The present study evaluated nuclear maturation after in vitro culture of oocytes originated from estrous and anestrous bitches. Two culture media were used, TCM 199 (tissue culture medium 199) and synthetic oviduct fluid (SOF). The females were divided in two groups. Group 1 consisted of 11 anestrous bitches, and Group 2 consisted of 6 estrous bitches. In each group, oocytes were harvested from sliced ovaries, and onehalf of them were immediately stained with Hoechst 33342. The oocytes were classified as in the GV (germinal vesicle) phase, GVBD (germinal vesicle breakdown) phase, MI (metaphase I), or as D/NI (degenerated/non identifiable). The other half of the oocytes were matured in SOF or TCM media for 24 hours, stained, and evaluated as previously described. There were 511 and 373 Grade I oocytes recovered in Groups 1 and 2, respectively. The mean number of oocytes obtained per anestrous bitch was 46.5 ± 25.2. For females in Group 2 (estrous), 62.2 ± 8.2 oocytes were obtained per bitch. Oocytes from estrous and anestrous bitches submitted to in vitro maturation had similar rates of GVBD (21.7%, TCM 199 and 23.6%, SOF – anestrous; 20%, TCM 199 and 23%, SOF - estrous). However, the MI rate was higher in the estrous group (9.8%, TCM 199 and 9.7%, SOF – anestrous; 25%, TCM 199 and 58%, SOF – estrous). The protocol that utilized estrous donor’s oocytes and in vitro maturation in SOF was superior to the other protocol tested, demonstrating that the association between estrous versus anestorus and maturation medium is beneficial for oocyte in vitro maturation (58% MI). It was demonstrated that 24 hours of in vitro maturation was insufficient for obtaining Metaphase II nuclear configurations in oocytes with all of the protocols tested.