Preparation of testes and spermatozoa

The mice were anesthetized with sodium pentobarbital. After skin disinfection (with 75% alcohol), an incision was made in the skin and peritoneum. After pushing away the bowels and fat and forcing the testes out of the scrotum (into abdomen), the testes and epididymis were surgically removed. The caput epididymis and cauda epididymis were cut into blocks and mechanically disrupted using a syringe (1 ml) under an anatomical microscope in Quinn’s Sperm Washing Medium (Cat. No. 1006, Quins’ SAGE IVF, USA) at 37 °C. Spermatozoa were obtained from the surface of the cell suspension after incubation at 37 °C for 20 min. The crabs were anesthetized by chilling on ice for 15 min. The carapaces were cut open cross-sectionally, and the heart and accessory sex gland were removed gently. Subsequently, the testes and seminal vesicles were exposed and surgically removed. To obtain free spermatozoa from crabs, we dissected the seminal vesicles into pieces, and spermatozoa were obtained via manual tissue homogenization in Ca²⁺-free artificial seawater (Ca2⁺-FASW; 475 mM NaCl, 12 mM KCl, 30 mM MgCl₂, 20 mM Tris, pH 8.2). After being left to stand for 20 min at 4 °C, the free spermatozoa were collected by centrifugation (10,000 rpm for 10 min, supernatants reserved; 500 rpm for10 min, supernatants reserved and 1000 rpm for 10 min, pellet collected).