Development of a combined mechanical and enzymatic method for the isolation of intact preantral follicles from fetal, calf and adult bovine ovaries

The isolation of preantral follicles from the ovaries of bovine fetuses, calves and adult cows was performed using a simple, rapid mechanical and enzyme method. The ovaries were cut into small pieces with a tissue chopper. Then, the suspension was filtered successively through 500 and 100 μm nylon mesh filters. This simple mechanical procedure resulted in large numbers of isolated preantral follicles: 2,142 ± 254; 512 ± 92 and 298 ± 54 from the ovaries of bovine fetuses, calves and cows, respectively. In addition, the ovarian fragments between 100 and 500 μm were suspended in 10 ml of M199 Hepes medium plus 5% FCS and divided into 2 equal parts: one portion was used for collagenase treatment (200 U/ml) for 20 minutes, while the other served as a control. Collagenase treatment resulted in 841 ± 161; 216 ± 51 and 52 ± 17 preantral follicles from fetuses, calves and cows, respectively, compared with 312 ± 86; 52 ± 15 and 10 ± 2 in the control group. The use of collagenase with ovarian fragments selected by filtration as a method for increasing the rate of recovery of preantral follicles is described here.