122 Prevention of polyspermy by sperm selection assay in pig IVF

The high proportion of embryo loss due to polyspermy is a problem not yet resolved in the protocols for in vitro production of porcine embryos. Polyspermic embryos are aneuploid and cannot develop to term. The sperm selection assay (SSA) method selects and concentrates capacitated spermatozoa (sp) based on the movement orientation toward a chemoattractant. This method has improved IVF outcome in other species but has not been tested in pigs. This study aimed to reduce the percentage of porcine polyspermic oocytes after IVF by incorporating the SSA procedure. Before IVF, the sp were subjected to SSA. This method consists of the introduction of sp in an SSA device at an initial concentration of 3 × 10⁶ sp/mL. It was implemented using porcine follicular fluid (PFF) as a chemoattractant, diluted at 1:10^{4 an} in an IVF medium. After a 30 min incubation, the sp are recovered at 1 × 10⁵ sp/mL, and this concentration was used for IVF. On the other hand, traditional IVF uses 1 × 10⁵ sp/mL. Cumulus-oocyte complexes (COC) were obtained from the ovaries of slaughterhouse sows and selected under magnification. Subsequently, they were subjected to IVM at 39°C, 5% CO₂, and a saturated humidity environment for 44 h, distributed randomly in groups of a maximum of 50 in four-well plates with 500 µL of M199 + hMG and cAMP during the first 22 h. The COC were then mechanically denuded and co-incubated with the sp. The sp were obtained from refrigerated semen in a long-term extender for artificial insemination. The semen was centrifuged (490 G, 5 min) in IVF medium (M199 + BSA, sodium pyruvate, sodium lactate, and caffeine) and adjusted to 3 × 10⁶ sp/mL. In total, four experimental groups were used: SSA + PFF (SSA with PFF 1:10⁴, n = 120), SSA control (SSA without PFF, n = 147), IVF minimum concentration (without SSA, sp concentration 1 × 10⁵ sp/mL, n = 117) and IVF control (without SSA, sp concentration 1 × 10⁶ sp/mL, n = 130). Five replicates were performed. 20 hours after starting IVF, presumptive zygotes were fixed in 4% paraformaldehyde and stained with Hoechst 33342 for DNA visualisation under UV light. The following parameters were determined: penetration (penetrated/mature oocytes), male pronuclear formation (MPN = oocytes containing MPN [male pronucleus]/penetrated) and monospermy (oocytes containing only one MPN or one decondensed sp head/penetrated). The data were analysed using chi-squared and Fisher’s exact test, considering significant P < 0.05. The percentage of monospermic oocytes increased in the SSA + PFF group compared with the IVF control (4 ± 2 vs 73 ± 6; P < 0.0005) due to a decrease in the polyspermy incidence. These results show that the incorporation of SSA into porcine IVF improves its performance, since polyspermy decreases. This tool is an easy-to-use method. We recommend its incorporation into routine porcine IVF protocols.