猪卵母细胞冷冻保存研究

【目的】本研究试图通过比较猪卵母细胞超低温冷冻保存方法、冷冻承载工具、冷冻卵母细胞的类型，从而有效地保存猪卵母细胞;【方法】利用屠宰场卵巢采集的卵母细胞，以台盼蓝染色、二乙酸荧光素（FDA）染色鉴定卵母细胞冷冻后的成活率，以体外成熟和体外受精鉴定卵母细胞冷冻后的发育能力，研究了不同冷冻方法和不同冷冻保护剂对猪卵母细胞的冷冻效果。【结果】（1）程序化法冷冻保存中，9%乙二醇（EG），10%二甲基亚砜（DMSO），10%甘油（Gly）均对猪MII期卵母细胞有冷冻保护作用，极显著高于对照组（FDA染色成活率33.8%，25.8%，23.5% vs 2.5%，P <0.01），且以9%EG的效果最好，显著高于另外两组（ 33.8% vs 25.8%，23.5%, P <0.05）。（2）玻璃微细管（GMP）法是猪卵母细胞超低温冷冻的较好方法，以普通的麦管（Straw）法进行的程序化冷冻为对照组，GMP管法显著提高猪卵母细胞的冷冻成活率（分别为63.3%和34.5%，P<0.05）。（3）在玻璃化冷冻方法中，不同的冷冻液载体对猪卵母细胞冷冻成活率有影响。以EFS40为冷冻液，Straw和GMP管作冷冻液载体，卵母细胞的成活率分别为45.0%和65.9%，二者差异显著（P <0.05）。（4）用Straw的程序化冷冻法和用GMP管的玻璃化冷冻法对猪GV期卵母细胞冷冻均有效，但二者差异显著（成活率分别为30.0%和59.7%，P<0.05）。冷冻后继续培养，分别有2.8%和6.3%的卵母细胞周围颗粒层发生扩散。（5）冷冻对MII期卵母细胞的发育潜能影响较大，用新鲜精液使其受精，仅有4.9%的受精卵分裂，极显著低于对照组的49.5%（P<0.01）；发育至4-细胞期的比例为1.7%，但未能发育至8-细胞期以上胚胎。【结论】选用MⅡ期卵母细胞、以GMP管为冷冻承载工具、应用玻璃化冷冻方法能够较好地保存猪卵母细胞，为进一步完善猪卵母细胞超低温冷冻保存技术体系提供了参考依据。

Studies on Cryopreservation of Porcine Oocyte

【Objective】The aim of the present study was to try to cryopreserve porcine oocytes efficiently, and to investigate the effect of cryopreservation method, cryopreservation tool and types of cryoprotectant on pig oocyte suvival and its in vitro maturation and cleavage following IVF and IVC. 【Method】Experiments in pig oocyte cryopreservation and in vitro fertilization（IVF） were conducted using oocytes collected from a slaughterhouse. The effects of different methods and different cryoprotectant solution on cryopreservation of porcine oocytes were examined. Survival was assessed by trypan blue (TB) staining, fluorescein diacetate (FDA) staining, maturation in vitro and cleavage after IVF. The results were showed as follows: 【Results】(1) Cryoprotectant solution (9%EG, 10%DMSO, 10%Gly) were effective to cryopreserve pig MII oocytes when in programmed freezing. The survival rates（FDA dyeing）of pig oocytes after frozen-thawed were very significantly higher than that of control (33.8%, 25.8%, 23.5% vs 2.5%, P <0.01). Among these three cryoprotectant solution, 9%EG was significantly superior to the other two in survival rate (33.8% vs 25.8%, 23.5%, P<0.05). (2)The freezing method by GMP (glass micropipette) was suitable and efficient to cryopreserve pig oocytes. Compared with programmed freezing method using Straw，GMP method could significantly promote the survival rate of pig oocytes (63.3% and 34.5%, respectively，P <0.05). (3) The vitrification solution carrier had a positive effect on survival rate of pig oocytes. When EFS40 was used as vitrification solution，Straw and GMP led to different survival rates, which were 45.0% and 65.9%, respectively (P <0.05). (4) The programmed freezing method by Straw and vitrification method by GMP were available in cryopreservation of pig oocytes, but these two methods resulted in different survival rates (30.0% and 59.7%, respectively, P <0.05). (5) Cryopreservation had a great effect on the developmental ability of pig oocytes after IVF and IVC. Vitrified MII oocytes were fertilized with fresh spermatozoa and only 4.9% eggs cleaved after 48 culture, which was very significantly lower than the cleavage rate of control(49.5%, P <0.01). The subsequent developmental rate to 4-cell stage was 1.7%, but none to 8-cell stage. 【Conclusion】The cryopreservervation of porcine oocytes with MII oocytes, vitrification and GMP were efficient.