140 Assisted activation of intracytoplasmic sperm injection porcine zygotes using zinc chelation

Pigs are considered an important model for biomedicine research. The frequent polyspermy problems when using IVF procedures in this species can be avoided by performing intracytoplasmic sperm injection (ICSI). However, ICSI is not yet efficient in pigs, and assisted activation can be used to improve developmental competence. Immediately upon fertilisation, orchestrated rises of intracytoplasmic calcium are followed by the release of zinc (Zn) into the perivitelline space. Both elements play an essential role in triggering oocyte activation and embryo development. Our work aimed to determine whether assisted activation using the novel Zn chelator 1,10-phenanthroline monohydrate (PHEN) improves pronuclear (PN) formation rates (Experiment 1) and developmental competence of porcine ICSI embryos (Experiment 2). Cumulus-oocyte complexes were collected from slaughterhouse ovaries and matured in vitro for 44 h. Matured and denuded oocytes were subjected to ICSI (ICSI) or ICSI followed by incubation in 1 mM PHEN in TALP-H for 30 min (ICSIp). Additionally, some eggs were parthenogenetically activated following the same PHEN protocol as a control group (PART). For Experiment 1, presumptive zygotes were fixed for 20 min in 4% paraformaldehyde solution 18 h after injection/activation and then mounted in Vectashield^® (Vector Laboratories Inc.) containing 1.5 μg/mL DAPI. Zygotes were classified according to the presence of PN: 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 formation (no activation). Embryos that exhibited a different pattern were included in the “other” category. For Experiment 2, ICSI embryos of each experimental group were cultured in vitro in PZM medium for 7 days. Cleavage and blastocyst rates were recorded. Data were analysed by Fisher’s exact test. Differences were considered significant at P < 0.05. For experiment 1, no significant differences were found in 2-PN formation rates among groups (ICSI n = 8/34, 23.53%; ICSIp n = 12/40, 30.00%; PART n = 11/66, 16.67%). Moreover, the ICSI group showed higher rates of non-activated eggs (ICSI n = 15/34, 44.12%; ICSIp n = 7/40, 17.50%; PART n = 10/66, 15.15%). For Experiment 2, no significant differences were found in cleavage (ICSI n = 54/62, 87.09%; ICSIp n = 72/100, 72.00%; PART n = 32/39, 82.05%) or blastocyst rates (ICSI n = 11/62, 17.74%; ICSIp n = 18/100, 18.00%; PART n = 7/39, 17.95%). In conclusion, although Zn chelator PHEN shows high rates of oocyte activation, it does not improve 2-PN formation or blastocyst rates when assisting ICSI at the timing and concentration tested.