122 A New Maturation Medium Improves Porcine Embryo Production In Vitro

Recently (Spate et al. 2017 Reprod. Fertil. Dev. 29, 150), a new medium [TCM-199 supplemented with hCG 10IU, pregnant mare serum gonadotropin (PMSG) 10 IUmL-1, fibroblast growth factor (FGF) 40ngmL-1, leukemia inhibitory factor (LIF) 2000UmL-1, IGF-1 20ngmL-1, epidermal growth factor (EGF) 10ngmL-1], termed FLI medium, was demonstrated to improve porcine oocyte maturation in vitro. The effects on embryo development and quality have not yet been investigated. The purpose of the present study was to compare the FLI medium in porcine in vitro embryo production (IVP) with our standard maturation medium (DMEM supplemented with 10IUmL-1 PMSG and hCG, 50ngmL-1 EGF, 100ngmL-1 IGF1, and 5ngmL-1 FGF). Briefly, gilt oocytes were collected via aspiration of follicles from abattoir ovaries and matured for 44h in either FLI or standard DMEM medium at 39°C, 5% CO2 in humidified air. In vitro fertilization was performed with freshly ejaculated sperm (250,000mL-1) of a multi-transgenic boar (GGTA1-KO/hCD46/hCD55/hCD59/hHO-1/hA20) by co-incubation with the matured oocytes in PGMTac4 medium for 4h. Zygotes were washed twice and then cultured for 6 days in PZM3 medium. Development to the blastocyst stage was recorded at Day 6 of culture. Blastocysts were fixed and Hoechst33342 stained for counting the nuclei. Each of the experiments was repeated 3 times. In a second step, Day 5 blastocysts derived from the FLI medium were transferred to synchronized pubertal gilts to test the in vivo developmental competence of the IVF embryos. Maturation of oocytes in FLI medium resulted in a significantly higher blastocyst rate (49.3 vs. 13.5; P≤0.001, Chi-squared test) and nuclei number (41.3±12.2 vs. 35.3±10.8; P ≤ 0.001, one-way ANOVA) compared with the standard medium, whereas the cleavage rate was not affected. Transfer of Day 5 blastocysts (average 35 embryos/recipient) derived from the FLI system using 8 recipients resulted in 7 pregnancies (87.5%) as determined by ultrasound scanning on Day 25 of gestation. At the time of writing, one recipient had delivered 5 healthy piglets after a gestation length of 114 days. Results indicate that the FLI medium significantly improves blastocyst rates and the cell number of the resulting blastocysts (Table 1) and yields pig IVF embryos with a high developmental capacity in vivo. By producing high-quality porcine embryos, this FLI-based IVF system provides an efficient method to modify the porcine genome by cytoplasmic microinjection of CRISPR/Cas molecules into IVF-derived zygotes. Table 1.Results of maturation of oocytes in FLI medium compared with DMEM