Evaluation of Calcium-Free Tyrode's Sperm Capacitation Medium for Use in Bovine In Vitro Fertilization

Our objective was to determine if a bovine sperm capacitation technique, developed with zona-free hamster oocytes, could be used for the in vitro fertilization of in vitro matured bovine zona-intact oocytes. Bovine cumulus-enclosed primary oocytes from 2- to 5-mm follicles were matured in tissue culture Medium 199 containing Earle's salts and bicarbonate and supplemented with 10% fetal calf serum, FSH (10 micrograms/ml), and estradiol-17 beta (1.5 microgram/ml) for 24 h at 37 degrees C under paraffin oil. Ejaculated bovine sperm, washed thrice in bovine serum albumin-saline (pH 7.6) and capacitated for 4 h in Ca(++)-free Tyrode's medium (pH 7.6), were diluted to 2 x 10(6) sperm/ml in Medium 199 supplemented with 10% fetal calf serum. Oocytes were added (10/500 microliters droplet) to this medium containing the capacitated sperm, freeze-thawed killed sperm, or no sperm and incubated for 8 h before transfer to fresh medium and then incubated for 40 h. At the end of each incubation, a portion of the oocytes were stained and evaluated for development or fertilization. After 24 h of culture, 49% of the oocytes had matured (metaphase II). Fertilization rates were 55.6% after exposure of all oocytes to Ca(++)-free Tyrode's capacitated sperm and 82.5% if only metaphase II oocytes were selected. The parthenogenetic controls were negative (1.4% and 0%). Therefore, the Ca(++)-free Tyrode's sperm capacitation technique can be used for bovine in vitro fertilization studies.