SELECTION OF FOLLICLES, PRECULTURE OOCYTE EVALUATION, AND DURATION OFCULTURE FOR IN-VITRO MATURATION OF EQUINE OOCYTES

Equine oocytes (n=537) were collected from slaughterhouse ovaries (n=1 18 mares) by scraping the internal follicular wall. Preculture record was made of the appearance of oocyte investments (no cumulus, corona r adiata only, compact cumulus, expanded cumulus), appearance of cytopla sm (homogeneous, condensed heterogeneous/fragmented), and nuclear matu ration stages (germinal vesicle, germinal-vesicle breakdown, metaphase I, metaphase II, degenerated). There was no difference between follic les >30 mm and follicles less than or equal to 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 fr om follicles 5 to 30 mm were cultured in modified TCM-199 for 18, 24, 36 and 48 h. Postculture nuclear maturation classifications were immat ure (germinal vesicle, germinal-vesicle breakdown, and metaphase I), m ature (metaphase II or secondary oocyte), and degenerated. The frequen cy distribution of oocytes among the 3 postculture maturation classifi cations 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 post culture results was the heterogeneous/fragmentation group which had a high proportion of postculture degenerated oocytes (67%); however, onl y 4% of oocytes were in this group. Luteal status of the mare had an e ffect (P<0.05) on the frequencies of the maturation classifications, b ut not enough to be useful in selecting oocytes. Consistency of the fo llicle and the type of oocyte investment did not alter significantly t he maturation frequencies. The frequency of degenerated oocytes after culture was high under the following conditions: 1) diameter of the fo llicle from which the oocyte was selected was 5 to 10 mm (44% degenera ted oocytes), 2) the largest follicle per pair of ovaries was less tha n or equal to 10 mm (63%), and 3) the mare was pregnant (66%). These r esults were probably related to the reported high frequency of atretic follicles in the 5- to 10-mm population. In summary, oocytes from ind ividual follicles less than or equal to 10 mm or from follicles in whi ch the largest follicle per mare was less than or equal to 10 mm were the poorest candidates for in vitro maturation.