Rooster Semen Cryopreservation Protocol

- 2005 -

Based on information from: Seigneurin, F, Blesbois, E., 1995. Effects of the freezing rate on viability and fertility of frozen-thawed fowl spermatozoa. Theriogenology. 43, 1351-1358.

Recipes: Lakes Low Temperature (LLT) Medium (1 liter)

Non-glycerol
Sodium glutamate-monosodium salt 169.1 mM
Fructose 33.3 mM
Magnesium Acetate (4H2O) 4 mM
Sodium Acetate (Anhydrous) 62 mM
Potassium citrate 3.7 mM
pH 7.5

Glycerol
Identical to the medium above except it includes 16% glycerol (v:v)

Procedure for Rooster semen cryopreservation

Semen is collected from roosters by digital manipulation and the sample is observed to make sure it is free of feces and urine. Following collection the sample is diluted to approximately 200µl with LLT non-glycerol medium which was pre-cooled to 5ºC. The samples are then placed in a rack in a styrofoam box containing ice and transported to the laboratory. Then the samples are diluted 1:2 (v:v; sample to cryopreservation medium) with LLT glycerol medium and loaded into 0.5ml straws. The samples are then frozen in liquid nitrogen vapor (2.5in above liquid nitrogen) for 10min and plunged into the liquid nitrogen for storage.

Procedure for Rooster semen thawing

Frozen samples are thawed in a 5ºC water bath for 5 min and then warmed to room temperature for use or analysis.

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