Page Banner

United States Department of Agriculture

Agricultural Research Service

Rainbow Trout Milt Protocol
headline bar

Rainbow Trout Milt Cryopreservation Protocol

 

 Milt Collecting Video

 

April, 2004

 

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

 

Upon receipt, the sperm motility is assessed visually using phase contrast microscopy (400 x) and rated with a motility score of 0 to 5, with 0 being no motile cells.  To assess motility, a drop of water (12ºC) 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 water.  The sperm motility is assessed immediately, without a coverslip.

 

Samples are then diluted 1:3 (v:v) with cryopreservation media at 12°C.  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: 5°C to -70°C at -30°C per minute and then plunged in liquid nitrogen for storage.

 

Samples are thawed for 1 minute in a 12°C water bath and motility analysis is performed as described previously.

 

Recipe:

 

Cryopreservation media

300mM Glucose

10% DMSO (by volume)

13.30% Egg Yolk (by volume)

pH of 8.0 to 8.5.

 

Reference:

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


Last Modified: 6/14/2010