and Kruskal–Wallis tests using GraphPad software. Differences were considered significant at P < 0.05. We found no significant differences in 2-PN (ICSI, 7/34, 20.58%; ICSI + IONO n = 6/30, 20.00%) or 1-PN (ICSI, 5/34, 14.70%; ICSI+IONO, 6/30, 20.00%) formation rates or γH2AX intensity levels between groups. Sham injection (oolema piercing and PVP injection without sperm) showed no activation (0/13, 0%). Pronuclear formation rate for the PA control group was 24/46 (52.2%) with cleaved 51/87 (58.6%) and blastocyst 35/87 (40.2%) rates. Our results demonstrate for the first time that koala sperm cells can induce bovine oocyte activation and pronuclear formation without the need of chemical activation assistance, allowing the potential use of this technology for assessing sperm fertilization ability and DNA quality.