9 First pregnancies from in vitro-produced sheep embryos at high altitude in the Peruvian highlands

The objective of this study was to assess the feasibility of producing pregnancies from in vitro-produced sheep embryos at high altitude in the Peruvian highlands by comparing two different maturation media. The research was conducted at the Casaracra Experimental Center of the Daniel Alcides Carrión National University (Paccha District, Yauli Province, Junin Region, Peru) in the Central Andes Mountains at an elevation of 3921 m above sea level (masl). Ovaries collected from a local slaughterhouse were transported for 2 h in a thermos flask in 38°C saline. A total of 101 cumulus–oocyte complexes (COCs) were collected; 47 COCs were matured in maturation medium (VitroMat Protect, ArtLabSolutions) in a humidified incubator with 9.1% CO₂ at 38.7°C (treatment 1), and 54 COCs were matured in a different maturation medium (Transport VitroMat Protect, ArtLabSolutions) in an incubator without CO₂ at 38.7°C (treatment 2) for 22–24 h. After maturation, oocytes were fertilized with refrigerated sperm collected from a Dohne Merino ram and capacitated in fertilization medium (VitroFert, ArtLabSolutions) with 2% estrous sheep serum (ESS) using the “swim up” method. Oocytes were fertilized using 0.5 × 10⁶ spermatozoa per 500-µL drop of VitroFert supplemented with 10 IU mL⁻¹ of heparin and 2% ESS. Sperm and oocytes were co-incubated for 18–20 h at 38.7°C in 9.1% CO₂ in a humidified incubator. Presumptive zygotes were transferred to 600-µL drops (20 zygotes per drop) of culture medium (VitroBlast Plus, ArtLabSolutions) and incubated at 38.7°C in 9.1% CO₂ and 7% O₂ for 5 days. Cleavage rates were evaluated after 24 h of culture, and embryo development was assessed on Day 5 of culture. Cleavage rate was lower (P < 0.05, chi-squared) for treatment 1 (20.8%; 10/47) than for treatment 2 (51.8%; 28/54). Likewise, the percentage of oocytes that developed into transferable-quality embryos on Day 5 was lower (P < 0.05, chi-squared) for treatment 1 (6.3%; 3/47) than for treatment 2 (46.3%; 25/54). From treatment 2, 20 embryos were selected for embryo transfer; six recipients received a single embryo, and seven recipients received two embryos. Pregnancy rates were 16.6% (1/6) and 43.8% (3/7) for single and twin embryos, respectively, 1.5 months after embryo transfer and were not different (P > 0.05, chi-squared). This study demonstrates that pregnancies can be established with in vitro–produced sheep embryos at high altitudes (>3900 masl) in the Peruvian highlands. Further studies are needed to optimize the incubation environment at high altitudes where atmospheric pressure is lower.

This research was supported by Universidad Nacional Daniel Alcides Carrión, Pasco, Peru and the ME Ensminger Endowment at Iowa State University.