Development to blastocysts of domestic cat oocytes matured and fertilized in vitro after prolonged cold storage

Abstract

Four experiments determined the kinetics of in vitro maturation and fertilization of cat oocytes and the effects of prolonged cold storage of ovaries before oocyte recovery on in vitro maturation/in vitro fertilization (IVM/IVF) success. Domestic cat ovaries were collected at ovariohysterectomy and stored at 4°C in PBS until oocyte recovery and culture in Eagle's minimal essential medium (EMEM) containing FSH, LH, oestradiol and BSA for maturation. In Expt 1, meiotic maturation was assessed at 0, 12, 24, 38 and 48 h of culture. After 24 h, > 61% of oocytes were in telophase I or metaphase II. In Expt 2, oocytes were recovered from ovaries stored for 24, 48 or 72 h and cultured in EMEM for 24 h. There was no difference among groups (P > 0.05) in the ability to achieve nuclear maturation (mean ± SEM, 57.1 ± 5.3%, 60.4 ± 5.4%, 55.4 ± 15.1% for 24, 48 and 72 h, respectively). Fertilization and embryo development after insemination at 16, 24, 32, 40 and 48 h of culture were examined in Expt 3. Of 98 oocytes inseminated at 32 h, 69% cleaved, 59% developed into morulae and 13% into blastocysts, more (P < 0.05) than those oocytes inseminated at earlier and later times. Development to blastocysts occurred after insemination at 16 (1.2%), 24 (9.1%) and 32 (13.3%) h of culture, but not after insemination at 40 or 48 h. Expt 4 involved cold storage of ovaries for 24, 48 or 72 h before oocyte recovery and insemination at 32 h of culture (the optimal time measured in Expt 3). Compared with storage for 24 h, fertilization success was lower (P < 0.05) in the 48 and 72 h groups, and, although 9.1% of inseminated oocytes from the 24 h storage group developed to blastocysts, none (P < 0.05) achieved this stage after 48 or 72 h of storage. These results indicate that domestic cat oocytes reach nuclear maturity by 24 h in culture and can be fertilized and develop to blastocyts optimally after insemination at 32 h. Oocytes recovered from ovaries stored at 4°C for up to 72 h are capable of reaching telophase I or metaphase II in vitro. However, only oocytes stored within the ovary for 24 h produce blastocysts, indicating that the ability to achieve nuclear maturation is an inadequate indicator of fertilization and developmental competence.