Estradiol

Total protein was extracted from mammary glands by using the radioimmunoprecipitation assay buffer (R2078, Sigma, St Louis, MO, USA) with protease and phosphatase inhibitors (33 (link), 34 (link)). Protein concentrations of mammary extracts were quantified by using the bicinchoninic acid protein assay (Thermo Scientific). Sandwich enzyme-linked immunosorbent assays were performed to quantify estradiol (Biovision, Milpitas, CA, USA), progesterone (MyBioSource, San Diego, CA, USA), estrogen receptor 1 and 2, and progesterone receptor (LifeSpan BioSciences, Seattle, WA, USA) in mammary glands. Samples were read within the linear range of the assay. The accuracy of the analysis was confirmed by the controls provided in each kit.

Plasma TC, HDL-cholesterol, and TG levels were analyzed using commercial enzymatic kits (Asan Pharmaceutical, Seoul, Korea). LDL-cholesterol was calculated by the Friedewald’s formula: LDL-cholesterol = TC − HDL-cholesterol − TG/5. Glucose oxidase-based kits (Asan Pharmaceutical) were used to determine FBG levels according to the manufacturer’s protocol. Enzyme-linked immunosorbent assay (ELISA) kits were used to determine leptin and adiponectin (R&D Systems, Minneapolis, MN, USA), insulin and C-peptide (Mercodia, Uppsala, Sweden), osteocalcin, ALP, CTX, and NTX (Cusabio Biotech, Wuhan, China), and estradiol (Biovision, Milpitas, CA, USA) levels in plasma according to the manufacturer’s protocol. HOMA-IR was calculated using the following equation [34 (link)]: HOMA-IR = Fasting plasma insulin (µU/mL) × Fasting blood glucose (mmol/L)/22.5.