Identification of the capacitating agent for bovine sperm in egg yolk-TEST semen extender

Bovine sperm can be capacitated in egg yolk-TEST buffer. To determine what constituent of the buffer was responsible, ejaculated semen was diluted 1:10 at 37 degrees C with the following 20% egg yolk (vol/vol)-containing buffers: TES-Tris, TES-tetramethylammonium hydroxide, taurine-Tris, citric acid-Tris, citrate, egg yolk salts, egg yolk proteins Tris, and citrate-taurine. Buffers were pH 7.6 and 321 to 325 mOsmol/kg. Extended semen was cooled slowly to 4 degrees C and stored 8 h. Sperm taken at 0 and 8 h were washed in pH 7.6 bovine serum albumin-saline and assessed for motility and capacitation using zona-free hamster eggs. Sperm motilities at 0 and 8 h were similar (60 to 73%) in all extenders except citric acid-Tris (54%) and egg yolk proteins Tris (15%). Bull sperm, stored 8 h in egg yolk-TEST, became capacitated. Because sperm storage in egg yolk-citrate did not result in penetration, both egg yolk and citrate were ruled out as capacitating agents. Capacitating activity resided in the TES and Tris molecules. The TES molecule contains a Tris component and this capacitated bull sperm. The TES molecule also contains a taurine component. However, taurine was not a capacitating agent for bull sperm. In conclusion, both TES- and Tris-containing buffers, alone or together (TEST), were equally effective in capacitating bull sperm.