Nuclear maturation of domestic cat ovarian oocytes in vitro

Using the domestic cat as a model for salvaging genetic material from rare Felidae, we collected oocytes from ovarian tissue and placed them in 1 of 3 treatments to observe time-related, meiotic changes of in vitro oocyte maturation. Oocytes obtained from ovaries collected at ovario-hysterectomy were assigned to 1 of 3 treatment groups: 1) modified Krebs-Ringer bicarbonate buffer (mKRB) + 4% BSA and 5 microgram/ml FSH (+FSH, n = 499); 2) mKRB + 4% BSA (-FSH, n = 502); or 3) mKRB + 5% natural estrus cat serum (NE, n = 873). They were placed in the respective media in a 5% CO2 humidified environment at 38 degree C. Beginning at 16 h, oocytes were removed at 4-h intervals through 48 h, and the meiotic status was evaluated by means of cytogenetic analysis. On the basis of chromosomal analysis, each cell was placed into one of the following categories: metaphase 11 (MII); metaphase 1 (MI); pre-MI (germinal vesicle [GV], GV breakdown, or diakinesis); degenerate or unidentifiable. The percentage of oocytes with degenerate chromatin increased over time in all culture treatments, but was always greatest (p < 0.05) in the NE group. In the +FSH and -FSH treatments, the proportion of oocytes with nuclear material reaching MII increased with time in culture to 32 h and was equal to or greater than the proportion of oocytes with pre-MI + MI chromatin at this time interval (-FSH, 55%; +FSH, 38%). After 32 h, the number of -FSH oocytes reaching MII status was not sustained, whereas in the +FSH group, the proportion of ova with MII material remained constant (45-48%, p > 0.05). While MII chromosomes were observed at all times in the NE treatment and peaked at 36 h, levels never became greater than those with pre-MI + MI chromatin, indicating a lack of support for nuclear maturation in this treatment. These results indicate that although oocytes in the -FSH and NE treatments can achieve nuclear maturation in vitro, this process generally is not supported under the described conditions. In contrast, supplementation of culture medium with FSH enhances nuclear maturation and allows MII chromosomes to be maintained throughout the 20-48-h culture interval.