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United States Department of Agriculture

Agricultural Research Service

Rainbow Trout Milt Protocol
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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.




Cryopreservation media

300mM Glucose

10% DMSO (by volume)

13.30% Egg Yolk (by volume)

pH of 8.0 to 8.5.



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

Last Modified: 6/14/2010