Sheep Fetal Hypoxia Exposure Protocol

Twelve pregnant Welsh mountain sheep carrying singleton fetuses were used in the study. In brief, using the protocol described in Allison et al. (2016), at 117 ± 1 days of gestation (0.8 of gestation; term ∼150 days), general anaesthesia was induced using 1.5–2.5 mg kg⁻¹i.v. alfaxalone (Jurox Ltd, Malvern, UK). The ewe was intubated with a cuffed endotracheal tube and anaesthesia was maintained by inhalation of 1.5% isoflurane in oxygen. Under aseptic surgical conditions, an arterial catheter was inserted into the fetal femoral artery and advanced into the descending aorta. Another catheter was placed in the amniotic cavity. Following surgery, ewes were housed in individual floor pens with a 12 h light–dark cycle with ad libitum access to hay, nuts and water. After 5 days post‐operative recovery, ewes and fetuses were randomly allocated to chronic normoxia (n = 6) or chronic hypoxia (n = 6). Chronically hypoxic animals were housed in bespoke isobaric hypoxic chambers (Fig. 1) for 2 days prior to the initiation of hypoxia, and remained in these chambers for a further 10 days under hypoxic conditions, before being returned to the individual floor pens and normoxic conditions. Hypoxia was induced incrementally over the first 24 h, then maintained at 10% inspired oxygen for the remainder of the experimental protocol, the full details of which have been previously described (Brain et al. 2015; Allison et al. 2016). Pregnancies allocated to the chronic normoxia group were housed in a barn in floor pens with the same floor area as that of the hypoxic chambers. Both the chronic normoxia and hypoxia groups of ewes were fed daily the same bespoke maintenance diet made up of concentrate pellets and hay (40 g nuts kg^–1 and 3 g hay kg^–1; Manor Farm Feeds Ltd, Oakham, UK) to facilitate the monitoring of food intake. Ambient room temperature was maintained between 20 and 24°C in both the hypoxic chambers and the normoxic floor pens. At the end of the experimental protocol the ewe and fetus were killed under Schedule 1 of the UK Animals (Scientific Procedures) Act 1986 using a slow i.v. injection into the maternal jugular vein of 120 mg kg⁻¹ pentobarbitone sodium (Pentoject; Animalcare Ltd, York, UK).