20 m filter

Feline testes were obtained from a local veterinary clinic. After removing external tissues from the testes, we separated the epididymides. The epididymides were placed in PBS(–), cut into small pieces, and incubated at 38.5°C under 5% CO₂ in humidified air for 10 min. After filtering through a 20-µm filter (Nipro, Osaka, Japan), the semen was centrifuged for 5 min at 500 × g, and the seminal plasma was removed by aspirating the supernatant.
The pelleted sperm was resuspended in EYT-FC solution, which contains egg yolk supplemented with 13 µg/ml citric acid (Nacalai Tesque), 10 µg/ml D-fructose (Nacalai Tesque), 24 µg/ml Tris aminomethane (Nacalai Tesque), 1000 IU/ml penicillin (Sigma-Aldrich), and 1 mg/ml streptomycin (Sigma-Aldrich), and stored at 4°C. After 1 h, an EYT-FC solution containing 14% (v/v) glycerol (Nacalai Tesque) was added to the existing solution to obtain a final cell density of 12.5 × 10⁶ cells/ml and a final glycerol concentration of 7% v/v. This solution was then loaded into 0.25-ml straws (Fujihira, Tokyo, Japan). After sealing, the straws were laid horizontally on a rack, placed 4 cm above the surface of liquid nitrogen for 5 min, plunged into liquid nitrogen, and stored in a liquid nitrogen storage tank until further analyses.

Testes were obtained from a local veterinary clinic. After removing external tissues from testes, we then separated the epididymides. The epididymides were cut into small pieces in
Dulbecco’s phosphate-buffered saline without Ca²⁺ and Mg²⁺ (PBS (–), Nacalai, Kyoto, Japan) and cultured at 38.5°C under 5% CO₂ in humidified air for 10 min.
After filtering through a 20-µm filter (Nipro, Osaka, Japan), the semen was centrifuged for 5 min at 500 × g, and the seminal plasma was removed by aspirating the
supernatant. The pelleted sperm were resuspended in m-HTF medium (Nippon Medical & Chemical Instruments, Osaka, Japan) to prepare a sperm suspension.
The sperm suspension was mixed with EYT-FC solution, which comprised egg yolk supplemented with 13 µg/ml citric acid (Nacalai), 10 µg/ml d-fructose (Nacalai), 24 µg/ml Tris aminomethane
(Nacalai), 1000 IU/ml Penicillin (Sigma-Aldrich), and 1 mg/ml streptomycin (Sigma-Aldrich), and stored at 4°C. After 1 h, EYT-FC solution containing 14% (v/v) glycerol (Nacalai) was added
the solution to obtain a final concentration of 12.5 × 10⁶ cells/ml, with a final glycerol concentration of 7%. Then, this solution was loaded into 0.25-ml straws (Fujihira,
Tokyo, Japan). After sealing, the straws were laid horizontally on a rack 4 cm above liquid nitrogen vapor for 5 min, plunged into liquid nitrogen, and stored in a liquid nitrogen storage
tank.