Selection of follicles, preculture oocyte evaluation, and duration of culture for in vitro maturation of equine oocytes

Equine oocytes (n = 537) were collected from slaughterhouse ovaries (n = 118 mares) by scraping the internal follicular wall. Preculture record was made of the appearance of oocyte investments (no cumulus, corona radiata only, compact cumulus, expanded cumulus), appearance of cytoplasm (homogeneous, condensed heterogeneous/fragmented), and nuclear maturation stages (germinal vesicle, germinal-vesicle breakdown, metaphase I, metaphase II, degenerated). There was no difference between follicles > 30 mm and follicles < or = 30 mm in the preculture frequency distribution among the 5 nuclear stages; 96% were at either the germinal vesicle or germinal-vesicle breakdown stages. Oocytes from follicles 5 to 30 mm were cultured in modified TCM-199 for 18, 24, 36 and 48 h. Postculture nuclear maturation classifications were immature (germinal vesicle, germinal-vesicle breakdown, and metaphase I), mature (metaphase II or secondary oocyte), and degenerated. The frequency distribution of oocytes among the 3 postculture maturation classifications changed (P < 0.05) at 18 h (15% mature oocytes), changed (P < 0.05) further at 24 h (55% mature oocytes), with no additional change for 36 or 48 h. The only preculture cytoplasm group that affected the postculture results was the heterogeneous/fragmentation group which had a high proportion of postculture degenerated oocytes (67%); however, only 4% of oocytes were in this group. Luteal status of the mare had an effect (P < 0.05) on the frequencies of the maturation classifications, but not enough to be useful in selecting oocytes. Consistency of the follicle and the type of oocyte investment did not alter significantly the maturation frequencies. The frequency of degenerated oocytes after culture was high under the following conditions: 1) diameter of the follicle from which the oocyte was selected was 5 to 10 mm (44% degenerated oocytes), 2) the largest follicle per pair of ovaries was < or = 10 mm (63%), and 3) the mare was pregnant (66%). These results were probably related to the reported high frequency of atretic follicles in the 5- to 10-mm population. In summary, oocytes from individual follicles < or = 10 mm or from follicles in which the largest follicle per mare was < or = 10 mm were the poorest candidates for in vitro maturation.