猪卵母细胞超低温冷冻保存与体外受精的研究
摘要
猪卵母细胞由于含有大量的脂滴，冷冻较为困难。通过对猪卵母细胞冷冻保存的研究，探讨建立猪卵母细胞冷冻保存的最佳条件和技术路线，具有重要的理论意义和实用价值。本试验以猪卵母细胞为研究对象，利用屠宰母猪卵巢采集卵母细胞，进行猪卵母细胞超低温冷冻保存和体外受精的研究。在试验中，以台盼蓝染色、FDA染色、体外成熟和体外受精等作为鉴定卵母细胞冻后存活率和发育能力的指标，研究了不同冷冻方法和不同冷冻保护剂对猪卵母细胞冷冻效果的影响；并用冷冻或未经冷冻的卵母细胞和精子进行体外受精，研究了不同培养系统对猪受精卵体外发育的影响。结果表明：
1．使用程序化冷冻法，三种冷冻保护剂EG、DMSO、Gly对猪卵母细胞均有冷冻保护作用，极显著高于对照组(冻后存活率33.8％，25.8％，23.5％ vs 2.5％，P＜0.01)；在该三种冷冻保护剂中，EG的效果最好，其冻后存活率显著高于另外两组(33.8％ vs 25.8％，23.5％，P＜0.05)。
2．采用自制的GMP管冷冻猪卵母细胞，证明所用GMP法是一种冷冻猪卵母细胞的有效方法，与普通麦管法相比，该法能显著提高猪MⅡ期卵母细胞冻后存活率(34.5％ vs 63.3％，P＜0.05)。
3．在玻璃化冷冻中，以EFS40为冷冻液，分别使用麦管和GMP管作冷冻载体，卵母细胞冻后存活率分别为45.0％和65.9％，两组间差异显著(P＜0.05)。
4．用程序化法和玻璃化法冷冻猪GV期卵母细胞，两组的冻后存活率分别为30.0％和59.7％，差异显著(P＜0.05)；冻后经体外培养，分别有2.8％和6.3％的卵母细胞发生卵丘扩散。
5．冷冻MⅡ期卵母细胞用鲜精受精后，仅有4.9％的受精卵发生卵裂，显著低于对照组的49.5％(P＜0.01)；发育至4细胞胚的比例为1.7％，无一能发育至8细胞期。
6．分别用冻精和鲜精与猪未经冷冻的MⅡ期卵母细胞进行体外受精，结果卵裂率分别为34.1％和49.5％(P＜0.05)；桑椹胚发育率分别为3.2％和13.3％(P＜0.05)。
7．TCM199(含15％FCS)、TCM199(含15％FCS)+颗粒细胞单层和NCSU-23三种培养系统均能支持猪4细胞期之前胚胎的发育，其卵裂率分别为44.7％，41.9％和49.1％(P＞0.05)。TCM199(含15％FCS)不能支持猪胚胎越过4细胞阻滞的发育；TCM199(含15％FCS)+颗粒细胞单层和NCSU-23能使猪胚胎发育至桑椹胚，但两组的8细胞发育率(14.3％和24.2％)和桑椹胚发育率(3.3％和11.7％)均有明显差异(P＜0.05)。表明NCSU-23培养液适用于猪早期胚胎的体外发育。
Porcine oocytes had been hard to be cryopreserved due to their high lipid content. The purpose of this study was to establish and optimize the procedures for the cryopreservation of porcine oocytes, which was significant either in theory or in practical use. In present study, porcine oocytes were collected from ovaries of slaughtered gilts in the slaughterhouse and used for cryopreservation and in vitro fertilization. Survival of oocytes was assessed by Trypan blue (TB) staining, Fluorescein diacetate (FDA) staining, maturation and fertilization in vitro and so forth. The effects of different freezing methods and different cryoprotectants on cryopreservation of porcine oocytes were investigated. Frozen or unfrozen oocytes and spermatozoa were respectively used to conduct in vitro fertilization and the impact of different culture systems on the developmental competence of porcine early stage embryos was also examined here. The results were shown as follows.
1. All of three cryoprotectants, EG, DMSO and Gly were effective to cryopreserve pig oocytes in programmed freezing, resulting in post-thawing survival rate of 33.8%, 25.8% and 23.5%, respectively, which were higher than that of control significantly (2.5%, P<0.01). Among these three cryoprotectants, EG group was superior to other two groups in survival rate significantly (33.8% vs 25.8%, 23.5%, P<0.05).
2. The self-made glass micropipettes (GMP) were adopted for oocyte freezing in pig and were confirmed to be a suitable and efficient method in vitrification of porcine oocytes. Compared with traditional programmed freezing method with plastic straws, GMP method could significantly improve the survival rate of porcine oocytes after freezing (34.5% vs 63.3%,P<0.05).
3. The vitrification solution carrier would affect the survival rate of oocytes. When EFS40 was used as vitrification solution, different carrier, straw and GMP, resulted in different survival rate significantly (45.0% and 65.9%, P<0.05).
4. Both programmed freezing method and vitrification method were available in cryopreservation of porcine GV stage oocytes. However, they yielded different survival rates (30.0% and 59.7%, P<0.05).
5. Vitrified MII stage oocytes were fertilized with fresh spermatozoa, producing only 4.9% cleavage embryos in 48h after culture. The cleavage rate derived from vitrified oocytes was significantly lower than that from control (49.5%, p<0.01). The subsequent developmental rate to 4-cell stage from frozen oocytes was 1.7% and none developed to 8-cell stage.
6. Matured oocytes in vitro were fertilized with frozen or fresh semen and then were cultured in vitro, resulting in different developmental percentage to 2-cell stage embryos (34.1% and 49.5%, P<0.05) and different developmental rate of morulae (3.2% and 13.3%, P<0.05).
7. All of three culture systems, TCM199 medium (containing 15%FCS), TCM199 (containing 15%FCS) + cumulus cell layer and NCSU-23 medium, could support the development of pig embryos before 4-cell stage with much similar cleavage rate (44.7%, 41.9% and 49.1%, P>0.05). TCM199 medium could not support the development of porcine fertilized eggs to pass the 4-cell stage block. TCM199 + cumulus cells and NCSU-23 medium could enable porcine embryos develop to morulae, but these two groups could yield significantly different developmental rate of 8-cell stage and morulae (14.3% vs 24.2% and 3.3% vs 11.7%, P<0.05), suggesting that NCSU-23 was more suitable to be used for the cultivation of early stage embryos in pig.
1．使用程序化冷冻法，三种冷冻保护剂EG、DMSO、Gly对猪卵母细胞均有冷冻保护作用，极显著高于对照组(冻后存活率33.8％，25.8％，23.5％ vs 2.5％，P＜0.01)；在该三种冷冻保护剂中，EG的效果最好，其冻后存活率显著高于另外两组(33.8％ vs 25.8％，23.5％，P＜0.05)。
2．采用自制的GMP管冷冻猪卵母细胞，证明所用GMP法是一种冷冻猪卵母细胞的有效方法，与普通麦管法相比，该法能显著提高猪MⅡ期卵母细胞冻后存活率(34.5％ vs 63.3％，P＜0.05)。
3．在玻璃化冷冻中，以EFS40为冷冻液，分别使用麦管和GMP管作冷冻载体，卵母细胞冻后存活率分别为45.0％和65.9％，两组间差异显著(P＜0.05)。
4．用程序化法和玻璃化法冷冻猪GV期卵母细胞，两组的冻后存活率分别为30.0％和59.7％，差异显著(P＜0.05)；冻后经体外培养，分别有2.8％和6.3％的卵母细胞发生卵丘扩散。
5．冷冻MⅡ期卵母细胞用鲜精受精后，仅有4.9％的受精卵发生卵裂，显著低于对照组的49.5％(P＜0.01)；发育至4细胞胚的比例为1.7％，无一能发育至8细胞期。
6．分别用冻精和鲜精与猪未经冷冻的MⅡ期卵母细胞进行体外受精，结果卵裂率分别为34.1％和49.5％(P＜0.05)；桑椹胚发育率分别为3.2％和13.3％(P＜0.05)。
7．TCM199(含15％FCS)、TCM199(含15％FCS)+颗粒细胞单层和NCSU-23三种培养系统均能支持猪4细胞期之前胚胎的发育，其卵裂率分别为44.7％，41.9％和49.1％(P＞0.05)。TCM199(含15％FCS)不能支持猪胚胎越过4细胞阻滞的发育；TCM199(含15％FCS)+颗粒细胞单层和NCSU-23能使猪胚胎发育至桑椹胚，但两组的8细胞发育率(14.3％和24.2％)和桑椹胚发育率(3.3％和11.7％)均有明显差异(P＜0.05)。表明NCSU-23培养液适用于猪早期胚胎的体外发育。
Porcine oocytes had been hard to be cryopreserved due to their high lipid content. The purpose of this study was to establish and optimize the procedures for the cryopreservation of porcine oocytes, which was significant either in theory or in practical use. In present study, porcine oocytes were collected from ovaries of slaughtered gilts in the slaughterhouse and used for cryopreservation and in vitro fertilization. Survival of oocytes was assessed by Trypan blue (TB) staining, Fluorescein diacetate (FDA) staining, maturation and fertilization in vitro and so forth. The effects of different freezing methods and different cryoprotectants on cryopreservation of porcine oocytes were investigated. Frozen or unfrozen oocytes and spermatozoa were respectively used to conduct in vitro fertilization and the impact of different culture systems on the developmental competence of porcine early stage embryos was also examined here. The results were shown as follows.
1. All of three cryoprotectants, EG, DMSO and Gly were effective to cryopreserve pig oocytes in programmed freezing, resulting in post-thawing survival rate of 33.8%, 25.8% and 23.5%, respectively, which were higher than that of control significantly (2.5%, P<0.01). Among these three cryoprotectants, EG group was superior to other two groups in survival rate significantly (33.8% vs 25.8%, 23.5%, P<0.05).
2. The self-made glass micropipettes (GMP) were adopted for oocyte freezing in pig and were confirmed to be a suitable and efficient method in vitrification of porcine oocytes. Compared with traditional programmed freezing method with plastic straws, GMP method could significantly improve the survival rate of porcine oocytes after freezing (34.5% vs 63.3%,P<0.05).
3. The vitrification solution carrier would affect the survival rate of oocytes. When EFS40 was used as vitrification solution, different carrier, straw and GMP, resulted in different survival rate significantly (45.0% and 65.9%, P<0.05).
4. Both programmed freezing method and vitrification method were available in cryopreservation of porcine GV stage oocytes. However, they yielded different survival rates (30.0% and 59.7%, P<0.05).
5. Vitrified MII stage oocytes were fertilized with fresh spermatozoa, producing only 4.9% cleavage embryos in 48h after culture. The cleavage rate derived from vitrified oocytes was significantly lower than that from control (49.5%, p<0.01). The subsequent developmental rate to 4-cell stage from frozen oocytes was 1.7% and none developed to 8-cell stage.
6. Matured oocytes in vitro were fertilized with frozen or fresh semen and then were cultured in vitro, resulting in different developmental percentage to 2-cell stage embryos (34.1% and 49.5%, P<0.05) and different developmental rate of morulae (3.2% and 13.3%, P<0.05).
7. All of three culture systems, TCM199 medium (containing 15%FCS), TCM199 (containing 15%FCS) + cumulus cell layer and NCSU-23 medium, could support the development of pig embryos before 4-cell stage with much similar cleavage rate (44.7%, 41.9% and 49.1%, P>0.05). TCM199 medium could not support the development of porcine fertilized eggs to pass the 4-cell stage block. TCM199 + cumulus cells and NCSU-23 medium could enable porcine embryos develop to morulae, but these two groups could yield significantly different developmental rate of 8-cell stage and morulae (14.3% vs 24.2% and 3.3% vs 11.7%, P<0.05), suggesting that NCSU-23 was more suitable to be used for the cultivation of early stage embryos in pig.
引文
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