Search

With the ultimate goal of establishing experimental protocols necessary for cloning male Sika deer to gain more pilous antler, the conditions for in vitro maturation of Sika deer oocytes were examined. Sika deer ovaries were collected from Slaughterhouse in oestrus season (October and November), placed into saline (25°C) supplemented with 1% (v/v) penicillin and streptomycin, and transported into the laboratory within 4h. The small vesicular follicles (diameter, 2-6 mm) on the ovarian surface were incised with a scalpel in a Petri dish containing mPBS (Dulbecco PBS supplemented with 0.1% [w/v] D-glucose, 36 mg/L of pyruvate, and 0.4% [w/v] BSA) to release the cumulus-oocyte complexes (COCs). The COCs with uniform cytoplasm and several layers of cumulus cells were selected, divided into black color (more lipid drops) oocytes and white color (little lipid drops) oocytes (more black color oocytes and less white color oocytes were collected), and cultured in two types of media: medium 1: bicarbonate-buffered medium TCM199 with 25 mM NaHCO3, 0.2 mM pyruvate, 0.086 mM kanamycin monosulfate, 10% (v/v) FCS, 10 µg/ml porcine FSH, 1µg/ml bovine LH, 1 µg/ml 17-β-estradiol, and 0.1 mM cysteamine), and medium 2: medium 1 without 17-β-estradiol, for 20, 24, 28 or 48h. In black color oocytes, after 28h of maturation in vitro, the maturation rate (63%, n=43) of cultured in medium 1 was significantly greater (P<0.01) than those of oocytes cultured in vitro for 20h (44%, n=34) and 24h (40%, n=30). And it (63%, n=43) was also higher than that of oocytes cultured in vitro for 48h (53%, n=53), but no significant difference was observed. Same trend was seen in black color oocytes when cultured in medium 2. The maturation rate at 28h (68%, n=37) was higher than those of oocytes cultured in vitro for 20h (53%, n=43), 24h (54%, n=38) and 48h (57%, n=47), but no significant difference was observed among them. In white color oocytes, the maturation rates of oocytes cultured in medium 1 were 17% (n=32) at 28h and 11% (n=38) at 48h. No matured oocytes cultured in vitro for 20h and 24h were seen. Same situation was seen in white color oocytes when cultured in medium 2, the difference was that 6% (n=38) oocytes cultured in vitro for 24h was matured. In this study, we also find that when COCs were cultured in medium 2 (without 17-β-estradiol), their cumulus cells expanded slightly faster and oocytes matured better than those cultured in medium 1. Our results indicate that (i) the best maturation condition of black color oocytes in Sika deer is achieved at 28h by medium 2; (ii) medium 2 (without 17-β-estradiol) is slightly better than medium 1 for in vitro maturation of Sika deer oocytes; (iii) the maturation rate of black color oocytes is much higher than that of white color oocytes when cultured in same medium and same time. These results have begun to elucidate parameters important for animal modeling and cloning with the Sika deer, should facilitate the development of genetically defined animal models in this species.*This work was supported by the National Nature Science Foundation of China (No. 30671510)