I recommend that you look at the composition of several semen extenders (diluents) and decide what is simplest and best. I doubt that physiological saline will work well. Some semen extenders are simple, such as early glucose and citrate solutions. The sperm need an energy source to survive very long. Even saline with a little glucose might be better, but also pH is important.

Dear Jack,

Thank you for your suggestions. I can try saline with glucose and guess temperature is also important for the motility.

I wish conveniences. Have a good day.

I am not familiar with rat sperm. For mouse sperms, several protocols have been used with good results, such as HTF or Ham's F10 with 10 to 15% FBS in 5% CO2 incubator. Normal saline plus glucose may be not enough for sperm survival, but I'd like to know the results. Please let me know your results after using normal saline. By the way, what method did you use to retrieve the rat semen?

Thank you for your answer Dear Tzu-Bou Hsieh ,

I will work saline with glucose and than you can be sure to let you know the results.

I did a drug study then I sacrificed the rats. After that, I separated the epididymis from the testis and isolated the sperms by making incisions the epididymis in a petri dish containing saline.

I wish conveniences.