蓝狐卵母细胞的体内外成熟

Study on the Maturation in vitro of Blue Fox Oocyte

对蓝狐卵母细胞成熟时间和超数排卵后卵母细胞的发育阶段进行了研究。采集间情期 (11～ 12月份 )蓝狐卵巢 ,用切割法回收卵泡中的卵母细胞 ,每个卵巢平均获得 8个卵丘卵母细胞复合体 (COCs:cumulus oocyte complexes,480个卵 / 6 0个卵巢 )。COCs在成熟液 (TCMI99+10 %FCS+10 μg/ L EGF+10 IU/ m L PMSG+10 IU/ m L h CG)培养 ,并在不同时间将卵母细胞固定、染色观察其成熟阶段。结果表明 ,核网期卵母细胞在培养前的比例为 80 .70 %(P<0 .0 1) ;GV期卵母细胞在培养 2 4h比例最高 (34 .0 4%,P<0 .0 1) ;GVBD期卵母细胞也是在培养 2 4h比例较高(4 4.6 8%,P<0 .0 5 ) ;M 期卵母细胞在培养 48～ 12 0 h比例较高 (P<0 .0 5 ) ,48h最高 (30 .0 0 %) ;M 期卵细胞在培养 72～ 96 h比例较高 (P<0 .0 5 ) ,72 h比例最高 (2 9.41%) ;卵母细胞在培养 12 0 h退化比例最高 (2 2 .2 2 %,P<0 .0 1)。因此 ,间情期卵母细胞体外培养 72～ 96 h为最佳。对繁殖季节 (3月份 )蓝狐进行超排处理 ,回收卵巢卵泡中卵母细胞及输卵管、子宫角的卵母细胞 ,固定、染色 ,观察其所处的发育阶段。母狐皮下注射 5 0 0 IU PMSG,48h后皮下注射 2 5 0 IU h CG,一组在 72 h后屠宰 ,结果表明 ,母狐卵巢体积虽然有所增大 ,但没有排卵 。

The present study concentrated on the blue fox oocyte maturation time in vitro and its maturation stage after superovulatio n.Cumulus oocyte complexes(COCs) without visible follicle were collected from ov aries in anoestrus season.About 8 COCs were collected per ovary by slicing methe d.COCs were cultured in TCMI99+10% FCS+10 μg/L EGF+10 IU/mL PMSG+10 IU/mL hCG, t hen fixed and dyed to observe the maturation stage in different maturation time. About 80.70% oocytes were in dictyotene stage(P<0.01) before cultivation.T he rate of GV stage was 34.04%(P<0.01) after 24 h cultivation while the rat e of GVBD stage was 44.68%(P<0.05).The MⅠ stage oocytes were observed from 48 to 120 h cultivation(P<0.05) and the highest rate of MⅠ oocyte was in 48 h(30.00%).The MⅡ stage oocytes were observed from 72 to 96 h(P<0.05),an d the highest rate were observed in 72 h(29.41%).The highest degeneration rate a p peared after 120 h cultivation was 22.22%(P<0.01).Therefore,the optimum ma turat ion time for fox foliicle oocytes in anoestrus season was from 72 h to 96 h.Superovulation treatment of blue fox in reproductive season(March) was administr ated.COCs were collected from visible follicles,oviduct and uterine horn.The fem ale foxes were injected 500 IU PMSG,and 250 IU hCG 48 h later respectively.On e was killed 72 h later and the ovaries were teken out.The volume of ovaries in creased and the follicles began to develop without ovulation.The oocytes were fi xed and dyed and it was found that most of the oocytes were in dictyotene stage( 72.22%),a few were in GV(11.11%),GVBD(11.11%) or MⅠ(5.56%) stage.The other fox diplayed estrus 144 h later and injected with 500 IU PMSG(148 h) and 250 IU hC G(167 h) again.Artificial insemination was carried out after hCG injection.This fox was killed 187 h later.Thirteen COCs were collected from oviduct and sixte en COCs from uterine horns.Oocytes were surrounded several many layers of cumulu s cells and more lipid appeared in cytoplasms.Most of the oocytes from visible f ollicles were in dicotyotene stage(33.33%) and GV stage(41.67%),a few were in GV BD(16.67%) or MⅠ(8.33%) stage.The oocytes from ovaries were in GV stage(100%) .T he ovulated oocytes reached uterine horn at the same day,most of them were in GV ,GVBD or MⅠ stage.The oocytes collected from uterine horn were cultured for 72 h in vitro,but they were still in GV,GVBD or MⅠ stage.It is concluded th at superovulation treatment should be done after hormone induction or natural es trous,and the ovulated oocytes get to uterine horns quickly.