Abstract: In the present study we investigated the effects of supplementing the in vitro maturation (IVM) medium with insulin on the incidence of maturation to MⅡ. In order to provide parameters for canine oocyte IVM system, cumulus oocyte complexes (COCs) were released by slicing; oocytes were cultured in TCM199 supplemented with 0.6% gulocse and different concentrations of insulin (0, 3, 6, or 9 IU/mL) at 38.5 ℃, 5% CO₂ in air. Observed cumulus cells which were removed from around oocytes using a small glass pipette, denuded oocytes were fixed in then Hoechst 33342 at room temperature for 15 min. Nuclear status was observed under UV light using a fluorescence microscope. Longer culture time to 72 and 96 hours, then statistical analysis by SPSS 14.0. There is no significant different on cumulus expansion. No oocyte development to MⅡbut the percentage of oocytes at GVBD stage was different among the groups 6 IU/mL, and the control. The percentage of oocytes get to M Ⅱin the 72 and 96 h treatment groups with 6 IU/mL insulin groups 20.8%±1.9% vs 13.33%±1.5%, respectively. These results indicated that the addition of insulin to the in vitro maturation medium of canine oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect cumulus expansion, however, the maturation rate of oocyte increased by delayed culture period.

Key words: canine; oocyte;  in vitro maturation; insulin