猪卵泡卵母细胞体外成熟与冷冻保存  

In vitro Maturation and Cryopreservation of the Porcine follicular Oocytes

作　　者：王锋[1] 蔡令波[1] 顾熟琴[1] 杭苏琴[1] 牛树理[1] 

机构地区：[1]南京农业大学动物胚胎工程技术中心,江苏南京210095

出　　处：《中国兽医学报》2004年第6期605-608,共4页Chinese Journal of Veterinary Science

基　　金：江苏省自然科学基金资助项目 ( BK2 0 0 3 0 75 )

摘　　要：研究了激素、猪卵泡液、不同类型血清对猪卵泡卵母细胞体外成熟的影响 ;比较了卵泡直径小于 2 mm、2～ 5 mm和大于 5 mm的卵母细胞体外成熟能力的差异 ,并对不同发育阶段猪卵母细胞的冷冻保存进行了研究。结果表明 :猪卵母细胞体外培养 4 8h时 ,培养的前 2 4 h培养液中加入激素 ,后 2 4 h去掉激素 ,卵母细胞的 A级成熟率 (5 1.73% )和总成熟率 (83.2 5 % )最高 ,极显著高于前 2 4 h不加激素 ,后 2 4 h添加激素培养的成熟率 (P<0 .0 1) ;也显著高于不含激素的培养液连续培养 4 8h的成熟率 (P<0 .0 5 ) ;但与添加激素连续培养 4 8h的成熟率差异不显著 (P>0 .0 5 )。在体外成熟培养液中 ,添加 10 % (体积分数 ) ECS的成熟率 (72 .86 % )显著高于添加 10 % (体积分数 ) NCS的成熟率(6 2 .2 1% ) (P<0 .0 5 ) ,而添加 10 % p FF则抑制卵母细胞的体外成熟。随着卵泡直径的增大 ,卵母细胞体外成熟能力逐渐增强。采用程序冷冻保存方法 ,成熟卵母细胞的成活率 (35 .5 9% )显著高于培养前 (2 4 .6 4 % )和培养 2 4 h(2 3.36 % ) (P<0 .0 5 )。培养 2 4 h(77.2 2 % )和培养成熟 (72 .81% )的卵母细胞解冻后的形态完整率均显著高于培养前(5 3.2 4 % ) (P<0 .0 5 )Factors affecting oocytes IVM,such as different type of serums,hormone,follicular fluid and size of follicle were studied.Cryopreservation of oocytes in different development phase was also studied in this paper.The results showed that when oocytes maturation for 48 h in vitro,removal of hormone supplements from mature media at 24 h after culture enhance oocytes maturation and cumulus expansion.And the maturation rate(83.25%) was very significant higher than oocytes that culture for 24 h in the media without hormone subsequent added hormone for another 24 h(63.94%)(P<0.01) and also higher than oocytes culture for 48 h in media without hormones(60.01%)(P<0.05).The maturation rate of IVM was very significantly higher in the culture medium contain 10% ECS(72.86%) than 10% NCS(62.21%)(P<0.05).Addition of 10% porcine follicular fluid(pFF) to mature medium inhabited the in vitro maturation of oocytes.Oocytes from different size of follicular had different development ability.Oocytes collected from big(φ>5 mm) and medium(φ2-5 mm) follicle had a higher maturation rate than oocytes from little(φ<2 mm) follicle(P<0.05).Oocytes in MⅡ stage had higher antifrreze ability than GV stage and oocytes culture for 24 h.The survival rate of oocytes after cryopreservation and thawing was 35.59%,significant higher than oocytes cryopreservation on GV stages(24.64%) and culture for 24 h(23.36%)(P<0.05).The rate of morphological normality after thawing was significantly lower in group of GV stages(53.24%) than in group of MⅡ stage(72.81%) and culture for 24 h(77.22%)(P<0.05).

关 键 词：猪 卵泡 卵母细胞 体外成熟 冷冻保存 

分 类 号：S828[农业科学—畜牧学]