Bring media and semen sample to the room temperature.

Analyse the sample before the process. Use 1:1 ratio of media to the sample. Mix well without forming the air bubbles. Keep the vial upright for 15 mins.

Notes:

1. Use well liquefied semen samples only

2. Mix occasionally during the 15 mins to make the contents homogenous

3. During this step media penetrates into the sperm cells

Keep the vial just above the liquid nitrogen using a string for 15 mins.

Notes:

1. Do not touch the liquid nitrogen directly

2. This is a first cooling step

After 15 mins, plunge the vial directly into the liquid nitrogen for long term storage

Notes:

1. This is vitrification step

2. Use multiple cryo-vials with 0.5ml volume to avoid repeated freeze and thaw cycles for the same sample.

For thawing, pull out the vial from liquid nitrogen and keep under running water till the contents liquify completely. Then in the room temperature for 15 mins followed by 37 C. Process the sample before use.

Notes:

1. Direct usage from the thawing is not advised

2. Washing the sample at least once will help reducing the glycerol content from the freezing media