This research is composed of three experiments: The influence of9-cis-retinoic acid on nuclear and cytoplasmic maturation and gene expression in canine oocytes during in vitro maturationRetinoids have important roles in the regulation of oocyte nuclear and cytoplasmic maturation. The present study investigated the effects of a retinoid metabolite on nuclear maturation, cytoplasmic maturation, and gene expression in canine oocytes during in vitro maturation IVM. COCs were harvested from ovaries by slicing. Only oocytes that were>120μm in diameter, with a homogeneous dark cytoplasm and three or more layers of compact cumulus cells were used. Varying concentrations of9-cis-RA (0,5,50, and500nM) were included in the maturation medium, and the following were measured:(ⅰ) oocyte nuclear maturation after culture for48h;(ⅱ) cytoplasmic granular migration by labeling of oocytes with fluorescein isothiocyanate labeled lectins; and (ⅲ) relative expression of genes related to apoptosis (BAX and Bcl2) in cumulus cells detached from oocytes, by semi-quantitative reverse transcriptase-polymerase chain reaction. After48h culture with IVM, the highest percentage of oocytes that had developed to the MII stage were in the5nM9-cis-RA treatment group (18.3±2.5%; P<0.05). Complete granular migration was observed in oocytes matured with5nM9-cis-RA, consistent with a commensurate gain in developmental competence. Treatment with5nM9-cis-RA had no effect on Bcl2gene expression, but down-regulated BAX expression. In conclusion, since5nM9-cis-RA was beneficial to nuclear and cytoplasmic maturation of canine oocytes, we inferred an important role for9-cis-RA during IVM.Influence of ovarian cortex cells co-culture during in vitro maturation on meiotic resumption of canine oocytesThe present study was carried out to evaluate the effects of COCCs monolayers co-culture on the meiotic resumption competence of canine immature oocytes during IVM. COCs were harvested from ovaries by slicing and evaluated under stereomicroscope. Only oocytes that were larger than120μ m in diameter, with a homogeneous dark cytoplasm and more three layers of compact cumulus cells were used and randomly allocated into two treatment groups and cultured for48h with the following culture systems:Control group (COCs were cultured in maturation medium); Co-culture group (COCs were co-cultured with COCCs monolayers). The COCCs was cultured in DMEM without hormone until the confluence, and then cultured in maturation medium supplemented with hormone for12h before the oocytes added. After cultured in vitro for48h, meiosis process of the oocytes was examined by Hoechst33342 staining under inverted fluorescence microscope. The proportion of oocytes meiotic resumption (GVBD to MII) cultured in co-culture group (58.2%) was higher than for oocytes cultured in control group. More GVBD developed in the co-culture group than that in the control group; however, the percentage of meiotic progression to MII stage showed no significant difference between the treat group and control group. These results indicate that co-culture with COCCs monolayers during IVM could positively influence the meiotic resumption competence of canine immature oocytes.Canine oocyte collection during cryopreservation of the ovarian tissuesThe aim of the present study was to evaluate the effect of canine ovarian tissues cryopreservation on the survival of oocytes from vitrified-warmed ovarian tissues. The ovarian tissues were harvested from bitch ovaries and cut into small fragments using a scalpel blade and then vitrified using solid-surface vitrification. Oocytes were also collected and vitrified following the procedure for ovarian tissues. After storage in liquid nitrogen and thawing, the vitrified oocytes and the oocytes in vitrified ovarian tissues were recovered and then the viability of the oocytes were examined by PI staining under inverted fluorescence microscope. Although oocytes survival rate in vitrified oocytes group was higher than oocytes collected from vitrified ovarian tissues group, no statistically difference in viability was observed between the two groups (22.7%versus25.3%). These results demonstrate that cryopreserved canine ovarian tissues could preserve canine oocytes and provide an alternative technology for cryopreserved canine oocytes for canine assisted reproductive programs and other studies.