10 Single births after embryo transfer of two IVP ovine embryos (fresh and frozen) diagnosed as twin pregnancies

In vitro embryo production (IVP) allows the production of progeny from nonfertile females, pregnant, lactating, and even dead or slaughtered females from high genetic donors. The IVP includes (1) IVM of oocytes, (2) IVF, co-culture of capacitated spermatozoa with in vitro matured oocytes, and (3) in vitro culture (IVC) of presumptive zygotes up to the blastocyst stage. At this stage, the blastocysts are directly transferred to a recipient or could be cryopreserved to be used in the future. In small ruminants, it is common to transfer two embryos to the same recipient. In this case report, we present the results of embryo transfers of ovine embryos produced in vitro, with the respective results in an embryo transfer program after the transfer of two embryos either fresh or frozen. The IVP was performed with a continuous in vitro culture system (IVF-Bioscience™) using ovaries (n = 179) collected from a slaughterhouse. For the IVM, the cumulus–oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24 h at 38.5°C in 5% CO₂ in air and 100% humidity. The IVF process was conducted with semen from the same ram at a concentration of 2 × 10⁶ sperm/mL, for 18 h in 38.5°C, 5% CO₂ in air and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO₂, 5% O₂, and 90% N₂ at 100% humidity. The expanded blastocysts (BX) on Day 7, were frozen in ethylene glycol (EG). Only BX quality 1 and 2 (IETS Manual 4th Ed) were subjected to a controlled-rate freezing curve after equilibration for 8 to 10 min in freezing medium (Ethylene Glycol Freeze Plus Vigro™) starting at −6°C (seeding), decreasing 0.5°C/min, ending at −32°C and then plunging into liquid nitrogen. The remaining blastocysts (grade 1 and 2) were transferred fresh to recipients. For embryo transfer (ET), 120 ewes (50 for fresh embryos and 70 for frozen embryos) with a body condition score of 2.5 (in a scale from 1–5) were used as recipients and synchronized with a CIDR-G (intravaginal device with 0.3 g of progesterone) on Day 0, on Day 12 CIDR-G was removed and a 400 UI dose of equine chorionic gonadotropin was applied, on Day 19 ET was conducted. Recipients were transferred (two embryos each), 70 with frozen embryos and 50 with fresh embryos. Pregnancy was detected using transrectal ultrasonography at 35 days of gestation, 36 from 50 recipients transferred with fresh embryos were diagnosed as pregnant, with only 26 diagnosed as a twin pregnancy. For ET with frozen embryos, 44 recipients were pregnant, of which 33 were twin pregnancies. For the ET conducted with fresh embryos, only 31 recipients from the 36 diagnosed as pregnant came to parturition, and none with twins. Regarding the ET with frozen embryos, just 40 lambs were born, but there were no twins. These results may be due to overdeveloped fetuses or from excessive competition.