The results of production and transplantation of IVEP embryos in sheep (Ovis aries)

In vitro embryo production (IVEP) in sheep is necessary to develop because of its use in breeding and conservation of valuable animals and possible creation of new genotypes by genomic editing. In the present work for the first time in national practice, full-fledged ovine embryos were produced in vitro and live lambs were born after their transplantation to recipient ewes. The aim of this work was to model the main steps of IVEP technology in this species, and to evaluate its efficiency in vitro and in vivo. Female germ cells were obtained post mortem from the ovaries of sexually mature ewes and sheep of various breeds and ages after slaughtering. Oocyte-cumulus complexes (OCC) ( n = 1028) were retrieved by dissecting of visible follicles and only high-quality OCC ( n = 620) were cultured for 24 h, 25-35 OCC per 500 ml of ТС-199 medium supplemented by 10 % fetal calf serum, 10 mg/ml of FSH and 10 mg/ml LH, 10 ng/ml of epidermal growth factor. A part of mature oocytes ( n = 96) was used for cytological analysis of nuclear maturation rate, and other oocytes ( n = 524) were transferred to BO-IVF medium (IVF Bioscience, UK) for in vitro fertilization. The granules of Katadin breed ram frozen semen were thawed and treated by “swim-up” method in Sperm-TALP medium (G.N. Singina, 2019). Mature oocytes were co-cultured with ram sperm in BO-IVF for 15-16 hours and then were transferred to BO-IVS medium (IVF Bioscience, UK) for in vitro embryo development. At day 2 of culture, cleavage rate was evaluated and a part of cleaved embryos was transplanted to recipient animals; at day 7, development to blastocyst occurred. Two-day embryos were transplanted synchronously to cycling Romanov breed ewes ( n = 6) by endoscopic surgical method (V.A. Lukanina et al., 2023) using two-port laparoscopy under local anesthesia. After 35-42 days of transplantation, recipient ewes were examined for pregnancy, and fetus development was monitored until live lamb birth. According to cytological analysis, oocyte nuclear maturation rate was 77.1 % (74/96), 316 out of 524 mature and fertilized oocytes were cleaved (60.3 %), and 92 cleaved embryos were transplanted to recipient animals. Remaining early embryos ( n = 224) continued in vitro development and 34.8 % reached blastocyst stage. According to ultrasound diagnostics after embryo transplantation, pregnancy rate was 50 % (3/6), and 33.3 % (2/6) transplantations resulted in live offspring. Thus, reported data demonstrated efficiency of IVEP technology in sheep: produced embryos were full-fledged and capable to develop to viable offspring. There is a good reason to believe that proposed technology of in vitro embryo production and transplantation to recipient ewes can be applied to reproduction technologies and gene editing in ovine.