Midazolam

At termination, animals were fasted for six hours. Afterwards, mice were anesthetized with 0.1 ml/10 g BW with 25% Midazolam® (5 mg/ml Midazolam, B.Braun, Melsungen, Germany) and 25% Hypnorm® (0.315 mg/ml of fentanyl citrate and 10 mg/ml of fluanisone, Skanderborg Pharmacy, Denmark) diluted in saline. Retro-orbital blood was collected into an autoclaved 1.5 ml Safe-Lock Eppendorf® microcentrifuge tube (Buch & Holm A/S, Herlev, Denmark) and stored on ice for several hours, and later stored at 4 °C for 1 day to let the blood coagulate. The samples were then centrifuged for 10 min at 10,000 rpm at 4 °C. Supernatants were subsequently stored at − 80 °C. Animals were euthanized by cervical dislocation. Caecum contents were collected immediately after euthanizing into sterile Eppendorf® tubes and stored on dry ice during collection, with subsequent storage at − 80 °C. Before post-mortem sample collection mice were randomized, so all parameters were blinded to the reader.

The pigs were premedicated with an intramuscular injection of 20 mg/kg ketamine (Pfizer AS, Oslo, Norway) and 1 mg of atropine (Nycomed Pharma, Oslo, Norway). Anesthesia was induced by intravenous injection of 10 mg/kg pentobarbital sodium (Abbott, Stockholm, Sweden) and 0.01 mg/kg fentanyl (Hameln Pharmaceuticals, Hameln, Germany). The animals were ventilated after intubation. Normal ventilation was defined as an PaCO₂ of 40 ± 2 mmHg. A central venous catheter was placed through the left internal jugular vein and anesthesia was maintained throughout the experiment by a continuous infusion of 4 mg·kg⁻¹·hr⁻¹ pentobarbitone sodium, 0.02 mg·kg⁻¹·hr⁻¹ fentanyl, and 0.3 mg·kg⁻¹·hr⁻¹ midazolam (B. Braun, Melsungen, Germany). The circulating volume was maintained by a 20 ml·kg^‐1·hr⁻¹ continuous infusion of 0.9% NaCl supplemented with 1.25 g·L⁻¹ glucose. Following sternotomy, the pericardium was removed, and the coronary arteries and pulmonary trunk were dissected free from connective tissue. The hemiazygos vein was then ligated.

Animals were anesthetized with 0.3 mL/100 g of bodyweight of Hypnorm (VetaPharma Ltd., Leeds, UK) and Midazolam (B. Braun, Melsungen, Germany) mixture subcutaneously. Initial analgesics were given, 0.2 mL/100 g of bodyweight of Temgesic (RB Pharmaceuticals Limited, Berkshire, UK). Once anesthetized, the animals were shaved on the lumbar part of the back. A midline dorsal skin incision was made. Blunt dissection of the connective tissue between skin and muscle layer gave access to the abdominal wall. The muscular layer was perforated 2 centimetres lateral of the dorsal midline to gain access to the abdominal cavity. The ovary, located surrounded by a large fat pad, was pulled out the incision and fixated. Two ligatures of absorbable 5.0 ethilon sutures were placed between the uterine horn and the ovary. The ovary was safely cut off and the uterine horn was placed back into the abdominal cavity. The muscle layer was closed with absorbable 5.0 sutures. Bilateral ovary resection was performed from the same initial median incision. After resection, the wound was carefully closed in layers with 4.0 ethilon sutures. After surgery, analgesic, Temgesic 0.2 mL/100 g of bodyweight, was administered subcutaneously three times a day for three days.