This study was conducted to evaluate the effects of different volume (100 μl vs. 2 ml) of microdrop culture on B6D2F1 mice oogenesis. In the present study, B6D2F1/CrljOri F1 mice were utilized in order to maximize oogenesis. Also we used TCM-199, Dulbecco's medified Eagle's medium (DMEM), embryo culture medium (Fertilization medium, Cleavage medium, Blastocyst medium), G series medium and One step medium. Blastulation rate was not different between groups (58.4±2.9% vs. 61.2±4.8%). Zona hatched rate (38±15.4% vs. 27±3.4%) and attached rate (55±13.9% vs. 46±3.9%) did not differ by the volume of culture media. Total cell numbers (59.8±9.7 vs. 70.3±8.7), ICM cell numbers (15.8±0.6 vs. 16.8±1.5), TE cell numbers (44.0±9.7 vs. 53.6±7.3), % ICM (26.4±2.9% vs. 23.8±3.3%) and ICM:TE ratio (1: 2.8±0.4 vs. 1: 3.2±0.6) were not different between groups (i.e., 100 μl vs. 2 ml). These results show that the capacity of the culture medium did not effect the cell numbers of B6D2F1 mice blastocysts. In summary, these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice.

This study was conducted to evaluate the effects of type of culture media (BM, G2, OS, TCM, and MEM) on B6D2F1 mice oogenesis. In the present study, B6D2F1/CrljOri F1 mice were utilized in order to maximize oogenesis. Also we used TCM-199, Dulbecco's medified Eagle's medium (DMEM), embryo culture medium (Fertilization medium, Cleavage medium, Blastocyst medium), G series medium and One step medium. In vitro maturation was highest in BM followed by the order of OS, MEM, TCM and G2 (90±2.8% > 88±3.2% > 85±4.9% > 78±10.2% > 64±7.7%, respectively). To note, the G2 group was statistically different compared to other groups (p<0.05). On the other hand the fertilization rate was highest in the G2 group followed by BM, OS, TCM, and MEM (87±7.2% > 85±6.9% > 74±14.0% > 71±13.8% > 2±1.4%, respectively). The MEM group was significantly lower compared to other groups (p<0.05). The developmental rate was highest in the OS group followed by the G2 group and the BM group albeit no statistical significance was noted (73±11.6% > 71±9.2% > 66±10.4%). Of note, all cells of the TCM and MEM groups were died during embryonic development. The zona hatched rate (51±9.8% vs. 50±9.1% vs. 47±7.2% for BM, G2, and OS respectively) and attached rate (45±12.3% vs. 38±16.1% vs. 37±11.5% for BM, G2, and OS respectively) were not different amongst groups. No difference was found in total cell numbers (74±13.9 vs. 64±9.2 vs. 76±6.7 for BM, G2, and OS respectively), ICM cell numbers (20±1.9 vs. 14±1.8 vs. 15±2.1), TE cell numbers (55±12.5 vs. 49±10.7 vs. 61±5.9), % ICM (30±2.8% vs. 24±7.0% vs. 22.8±2.2%) and ICM:TE ratio (1:2±0.5 vs. 1:3.1±0.8 vs. 1:3.1 ±0.5) amongst groups. In summary, these results can provide fundamental data to maximize culture condition for in vitro fertilization on B6D2F1 mice.

본 연구는 인간 난관액 또는 자궁액 내에 존재하는 에너지원이 생쥐 2-세포기 배의 체외 발달에 미치는 영향을 조사하기 위하여 실시하였다. ICR 암 생쥐에 5 IU hCG 주사 후 46～50시간에 2-세포기 배를 회수하였다. 회수된 배는 3가지 배양 조건 [대조군: 0 mM, Group A: glucose(G) 0.5 mM + pyruvate(P) 0.32 mM + lactate(L) 10.5 mM, Group B: G 3.15 mM + P 0.1 mM + L 5.83 mM]에서 72시간 배양하였다. 배양 24 시간에 상실배 출현율은 group A (72.3%)와 group B (56.6%)가 대조군(34.9%)보다 유의하게 높았다(p<0.05). 그러나 48시간에 배반포기 배 출현율은 대조군(51.8%)이 group A (39.8%)와 group B (28.9%)보다 유의하게 (p<0.05) 높았다. 72시간에 투명대 부착 (ZiB, 41.0～51.8%), 투명대 탈출 (ZeB, 18.1～32.5%) 및 총 배반포기 배 출현율 (68.7～73.5%)은 실험군 간에 통계적인 차이가 없었다. 배반포기 배의 평균 세포수와 ICM 세포수는 group A (70.8, 13.4)와 group B (64.4, 11.8)가 대조군 (53.1, 5.7)보다 유의하게(p<0.05) 많았고, 통계적인 유의차는 없었으나 group A가 group B보다 많은 경향이었다. 총 세포수에 대한 ICM 비율은 group A(22.9%)와 group B(23.7%)가 대조군(14.2%)보다 유의하게(p<0.05) 높았다. 영양배엽(TE) 세포수(34.1～45.1)는 실험군 간에 통계적인 차이가 없었다. ICM에 대한 TE 비율(ICM:TE ratio)은 대조군(1:6.0)이 group A(1:3.4)나 group B(1:3.4)보다 유의하게(p<0.05) 높았다. 생쥐 2-세포기 배를 배양하여 72시간까지의 배 발달율을 살펴보면 배양액에 에너지원을 첨가하는 것이 효과적이었으며, 자궁액 농도보다는 난관액 농도로 에너지원을 조절했을 때 배 발생 능력이 높은 경향을 보였다.

This research was conducted to investigate the interrelationship among methods of injection of PMSG-hCG to the number of ovulated eggs, percentage of matured oocytes and in vitro fertilization using out-bred ICR mice. The results obtained are as follows, 1) The optimurn dose was 5 IU for both PMSG and hCG, while the number of ovulated eggs was 428, percentage of M II was 73% and in vitro fertilization rate was 81 %. 2) The optimum injection interval of PMSG-hCG was 48 hours, while the number of ovulated eggs was 48 8, percentage of M II was 80% and in vitro fertilization rate was 81%. 3) The optimum time for collecting eggs was between 16 and 18 hours after hCG injection, while the numbers of ovulated eggs were 448, 427 and 437 in 14,16 and 18 hours after hCG injection respectively, and percentages of M II were 79 and 81 %, and in vitro fertilization rates were 81 and 80% in 16 and 18 hours after hCG injection, respectively. 4) The repeat of superovulation decreased with the number of ovulated eggs, percentage of M II and in vitro fertilization rate, than in control. But it was recovered by increasing the repeat interval.