Effects of serum and follicular fluid on the in vitro maturation of canine oocytes.
The effects of gonadotropin, serum and follicular fluid on the in vitro maturation of canine oocytes were examined. Additionally, spindle size and spindle migration in MI-stage oocytes derived by in vivo or in vitro maturation were evaluated for the first time. Mature oocytes collected from beagle dog ovaries were divided into two experiments. In experiment I, oocytes were cultured in basic TCM 199 medium supplemented with different levels of P4, E2 and FSH. In experiment II, oocytes in the estrus or anestrus stage were cultured in basic medium supplemented with 30% or 40% canine serum plus 20% or 10% follicular fluid. Our results showed that in experiment I, more oocytes reached MI-MII (18.57%) after supplementation with 1 IU/ml FSH+ 5 IU/ml P4 + 5 IU/ml E2 than after supplementation with other levels of reagents. However, there were no significant differences among the groups (three different concentration groups and a control group) with respect to the proportions of oocytes that resumed meiosis, completed meiosis or degenerated. In experiment II, the number of oocytes from the estrus stage that reached MI-MII in TCM 199 medium supplemented with 40% canine serum and 10% follicular fluid (46.72%) was significantly higher (p < 0.01) than the number of oocytes from the anestrus stage that reached MI-MII in medium supplemented with 30% canine serum and 20% follicular fluid (21.84%). In addition, the degeneration rate was significantly lower (p < 0.05) in the 40% canine serum/10% follicular fluid group from follicular stage than in the other three groups. The average spindle length of the MI-stage oocytes that matured in vivo was significantly (p < 0.01) longer than that of the MI-stage oocytes that matured in vitro (21.75 vs. 14.39 μm). These results suggest that supplementation of the culture medium with 40% estrus serum and 10% follicular fluid had a positive influence on the in vitro maturation of canine oocytes and greatly affected spindle size in MI-stage oocytes.