18 Hybrid lamb of domestic sheep and argali produced by somatic cell nuclear transfer

This study aimed to assess embryo developmental rates and viable offspring production of somatic cell nuclear transfer (SCNT) embryos using as donors of nuclei fetal fibroblast of a hybrid fetus produced by crossing domestic sheep (Ovis aries) × wild argali (Ovis ammon). Fibroblasts were isolated from a 37-day-old hybrid fetus cultured in DMEM supplemented with 15% FBS, cryopreserved using DMSO-containing medium, and stored in liquid nitrogen. For SCNT, cells were thawed and cultured until reaching 100% confluency and maintained for two days. Ovine cumulus-oocyte complexes (COCs) were obtained from slaughterhouse ovaries and were matured for 19–21 h in 500-µL drops of BO-IVM medium (IVF Bioscience). After maturation, COCs’ (n = 789) cumulus cells were removed by pipetting into a 0.1% hyaluronidase media. Only oocytes with an extruded polar body (PB) were used for SCNT (maturation rate was 79.8%, 630/789). Enucleation was done by aspiration of the first PB and nearby cytoplasm by micromanipulation in medium TC-199 containing 3 mg mL⁻¹ bovine serum albumin. Somatic cells with a diameter of ∼15 to 19 µm were transferred individually into the perivitelline space of the enucleated oocytes in the same medium and subsequently fused with the recipient oocytes by two pulses of 40 V for 20 µs. Fusion was carried out in 0.2-mm fusion chamber filled with 0.27 M mannitol fusion medium (0.1 mM calcium, 0.1 mM magnesium) using an Eppendorf Multiporator. Fused oocytes (n = 261/630, 41.4%) were activated 1–1.5 h after fusion by exposure to 5 mM ionomycin for 5 min, followed by incubation in 2 mM 6-(dimethylamino) purine and 10 mg mL⁻¹ cycloheximide for 4 h. Following activation, cloned embryos were cultured in CR1aa medium before transfer into the uterus horn synchronised recipients within 2 days after fusion. Peripubertal recipient gilts were synchronised by injections of prostaglandin F2α on the 15th and 4th day before the transfer of cleavage-stage 2-day embryos. In total, 153 cleaved embryos (58.6% of the number of fused oocytes) were transferred into 11 gilts (average 13.9 [range 8–20] embryos/recipient). Five gilts (45.5%) were pregnant by ultrasonography five weeks after transfer, and one (9.1%) delivered a single healthy cloned hybrid. Thus, to our knowledge, we demonstrated the successful production of the first cloned domestic sheep × argali hybrid lamb.

This research was supported by the Russian Science Foundation (project No 21-66-00007).