Rainbow Trout Milt Cryopreservation Protocol

- April 2, 2004 -

Rainbow trout milt is obtained by stripping the males and collecting the sample in a zip lock sandwich bag. The bag is filled with oxygen gas and placed in a 5ºC cooler for transport to the laboratory.

Upon receipt, the sperm motility is assessed visually using phase contrast microscopy (100 x) and rated with a motility score of 0 to 5, with 0 being no motile cells. To assess motility, a drop of swimming media (please see recipe section) is placed on a microscope slide and secured on the microscope stage. A glass Pasteur pipette is then dipped into the milt and quickly mixed into the swimming media. The sperm motility is assessed immediately, without a coverslip.

Samples are then taken to the 5ºC room and diluted 1:3 (v:v) with cryopreservation media at 5ºC. The samples are loaded into 0.5ml straws and frozen using the Cryo Bio System Mini Digitcool UJ400 (IMV Corporation, Minneapolis, MN) with the following curve: 5ºC to -70ºC at 30ºC per minute and then plunged in liquid nitrogen for storage.
Samples are thawed for 1minute in a 12ºC water bath and motility analysis is performed as described previously.

Recipes:
Swimming media
NaCl 125mM
CaCl2 0.1mM
Tris-Cl 30mM

Cryopreservation media
Glucose 300mM
DMSO 10% (v:v)
Egg Yolk 13.30% (v:v)

Both of the above media need a pH of 8.0-8.5.

References:
Coson, J. et al. 1999. In: The Male Gamete. Ed: C. Gagnon

Obtain information from Harvey Blackburn at: hblackbu@lamar.colostate.edu