Freeze-drying of spermatozoa.

Bull semen was diluted to a concentration of 2 X 10(8) cells/ml, cooled to 5 degrees C in 5 h, frozen in 0.025 ml spheres on the surface of solid carbon dioxide, and stored in liquid nitrogen. 50% by volume of the diluent was 325 m0sm Tes:N-tris (hydroxymethyl) methyl-2-amino ethane sulfonic acid titrated to pH 7.2 with 325 m0sm Tris:tris (hydroxymethyl) amino methane. The diluent also contained 30% by volume isotonic sodium citrate and 20% by volume egg yolk. The frozen spermatozoa were freeze-dried in 400 mg quantities in test tubes at -50 degrees C with a condenser at -196 degrees C. Moisture content was determined by weighing the individual samples before and after freeze-drying. Drying the samples for several days at 20 degrees C removed 868 mg from each gram of frozen material and this was considered the zero moisture level. Samples were stored at 20 degrees C or -196 degrees C after freeze-drying. The freezer-dried samples were rehydrated by flooding with five times their original volume of isotonic sodium citrate. Tests of the recovered spermatozoa included percentage motile cells, acrosome damage, enzyme release, protein denaturation, hypotonic swelling and fertility testing. Sperm motility decreased with dryness until it reached zero at 3% moisture. Acrosome morphology and enzyme release appeared normal down to 1% moisture. Preliminary results showed some fertility at all levels of dryness with -196 degrees C storage, and fertility at less than 1% moisture with 20 degrees C storage. Additional fertility testing is underway.