Orchiectomy

Orchiectomy was conducted using a castration rat model as described previously [30]. All surgical procedures were performed under sterile conditions and general anesthesia, which was comprised of inhalation of 1%~2% isoflurane. For the orchiectomy, a 10-mm incision was made in the skin of the lower abdomen over one testis of a rat. The testis and associated fat pad were pulled with forceps. The connective tissues leading to the testis which included the fat pad, testicular vein, and vas deferens were tied using surgical silk. Connective tissues between the testis and knot around the connective tissue were cut. Finally, the connective tissues were returned to the body cavity. The wound was cleaned and sutured. In the sham group, animals received a 3-cm wound, which was immediately stitched closed. Four weeks after surgery, rats were euthanized via terminal bleeding under isoflurane-anesthesia, and femurs and tibias were collected. Following the removal of adherent soft tissues, bone tissues were washed with phosphate-buffered saline (PBS, 0.14 M NaCl, 2.6 mM KCl, 8 mM Na₂HPO4, and 1.5 mM KH₂PO₄).