2.1. Study Design

The rats that were included in the experiment had intact circadian rhythm and ad libitum feeding within their natural environment. Forty-two female preadolescent Wistar-Albino rats (mean age: 4 weeks, weight range: 40–45 g) were divided into 6 equal groups randomly.

All surgical operations were performed under intramuscular 50 mg/kg ketamine (Ketasol 10%, Richter Pharma AG, Wels, Austria) and 10 mg/kg xylazine (Alfazyne 2%, Alfasan International BV, Woerden, Netherlands) anesthesia. Rats were placed in the dorsal recumbent position. A 1.5 cm midline laparotomy incision was made under sterile conditions so that the abdomen was opened and the large intestines were separated gently. In order to visualize the left ovary, large intestines are placed on the left side of the rat's abdomen. After the left ovary was found, it was made to undergo torsion of 720 degrees clockwise and then fixated to the anterior abdominal wall with a single 5/0 prolene suture. Afterwards, the abdominal incision was repaired with continuous sutures. Rats were covered with gauze to be protected from hypothermia and they recovered from the effects of anesthesia in approximately 30 minutes. Medication was given orally with gavage after an hour postoperatively; detorsion was performed at the 3rd hour and bilateral oophorectomy and blood samples were taken at the 9th hour postoperatively. The reason for choosing postoperative 9th hour for biochemical, histopathological, and immunohistochemical evaluation is based on pharmacokinetic properties of the molecules. When ALA is taken orally, it is rapidly absorbed and reaches plasma peak levels in 30 minutes to 1 hour [17]. In contrast, coenzyme Q₁₀ reaches peak plasma levels in about 4–6 hours after its oral administration [18].

Group 1 (sham group) included 7 rats that had laparotomy without torsion. Group 2 (saline group) consisted of 7 rats that underwent T/D and received saline orally (Polifleks 0.9% NaCl, Eczacıbaşı-Baxter, Istanbul, Turkey). Group 3 (olive oil group) included 7 rats that had T/D and received olive oil orally (Taris, Izmir, Turkey). Group 4 (ALA group) consisted of 7 rats that underwent T/D and received ALA orally (100 μM/kg/day, ≥99% titration in olive oil, Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Group 5 (CoQ₁₀ group) included 7 rats that had T/D and received CoQ₁₀ orally (10 mg/kg/day, ≥98% titration in olive oil, Sigma-Aldrich Chemie GmbH, Steinheim, Germany). Group 6 (ALA + CoQ₁₀) included 7 rats that underwent T/D and received both ALA and CoQ₁₀ orally. As both ALA and CoQ₁₀ were administered in olive oil solutions in the treatment groups, an olive oil group was included to rule out whether olive oil itself would exert antioxidant effects.