波尔山羊胚胎移植及配子移植研究
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摘要
波尔山羊原产于南非，是目前世界上最优秀的肉用山羊品种。它具有体形大、适应性强、生长速度快，产肉率高、抗病力强等特点，在世界山羊品种改良中起着非常重要的作用。而目前波尔山羊的数量尚少，发展太慢远远跟不上生产的需求。运用超数排卵-胚胎移植技术可以加速国内波尔山羊扩大种群。由于受生殖器官解剖学特点的限制，至今尚未建立一种令人满意的山羊超数排卵、胚胎采集、移植方法。本研究对胚胎移植中超数排卵、腹腔镜检查的准确性、胚胎采集与移植方法等方面进行了探讨，试图建立起一套完整的胚胎移植程序，为以后分割胚胎、细胞核移植、体外受精、转基因动物等技术建立基础。
论文首先对波尔山羊胚胎移植方法进行探讨。用三种方法对供体波尔山羊进行超数排卵处理。方法一，供体羊D0天置栓同时注射雌二醇，D12天开始用国产FSH进行超排连续四天，D13天下午撤栓，FSH使用总剂量为280IU．方法二，供体羊不用栓处理，于D0天注射PG0.6ml／只，D15天开始用国产FSH进行超排连续四天，于D17天下午注射PG0.6ml／只，FSH使用总剂量为280IU．方法三，供体羊于D0天置进口栓CIDR，于D12天换栓，D12天下午开始超排，超排所用FSH为进口试剂，一种澳大利亚产，所用剂量为25NIH／只，另一种加拿大产，所用剂量为300ms／只。通过腹腔镜技术检查供体羊卵巢超排情况，其中方法一、二超数排卵效果不佳，与不用激素处理没有差异。方法三中，用加拿大产FSH共处理5只供体羊，其中有三只回收到的是未受精卵，其余两只共回收到20枚胚胎，移植到受体徐淮白山羊体内，最后仅产仔2只，表明卵母细胞的受精质量不好，这可能与所用激素剂量过大有关。用澳大利亚产FSH处理的7只供体羊，共回收到可用胚胎91枚，移植到54只受体羊中，最后产仔43只，出生率达47.3％。取得了较好的结果。
尝试利用配子输卵管移植技术解决胚胎移植中遇到的波尔山羊不育问题。将从两只供体波尔山羊采集到的9枚卵母细胞和获能精子移植到4头受体徐淮白山羊中，另30枚采自当地羊和杂交波尔山羊的卵母细胞移植到8只受体中，结果9枚波尔山羊卵母细胞移植受体羊均未受孕，而当地羊和杂交羊卵母细胞移植受体羊有三只受孕产羔，共产仔5只，配子受精发育率为13％，初步验证了配子移植术在山羊上开展的可能性。
硕士学位论文 徐志鹏：波尔山羊胚胎移植与配子移植研究
前次配于移植研究结果未能有纯种波尔山羊出生，这可能与卵母细胞移植到
输卵管中时的成熟状态以及精子活力情况有关。因此我们将采得的波尔山羊卵母
细胞进行成熟培养，成熟培养系统采用TCM199＋10pg／ml FSH＋20pg／ml LH
＋lpg／m 176－EZ＋10％ EGS，共采集到9枚卵母细胞，26’J＼时后，有6枚卵丘细胞扩
散良好，判断为成熟。精子于含有咖啡因和肝素钠的培养系统中进行获能处理，
将成熟培养后的卵母细胞和获能精于一起移植到四只受体羊的输卵管中，结果有
两只羊已经妊娠。初步表明卵母细胞的成熟培养有利于提高配子移植的成功率。
本研究中成功的建立了波尔山羊胚胎移植及配子移植系列技术，为以后大规
模的开展胚胎移植工作奠定基础，同时，为开展山羊胚胎工程其它研究提供借鉴。
Boer goat, being from South Africa, is considered to be the premier goat meat producing breed. It owns so many good traits as large form size, high adaptability to environmental conditions and high fertility, and it play the important role in goat breeding process. But the number of Boer goat holds back the development of the meat goat industry. Now superovulation and embryo transferring techniques are used to solve this problem. But the results of it are various and no succeeding progress is formed. In our research, we focused our study on such aspects as superovulation, laparoscopic inspecting, embryo recovering and embryo transferring , and tried to establish a good procedure of embryo transfer. These will make the base to the next research in nuclear transferring, embryo splitting, in vitro fertilization and transgenic animal production.
In this paper, we studied the techniques of embryo transferring at first. In the embryo transfer experiments on Boer goat, three methods were used. In method one, the donor goats were plugged with progestagen sponge in DO, and at the same time, estradiol was injected. Then a uniform cohort of follicles is recruited by the administration of FSH eight times beginning at D12 (total does of 280IU), the progestagen sponge were removed in D13. In the method two, the donor goats were administrated with PG (does, 0.6ml per goat), then FSH was used for superovulation beginning at D15(total does of 280IU), PG was admistrated at D17(does,0.6ml per goat), FSH being used of method one and two was from native .In the method three, CIDR was plugged to improve superovulation at DO, then at D12 fresh CIDR was used to change the old one. In the afternoon of D12, The donor was administrated with FSH for superovulation. In this method , two kinds of FSH were used ,one is from Australia (total dose 25NIH per goat),the other from Canada (total dose 300mg per goat). Laparoscopic was used to check the status of donor's ovary after superovulation. We found that the result of method one and two is so poor, and FSH we used may be with this result. In the method three, 5 donors were superovulated with FSH from Canada. Fertilization failure was found in three goats. We recovered
-6-
20 embryos from the other two, and then we transfer these embryos into native goat. 5 months later, 2 baby goats were born. We can conclude that the fertilization rate maybe with the dose of FSH used for superovulation.7 donors were superovulated with FSH from Australia, and 91 embryos were recovered, then we transferred these embryos into 54 recipients. 40 Boer goats were born, and the birth rate was 43.9%.
In the second experiment, we tried to use the gamete intrafallopian transfer technique to solve the infertile problem in the goats. 9 oocytes were recovered from 2 donor Boer goats and they were transferred into 4 recipients, the result shows no recipient of it was pregnant. The other 30 oocytes were recovered from native goats and hybrid goats. They were transferred into 8 recipients, 3 of them were pregnant, and 5 goats were born, the development rate of oocytes was 13%. From this experiment, the possibility of GIFT in the goat is proved.
No gamete developed into baby Boer goat in the last GIFT procedure. This may has the relationship with the status of oocytes maturation. So we cultured the oocytes before transfer, and the culture system was TCM199 (1 Dug/ml FSH +20ug/ml LH +lug/m 1 1?P-E2+10% EGS). 9 oocytes were recovered from two donors .26 hours later, the cumulus cells of six oocytes spended well and We defined they were matured. Sperm were capacitated in the capacitation medium with heparin and coffein. Then the matured oocytes and capacitation sperm were transferred into 4 recipients fallopians . Now, two of them were pregnant. So we can conclude that the oocytes maturation can improve the success rate of GIFT procedure.
In our research, we established a series techniques about embryo transfer and gamete intrafallopian transfer. These can serve for embryo transfer in large scale and will provide
论文首先对波尔山羊胚胎移植方法进行探讨。用三种方法对供体波尔山羊进行超数排卵处理。方法一，供体羊D0天置栓同时注射雌二醇，D12天开始用国产FSH进行超排连续四天，D13天下午撤栓，FSH使用总剂量为280IU．方法二，供体羊不用栓处理，于D0天注射PG0.6ml／只，D15天开始用国产FSH进行超排连续四天，于D17天下午注射PG0.6ml／只，FSH使用总剂量为280IU．方法三，供体羊于D0天置进口栓CIDR，于D12天换栓，D12天下午开始超排，超排所用FSH为进口试剂，一种澳大利亚产，所用剂量为25NIH／只，另一种加拿大产，所用剂量为300ms／只。通过腹腔镜技术检查供体羊卵巢超排情况，其中方法一、二超数排卵效果不佳，与不用激素处理没有差异。方法三中，用加拿大产FSH共处理5只供体羊，其中有三只回收到的是未受精卵，其余两只共回收到20枚胚胎，移植到受体徐淮白山羊体内，最后仅产仔2只，表明卵母细胞的受精质量不好，这可能与所用激素剂量过大有关。用澳大利亚产FSH处理的7只供体羊，共回收到可用胚胎91枚，移植到54只受体羊中，最后产仔43只，出生率达47.3％。取得了较好的结果。
尝试利用配子输卵管移植技术解决胚胎移植中遇到的波尔山羊不育问题。将从两只供体波尔山羊采集到的9枚卵母细胞和获能精子移植到4头受体徐淮白山羊中，另30枚采自当地羊和杂交波尔山羊的卵母细胞移植到8只受体中，结果9枚波尔山羊卵母细胞移植受体羊均未受孕，而当地羊和杂交羊卵母细胞移植受体羊有三只受孕产羔，共产仔5只，配子受精发育率为13％，初步验证了配子移植术在山羊上开展的可能性。
硕士学位论文 徐志鹏：波尔山羊胚胎移植与配子移植研究
前次配于移植研究结果未能有纯种波尔山羊出生，这可能与卵母细胞移植到
输卵管中时的成熟状态以及精子活力情况有关。因此我们将采得的波尔山羊卵母
细胞进行成熟培养，成熟培养系统采用TCM199＋10pg／ml FSH＋20pg／ml LH
＋lpg／m 176－EZ＋10％ EGS，共采集到9枚卵母细胞，26’J＼时后，有6枚卵丘细胞扩
散良好，判断为成熟。精子于含有咖啡因和肝素钠的培养系统中进行获能处理，
将成熟培养后的卵母细胞和获能精于一起移植到四只受体羊的输卵管中，结果有
两只羊已经妊娠。初步表明卵母细胞的成熟培养有利于提高配子移植的成功率。
本研究中成功的建立了波尔山羊胚胎移植及配子移植系列技术，为以后大规
模的开展胚胎移植工作奠定基础，同时，为开展山羊胚胎工程其它研究提供借鉴。
Boer goat, being from South Africa, is considered to be the premier goat meat producing breed. It owns so many good traits as large form size, high adaptability to environmental conditions and high fertility, and it play the important role in goat breeding process. But the number of Boer goat holds back the development of the meat goat industry. Now superovulation and embryo transferring techniques are used to solve this problem. But the results of it are various and no succeeding progress is formed. In our research, we focused our study on such aspects as superovulation, laparoscopic inspecting, embryo recovering and embryo transferring , and tried to establish a good procedure of embryo transfer. These will make the base to the next research in nuclear transferring, embryo splitting, in vitro fertilization and transgenic animal production.
In this paper, we studied the techniques of embryo transferring at first. In the embryo transfer experiments on Boer goat, three methods were used. In method one, the donor goats were plugged with progestagen sponge in DO, and at the same time, estradiol was injected. Then a uniform cohort of follicles is recruited by the administration of FSH eight times beginning at D12 (total does of 280IU), the progestagen sponge were removed in D13. In the method two, the donor goats were administrated with PG (does, 0.6ml per goat), then FSH was used for superovulation beginning at D15(total does of 280IU), PG was admistrated at D17(does,0.6ml per goat), FSH being used of method one and two was from native .In the method three, CIDR was plugged to improve superovulation at DO, then at D12 fresh CIDR was used to change the old one. In the afternoon of D12, The donor was administrated with FSH for superovulation. In this method , two kinds of FSH were used ,one is from Australia (total dose 25NIH per goat),the other from Canada (total dose 300mg per goat). Laparoscopic was used to check the status of donor's ovary after superovulation. We found that the result of method one and two is so poor, and FSH we used may be with this result. In the method three, 5 donors were superovulated with FSH from Canada. Fertilization failure was found in three goats. We recovered
-6-
20 embryos from the other two, and then we transfer these embryos into native goat. 5 months later, 2 baby goats were born. We can conclude that the fertilization rate maybe with the dose of FSH used for superovulation.7 donors were superovulated with FSH from Australia, and 91 embryos were recovered, then we transferred these embryos into 54 recipients. 40 Boer goats were born, and the birth rate was 43.9%.
In the second experiment, we tried to use the gamete intrafallopian transfer technique to solve the infertile problem in the goats. 9 oocytes were recovered from 2 donor Boer goats and they were transferred into 4 recipients, the result shows no recipient of it was pregnant. The other 30 oocytes were recovered from native goats and hybrid goats. They were transferred into 8 recipients, 3 of them were pregnant, and 5 goats were born, the development rate of oocytes was 13%. From this experiment, the possibility of GIFT in the goat is proved.
No gamete developed into baby Boer goat in the last GIFT procedure. This may has the relationship with the status of oocytes maturation. So we cultured the oocytes before transfer, and the culture system was TCM199 (1 Dug/ml FSH +20ug/ml LH +lug/m 1 1?P-E2+10% EGS). 9 oocytes were recovered from two donors .26 hours later, the cumulus cells of six oocytes spended well and We defined they were matured. Sperm were capacitated in the capacitation medium with heparin and coffein. Then the matured oocytes and capacitation sperm were transferred into 4 recipients fallopians . Now, two of them were pregnant. So we can conclude that the oocytes maturation can improve the success rate of GIFT procedure.
In our research, we established a series techniques about embryo transfer and gamete intrafallopian transfer. These can serve for embryo transfer in large scale and will provide
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