Ram Semen Processing and Cryopreservation Protocol
The concentration and motility of the semen sample are determined using spectrophotometry and a Hamilton Thorne motility analyzer (Beverly, MA), respectively (at least 7 fields of analysis and 1000 cells). Semen samples are diluted to 200 x 106 cells per ml in egg yolk-Tris media (see recipe below).
The samples are then cooled to 5°C over 2 to 2.5 hours and loaded into 0.5ml straws. The samples are frozen using the Cryo Bio System Mini Digitcool UJ400 (IMV Corporation, Minneapolis, MN) with the following curve: 5°C to -5°C at -4°C per minute, -5°C to -110°C at -25°C per minute, -110°C to -140°C at -35°C per minute, and plunged into liquid nitrogen for storage.
Cryopreserved samples are thawed for 30 seconds in a 37°C water bath and analyzed for motility as described previously.
Recipe: (Sanchez-Partida et al., 1998)
Egg yolk-Tris media
95mM citric acid
5% glycerol (by volume)
15% egg yolk
1mg/ml streptomycin sulfate
Sanchez-Partida, L.G., Setchell, B.P., and Maxwell, W.M.C. Effect of compatible solutes and diluent composition on the post-thaw motility of ram sperm. Reprod. Fertil. Dev. 1975; 10, 347-357.