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

Departments of Animal Science and Large Animal Clinical Sciences, University of Minnesota, St. Paul 55108

ABSTRACT

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 µg/ml),and estradiol-17ß (1.5 µg/ml) for 24 h at 37°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 10⁶ sperm/ml in Medium 199 supplemented with 10% fetal calf serum. Oocytes were added (10/500 µ1 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.