초록
본 연구는 mTCM-199, mWaymouth MB 752/1 그리고 NCSU-23 성숙배지에서 성숙시킨 난포란을 mTBM 수정배지에서 액상정액의 정자를 이용하여 주입정자 농도별로 체외 수정시킴으로써 액상정액을 이용한 새로운 체외수정 방법을 개발하고자 실시하였다. 미성숙 난포란은 0.5 $m\ell$의 성숙배지에 각 well 당 30~40개씩 적하하였다. 액상정액 제조용 정액은 90% 이상의 운동성을 가진 농후정자부분을 사용하였으며 정액채취 후 2시간 동안에 22~24$^{\circ}C$의 실온까지 냉각시켰다. 실온까지 냉각한 정액은 BTS 희석액으로 2$\times$$10^{8}$ $m\ell$ 정자농도로 조정하여 100$m\ell$ 플라스틱병에 30 $m\ell$씩 주입하여 17$^{\circ}C$에서 5일 보관하였다. 5일 보관후 운동성이 70% 이상인 정자를 체외수정에 이용하였다. 38.5$^{\circ}C$, 5% $CO_2$, 95% 공기로 조절된 CO2 배양기에서 44시간 성숙 후 cumulus cell들이 제거된 성숙 난포란은 0.5 $m\ell$의 mTBM 수정배지에 30~40개씩 적하하고 최종정자농도를 1, 2, 4, 6 그리고 10$\times$$10^{6}$$m\ell$되도록하여 주입하고 6시간 동안 수정시켰다. 체외수정시킨 수정란들은 수정 후 6시간 동안 0.5$m\ell$의 NCSU-23 배양배지에서 배양한 후 정자침입율, 다정자침입율 그리고 웅성전핵 형성율을 조사하였다. mTCM-199, mWaymouth MB 752/1 그리고 NCSU23 성숙배지에서 성숙시킨 난포란을 mTBM 수정배지에서 액상정 액으로 체외수정 한 결과 NCSU-23 성숙배지에서 성숙한 난포란이 웅성전핵형성율이 높았고 다정자침입율이 낮았다. NCSU-23 성숙배지에서 성숙한 난포란을 mTBM 수정배지에서 수정할 경우 최적정자농도는 2~4$\times$$10^{6}$$m\ell$이었으며, 정자침입율은 50.8~50.9%, 다정자침입율은 13.3~19.5% 그리고 웅성전핵형 성율은 43.9~45.4%였다. 결론적으로 NCSU-23 성숙배지에서 성숙되고 mTBM 수정배지에서 수정된 난포란은 mTCM-199이나 mWaymouth MB 752/1 성숙배지에서 성숙되고 mTBM 수정배지에서 수정된 난포란보다 우수한 체외수정 결과를 나타내었다. 17$^{\circ}C$에서 5일 동안 보존한 액상정액으로 NCSU-23 배지에서 성숙한 난포란을 체외수정하기 위한 최적정자농도는 2~4$\times$$10^{6}$$m\ell$이었다.
The present study was carried out to investigate the effects of maturation media on penetrability of pig oocytes by liquid boar sperm coincubated with different sperm concentrations in a modified Trisbuffered medium (mTBM). Follicular oocytes collected from ovaries of prepubertal gilts were matured in a modified TCM-199 (mTCM-199) medium, modified Waymouth MB 752/1 (mWaymouth MB 752/1) medium or NCSU-23 medium. Oocytes (30~40) were transferred into each well of a Nunc 4-well multidish containing 0.5 $m\ell$ maturation medium. The spermich portion of ejaculates with greater than 90% motile sperm were used in the experiment. The semen was cooled 22 to 24$^{\circ}C$ over 2 h period. The semen was diluted with Beltsville Thawing Solution (BTS) extender at room temperature to give 2$\times$10$^{8}$ sperm/$m\ell$ in 100 $m\ell$ plastic bottle. Liquid boar semen of 30 $m\ell$ in 100 $m\ell$ plastic bottle was kept at 17$^{\circ}C$ far 5 days. The sperm with greater than 70% motility after day 5 of storage were used for in-vitro fertilization (IVF). After 44 h maturation of immature oocytes in 5% $CO_2$in air at 38.5$^{\circ}C$, cumulus cells were removed and oocytes (30~40) were coincubated for 6 h in 0.5 $m\ell$ mTBM fertilization medium with five different (1$\times$10$^{6}$ , 2$\times$10$^{6}$ , 4$\times$10$^{6}$ , 6$\times$10$^{6}$, 10$\times$10$^{6}$ $m\ell$) sperm concentrations. At 6 h after IVF, oocytes were transferred into 0.5 $m\ell$ NCSU-23 culture medium fur further culture of 6 h. At 12 h after IVF, sperm penetration, polyspermy and male pronuclear formation of oocytes were evaluated. Oocytes of NCSU-23 maturation medium decreased polyspermy and increased male pronuclear formation compared to those of mTCM199 and mWaymouth MB 752/1 maturation media. Of oocytes matured in NCSU-23 medium and inseminated in mTBM medium with 2~4$\times$10$^{6}$ $m\ell$ sperm concentrations, 50.8~50.9% showed sperm penetration, 13.3~19.5% polyspermy and 43.9~45.4% male pronuclear formation. In conclusion, we found out that oocytes matured in NCSU23 medium and inseminated in mTBM medium showed superior invitro fertilization compared to those matured in mTCM199 and mWaymouth MB 752/1 maturation media and inseminated in mTBM medium. The optimum sperm concentrations for in-vitro fertilization of oocytes matured in NCSU-23 medium by liquid boar semen stored at 17$^{\circ}C$ for 5 days were 2~4$\times$10$^{6}$ $m\ell$.
