In vitro fertilization of bovine oocytes by spermatozoa capacitated in vitro

In vitro fertilization of ovulated bovine oocytes was attempted with fresh or frozen semen after capacitation in vitro. Sperm incubations and oocyte cultures were performed in a Krebs Ringer bicarbonate medium containing Na pyruvate, glucose and .3% bovine serum albumin. High ionic strength (HIS) medium was prepared by adding NaCl to provide an osmolarity of 370 to 380 nOsmol/kg. Donor cows were treated with prostaglandin F2 alpha and a series of follicle-stimulating hormone injections and ovulated oocytes were recovered 72 h after prostaglandin treatment. Fresh or frozen semen was: 1) placed directly into a microdrop of standard medium (SM) under oil; 2) washed by centrifugation with SM and placed in a microdrop of SM or 3) pretreated with HIS for 10 min, washed and placed into a microdrop of SM. In all cases, spermatozoa were preincubated for 3 h at a concentration of approximately 10(6) cells/ml before addition of oocytes. Oocytes were incubated with spermatozoa for 6 h, transferred to fresh medium and cultured for 24 h. When spermatozoa were placed directly into a microdrop, three of 34 (9%) oocytes were penetrated, but none divided. Spermatozoa washed with SM penetrated 20 of 45 (44%) oocytes and three (7%) divided. Spermatozoa incubated in HIS penetrated 14 of 47 (30%) oocytes and five (11%) divided. The washing of spermatozoa with standard medium was equally as effective as incubation with high ionic strength medium in inducing in vitro capacitation of bovine spermatozoa.