90 Effect of sediment from spontaneously immortalized oviduct epithelial cells on the development and fertility of bovine OPU-IVF embryos

The oviduct is the site of fertilization and early embryonic development. Recently, conditioned medium derived from spontaneously immortalized bovine oviduct epithelial cells (SI-BOECs) and sediments obtained by ultracentrifuging this medium were shown to be effective in improving embryonic development (Miyashita et al. 2024 J. Reprod. Dev. 70, 42–48). In this study, we investigated whether the addition of sediment to a commercial medium different from that used previously improves the developmental rate and the conception rate of ovum pickup (OPU)-derived bovine embryos. Additionally, we also evaluated the cell count of embryos produced with or without the sediment. SI-BOECs that had reached ~80% confluence were cultured in KSOM for 48 h, and the resulting medium was centrifuged at 2000g for 10 min to remove cells. The conditioned medium (30 mL) was ultracentrifuged at 100 000g for 2 h at 4°C twice. The sediment was resuspended in 100 µL of PBS, which was added to in vitro culture medium (BO-IVC [IVF-Bioscience^TM]) to achieve 0.10 ng μL⁻¹ as a final concentration of protein. OPU-derived oocytes were collected from 104 Japanese Black cows, and IVF was performed using Japanese Black semen. At 6 h post-insemination (hpi), presumed zygotes were transferred to BO-IVC with or without sediment. The number of embryos that cleaved two or more (2-cell rate) and eight or more (8-cell rate) cell stages were measured at 48 hpi. The numbers of all blastocysts and blastocysts that were excellent by IETS standards were measured at 168 hpi. Only excellent grade embryos were transferred as fresh or frozen embryos to Holstein, Japanese Black, or crossbred heifers. Conception was evaluated using ultrasonography on Day 23 after embryo transfer. To determine the effect of sediment on the cell number of embryos, IVF and in vitro culture was performed in the same manner described above, using oocytes derived from slaughterhouses. Blastocysts obtained at 168 hpi were fixed and Hoechst stained, and the cell number was counted. In the development of OPU-derived embryos (sediment group, n = 1587; non-sediment group, n = 752), the 2-cell (57.9% vs. 53.2%) and 8-cell rates (50.2% vs. 39.4%), blastocyst rate (34.0% vs. 27.7%), and excellent embryo rate (18.7% vs. 14.0%) were significantly higher in the sediment group than in the non-sediment group (P < 0.05, chi-squared test). In contrast, there was no significant difference in the conception rate between the two groups (64.8%, n = 54 vs. 67.6%, respectively, n = 139; P > 0.05, chi-squared test). There was no difference in the number of cells in blastocyst stage embryos between the sediment and non-sediment groups (331.7 ± 117.7 vs. 258.0 ± 67.0 [t-test]). These results indicate that the addition of SI-BOEC-derived sediment to commercial culture medium improves the developmental rate of IVF embryos. Furthermore, the conception rate and cell number of embryos cultured with this sediment are not affected. Therefore, SI-BOEC-derived sediment is expected to be a beneficial tool in bovine in vitro embryo production.