Ram semen collection and processing Protocol

- April 2, 2004 -

The concentration and motility of the semen sample are determined using spectrophotometry (Hammerstedt, 1975) and a Hamilton Thorne motility analyzer (Beverly, MA), respectively (at least 5 fields of analysis and 1000 cells). Semen samples are diluted to 200 x 106 cells per ml in egg yolk-Tris media (300mM Tris, 28mM glucose, 95mM citric acid, 5% (v:v) glycerol, 15% egg yolk, 1mg/ml streptomycin sulfate and 0.06mg/ml benzylpenicillin; Sanchez-Partida et al., 1998).

The samples are then cooled to 5°C over 2 to 2.5 hours and loaded into 0.5ml straws. The samples frozen using the Cryo Bio System Mini Digitcool UJ400 (IMV Corporation, Minneapolis, MN) with the following curve: 4ºC to -5ºC at 4ºC per minute, -5ºC to -110ºC at 25ºC per minute, -110ºC to -140ºC at 35ºC per minute, and plunged in liquid nitrogen for storage. Cryopreserved samples are thawed for 30 seconds in a 37°C water bath and analyzed for motility as described previously.

References:
Hammerstedt, R. H. Tritium release from [2-3H] D-glucose as a monitor of glucose consumption of bovine sperm. Biol Reprod 1975; 12:545-551.
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.

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