Creatine monohydrate

The creatine supplementation and UCO protocol are described in detail elsewhere (Tran et al., 2021 (link)). Briefly, sterilised and filtered creatine monohydrate (Sigma‐Aldrich, St Louis, MO, USA) in saline was prepared at a concentration of 12 mg ml⁻¹ and was delivered intravenously at 1.5 ml h⁻¹ from 121 dGA at 09.00 h until 134 dGA; fetal weight was estimated to be 3 kg, thus, the infusion rate was ∼6 mg kg⁻¹ h⁻¹. Saline fetuses received an isovolumetric administration of 0.9% NaCl, pH 7.4 for the same duration. At 131 dGA, at 09.00 h, the Silastic cuff was inflated for 10 min using a predetermined volume of sterile water that would cause complete occlusion of the blood vessels in the umbilical cord; the internal cuff pressure was >100 mmHg. Successful UCO was determined by immediate hypertension and bradycardia (Tran et al., 2021 (link)). Control fetuses were not subjected to UCO.
Ewes were euthanised at 134 dGA with intravenous pentobarbital sodium (100 mg kg⁻¹; Lethabarb; Virbac, Milperra, NSW, Australia) and allowing approximately a further 3 min for euthanasia of the fetus. The right hemisphere of the fetal brain was immediately immersion fixed in 4% paraformaldehyde (Merck) in 0.1 M phosphate buffer for 5 days and then coronally sectioned into 5 mm blocks for paraffin embedding.