DEVELOPMENT OF A COMBINED NEW MECHANICAL AND ENZYMATIC METHOD FOR THEISOLATION OF INTACT PREANTRAL FOLLICLES FROM FETAL, CALF AND ADULT BOVINE OVARIES

The isolation of preantral follicles from the ovaries of bovine fetuse s, calves and adult cows was performed using a simple, rapid mechanica l and enzyme method. The ovaries were cut into small pieces with a tis sue chopper. Then, the suspension was filtered successively through 50 0 and 100 mum nylon mesh filters. This simple mechanical procedure res ulted 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 mum were suspended in 10 ml of M199 Hepes medium plus 5% FCS and divided into 2 equal parts: one portion was used for collagenase t reatment (200 U/ml) for 20 minutes, while the other served as a contro l. 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 follicle s is described here.