Bring all medias to 37 C temperature.

Mix the liquefied semen sample to 2ml of the Sperm Wash Media. Centrifuge at 1600 rpm for 8 mins

Notes:

1. Use well liquefied semen samples only

2. Use 1 to 1.5 ml of the semen sample

Discard the supernatant of the Wash Step 1

Notes:

1. Pellet is nothing but concentrated sperms

2. Disturbing the pellet may lead to loss of sperms

Add 2ml of Sperm Wash media to the pellet and mix well. Centrifuge again at 1600 rpm for 8 mins.

Notes:

1. This is wash step 2

2. Wash step removes other semen contents from sperm and non sperm cells

Discard 1.5ml of supernatant from the Wash Step 2

Notes:

1. Pellet is nothing but concentrated sperms

2. Disturbing the pellet may lead to loss of sperms

Add rest of the 0.5 ml of Sperm Wash media to the pellet in a very gentle manner without disturbing the pellet. Leave the tube at 45 degrees inclination for the incubation at 37 C temperature for 30 to 45 mins

Notes:

1. During the swim-up sperms from pellet reach the top fraction of the media

2. After the incubation remove the top fraction and keep at 37 C until use.