128 Time of sperm pre-incubation affects fertilization rates after standard IVF with frozen-thawed semen in the horse

The development of a repeatable method for equine standard IVF (Felix et al. 2022 Biol. Reprod. 107, 1551–1564) holds promise for clinical use. The initial 2022 study, performed with fresh semen, found that sperm pre-incubated for 6 h before addition of cumulus–oocyte complexes (COCs) yielded 0% fertilization versus 47–100% fertilization for sperm pre-incubated 20–22 h. Subsequently (Felix and Hinrichs 2023 J. Equine Vet. Sci. 125, 104648) we reported successful IVF with frozen-thawed sperm selected via a commercial sperm separation device (Zymot, Cooper Surgical). In that study, sperm were pre-incubated for 10 h before co-culture with COCs, as frozen-thawed sperm are known to exhibit some capacitation-related changes. However, the time required for frozen-thawed equine sperm to gain fertilizing ability in the developed system is unknown. The objective of this study was to investigate the minimum pre-incubation time (capacitation time) required for frozen-thawed equine sperm to gain the ability to fertilize oocytes in vitro. Semen from one stallion was frozen in a commercial freezing extender (MFR5) in 0.5-mL straws. One straw was thawed for each replicate. The contents of the straw (~400 µL) were diluted with 500 µL of modified TALP (FT-PHE; composition from Felix et al., 2022), and 850 µL of this suspension was placed into the loading chamber of the device. The surface of the filter was covered with 700 µL of FT-PHE and the device was incubated in 5% CO₂ in air at 38°C for 30 min. Next, 500 µL of sperm suspension was collected from the outlet port, diluted with an additional 500 µL of FT-PHE, and centrifuged. The resulting pellet was resuspended with TALP-R (Felix et al. 2022), and the suspension was added to droplets of equilibrated FT-PHE to a final concentration of 1 × 10⁶ sperm mL⁻¹. After 15 min, 3 h, 6 h, or 9 h of sperm pre-incubation, IVM equine COCs were added to the sperm droplets and the dishes returned to incubation. After 3 h co-incubation, COCs were transferred to embryo culture medium for 32 h. At that time, uncleaved structures were denuded of cumulus and stained with DAPI to assess chromatin status. Three replicates were performed (total of 20–23 COCs co-incubated per sperm treatment). Data were analyzed by Fisher’s exact test. The fertilization rate (cleaved embryos per fertilizable [non-degenerated] oocyte), increased over time, from 7.1% for sperm pre-incubated for 15 min before COC addition to 22.2%, 38.5%, and 73.3% for sperm pre-incubated for 3, 6, or 9 h. The fertilization rate at 9 h was significantly higher than that for 15 min or 3 h (P < 0.01). These results show that frozen-thawed sperm are capable of fertilization in vitro after only 3 h pre-incubation, but the fertilization rate increases with a longer pre-incubation time. Further studies are needed with additional stallions. These findings start to define factors affecting capacitation of equine sperm and advance the possibility of using standard IVF as a clinical procedure in equine reproductive management.