IN-VITRO MATURATION OF DOMESTIC DOG OOCYTES CULTURED IN ADVANCED PREANTRAL AND EARLY ANTRAL FOLLICLES

Initial studies in our laboratory demonstrated that a large proportion of domestic dog advanced preantral (APAN) and early antral (EAN) foll icles contained grown oocytes that had acquired the dense cytoplasmic lipid characteristic of preovulatory oocytes. The objective of this st udy was to assess nuclear maturation of those oocytes after in vitro c ulture. Both APAN and EAN follicles (152 to 886 mu m in diameter) were isolated from ovaries by treatment with collagenase and DNase. The fo llicles were cultured in Dulbecco's Modified Eagle's medium/nutrient m ixture F-12 Ham culture medium supplemented with 20% (v/v) fetal bovin e serum (FBS), 2 mM L-glutamine, 1% (v/v) antibiotic-antimycotic, 1 mu g FSH/ml, 10 IU hCG/ml and 1 mu g estradiol/ml. Within each group (AP AN or EAN), control follicles were not cultured (0 h), and 2 to 12 fol licles per well were incubated under a humidified atmosphere of 5% CO2 in air at 37 degrees C for 24, 48 or 72 h. After 24 h of culture, sig nificantly more (5.3%, 20/374; P<0.05) oocytes from APAN follicles rea ched the metaphase I to metaphase II stages (MI to MII) than the perce ntage of control follicles observed at 0 h (0.9%, 3/318). Continued cu lture resulted in a further increase (P<0.05) in the percentage of ooc ytes reaching MI to MII by 48 h (11.5%, 47/407), which remained unchan ged at 72 h (9.9%, 40/404). The percentage of oocytes from EAN follicl es reaching MI to MII did not significantly increase after 24 h of cul ture. However, there was an increase (P<0.05) by 48 h of culture (8.7% , 11/126), which remained unchanged at 72 h (7.5%, 8/106). These resul ts show that dog oocytes cultured within advanced preantral and early antral follicles in vitro are competent to resume meiosis to the metap hase stage. (C) 1998 by Elsevier Science Inc.