137 Incubation conditions to improve equine sperm in vitro capacitation and fertilising ability after intracytoplasmic sperm injection

Due to the failure of stallion spermatozoa to penetrate the zona pellucida during conventional IVF, intracytoplasmic sperm injection (ICSI) is currently the only repeatable technique to produce in vitro equine embryos. Specific sperm culture conditions could improve the understanding of capacitation-associated events in this species and potentially increase the success of these technologies. The aim of this study was to evaluate the effect of a defined medium for in vitro sperm incubation on sperm kinematic parameters, capacitation-associated events, and the ability to induce oocyte activation after heterologous ICSI using pig eggs. Frozen-thawed sperm (n = 4) were incubated in non-capacitating (modified TALP medium, pH: 7.5, NC) and capacitating (modified TALP supplemented with 25 mM sodium bicarbonate and 1 mg/mL PVA, pH: 7.5, CAP), for 45 min at 38.5°C in air. Aliquots of sperm (17 × 10⁶/mL) were used to evaluate sperm kinematic parameters by computer-assisted sperm analysis (CASA). Capacitation associated events, residues phosphorylated by PKA (pPKA) and tyrosine phosphorylation (pTyr), were evaluated by Western blot. For heterologous ICSI, motile equine sperm cells incubated for 45 min in NC or CAP medium were injected into matured pig oocytes. Presumptive zygotes were fixed for 20 min in 4% paraformaldehyde solution and stained with DAPI 18 h after injection/activation. Zygotes were classified according to the presence of PN: two PN (2PN), one PN with the presence of a semi-condensed or condensed sperm (1PN), and semi-condensed or condensed sperm with no evidence of PN formation (no activation). A t-test for the difference between treatments or Fisher’s exact test for egg activation analyses was performed; a P-value < 0.05 was considered significant. Sperm incubation in CAP conditions showed an increase in total (25.6% ± 5.5 vs 12.1% ± 2.3) and progressive (23% ± 5.2 vs 9.2% ± 1.8) motility (P < 0.05). Moreover, incubation in CAP conditions induced an increase in pPKA and pTyr levels (P < 0.05). Interestingly, two parameters associated with hyperactivation, VCL (115.74 ± 11.4 vs 71.85 ± 9.1 µm/s) and ALH (1.16 ± 0.1 vs 0.75 ± 0.1 µm) were improved under CAP condition (P < 0.05). Finally, sperm incubated under CAP conditions before ICSI induced a higher proportion of 2PN than those incubated in NC conditions (76.4% ± 7.28; 47.6% ± 6.98, respectively; P < 0.05). These results confirm that incubation of sperm cells in our capacitation medium supports crucial events associated with sperm capacitation and improves fertilising ability after ICSI. Future studies are needed to confirm these results in homologous ICSI and to elucidate the associated-signalling pathways involved.