In vitro maturation of equine oocytes collected by follicle aspiration and by the slicing of ovaries

Abstract

The aim of this study was to examine 2 techniques for oocyte recovery from equine ovaries at slaughter: by aspiration of follicles and by additional slicing of ovaries. The morphology and nuclear configuration of oocytes recovered with either technique, and the time course of nuclear maturation during in vitro maturation were evaluated. Recovery rates were 1.75 and 4.14 oocytes per ovary for aspiration and slicing (total 145 and 344 oocytes from 83 ovaries), respectively. The oocytes were classified according to their cumulus/ooplasm morphology into 4 groups: compact/circular(A), compact/semicircular(B), expanded(C) and others(D). The percentages of oocytes in Groups A, B, C and D were 34, 38, 25 and 3% (aspiration) and 55, 26, 17 and 3% (slicing), respectively. The proportions of oocytes with a germinal vesicle in Groups A, B, C and D were 28 29 (97%), 23 35 (66%), 11 23 (48%) and 2 4 (50%) in oocytes from aspiration and 91 100 (91%), 52 65 (80%), 15 29 (52%) and 1 2 (50%) in oocytes from slicing, respectively. Group A and B oocytes recovered by aspiration (n=212) and slicing (n=312) were cultured in TCM199 supplemented with 10% fetal bovine serum, 1 μg/ml estradiol-17β, and 0.02 AU/ml FSH at 38.5°C in 5% CO 2 in air (5 to 10 oocytes per 50- μ l microdrop). At 8, 16, 24, 32 and 40 h of culture, the oocytes were fixed and stained. There were no significant differences in the percentages of Metaphase II stage (MII)-oocytes between recovery techniques at any time points examined. The proportions of MII-oocytes were 1 42 (2%), 4 43 (9%), 21 42 (50%), 28 45 (62%), and 28 40 (70%) at the respective time point in oocytes from aspiration and 0 51 (0%), 3 54 (6%), 22 59 (37%), 43 72 (60%), and 51 76 (67%) in oocytes from slicing, respectively. In most of the oocytes, resumption of meiosis occurred between 8 and 16 h of culture. The proportions of MII-oocytes increased significantly between 16 and 24 h and between 24 and 32 h of culture.