摘要
本试验对应用胚胎工程技术提高奶牛繁育效率进行了系统的研究,包括MOET 技术、IVP 技术、性别控制技术以及引起奶牛繁殖障碍疾病的防治等几方面,得到如下结果与结论:
1 共超排供体奶牛1127 头次,回收胚胎共10900 枚,可用胚胎数量为6942 枚,平均每头次获得胚胎数量为6.73 枚。应用3 种超排处理方案,即PSO1、PSO2 和PSO3,超排成功率分别为89.50%、90.02%和98.54%,平均回收可用胚胎数分别为6.12 、5.98 和7.56 枚,利用发情周期9～11 d 的自然发情奶牛直接超排,效果最好。
2 对青年奶牛进行超排处理,应限制超排起始时间,15 月龄以上的青年奶牛超排效果较好;供体牛连续重复超排控制在3 次以内,连续4 次超排处理后极大降低胚胎可利用率;另外超排处理的间隔最短时间应该选择46～60 d;经产牛1～3 胎次超排效果较好,7 胎次以上差;产后间隔时间选取80～90 d 为宜。
3 供体奶牛采用性控精液进行超排处理,平均回收胚胎数为9.02 枚,平均可用胚胎为4.70 枚,受胎率与正常体内胚胎移植后的受胎率接近,说明采用性控精液生产性控胚胎可行。
4 研究中共处理受体牛5083 头,二次PG 法、CIDR 法和一次PG 法处理受体牛同期发情率分别为75%、78.33%和72%,受体移植后的妊娠率鲜胚达到55%以上,冻胚受胎率也在46%～47%,3 种处理方法结果相近。同期发情处理后青年受体牛比经产牛的利用率高,同时受体的营养水平严重影响受胎率。认为受体牛的同期发情以较为简便的二次PG 法处理为宜。自然发情受体的利用率达到85%,显著高于二次PG 法注射同期处理组,选取自然发情牛作为受体显著提高胚胎移植成功率。
5 采用直接解冻和分步解冻2 种方法解冻胚胎,移植后受体受胎率分别为45.22%和47.64%,表明以EG 作为冷冻介质冷冻保存胚胎并于35℃解冻后可以直接移植。
6 未孕受体牛以中医中药调理后进行同期发情处理,受体利用率显著提高,受胎率也有所增加;经产牛在产后20～25 d 应用GnRH 联合PG 调节激素水平,受体利用率和自然恢复组相近,而分娩到同期发情处理间隔缩短。
7 直径为3~6 mm的卵泡所获得卵母细胞体外成熟,受精率和囊胚形成率较高;在成熟液中添加10 ng/mL的EGF,利于核质成熟;卵母细胞成熟过程中,颗粒细胞保持至少3层以上,可以为其成熟提供必要条件,并能促进精子获能和正常受精。
8 Percoll法速度快,活精子数量多,但是多精子受精多,而上浮法回收精子数量少,可是精子的总体活力要高于Percoll法;在获能液中添加咖啡因对精子的活力和受精能力无显著的影响,甚至降低了精子的存活时间。
This dissertation studed systematically how to utilize embryo engineering technology to improve reproductive efficiency of dairy cows, including the techniques of MOET, IVP, sex control and the prevention and cure of reproductive disorder. The result showed:
1. 1127 donor cows were superovulated. The total number of recovered embryos was 10900 ,available embryos 6942,and average number of available embryos 6.73 per cow per time for successful superovulation. Three protocols including PSO1, PSO2 and PSO3 were applied in this experiment and the success rates of superovulation were 89.50%, 90.02% and 98.54%, respectively. The best result of superovulation was obtained in cows when initial injection of FSH directly without pre-treatment on day 9 to 11 of spontaneous estrus.
2. The age of the first time for superovulation of heifers should be restricted and optimal age of superovulation should be above the 15 months old. The continuous repeat superovulation to donor cows should be less than 3 times because the proportion of available embryos decreased evidently after treatment over 4 times. In addition, the interval between two superovulations should be 46 to 60 days. In multiparous cows, 1 to 3 parities were optimal to superovulation and it was not suitable for cows more than 7 parities. The interval between parturition and superovulation should be 80 to 90 days.
3. Superovulated donor cows were inseminated artificially by sexed semen. The average number of recovered embryos and available embryos was 9.02 and 4.70, respectively. The ET pregnancy rate of sexed embryos was closed to that of normal embryos. The result suggested that the sexed embryo might be produced by sexed semen.
4. In the study, 5083 recipient cows were treated by three synchronization procotols: double PG injection, CIDR+PG treatment and the single injection of PG. The synchronization rates were 75%, 78.33% and 72% correspondingly. The pregnancy rate of fresh embryo was 55% after embryo transfer, and that of frozen embryos was 46~47%, the result of the three groups was closed. The utilization rate of heifer recipients was higher than multiparous recipients after treatment of estrus synchronization. It was concluded that the protocol of double PG injection was better than the two others. However, the recipient utilization rate of spontaneous estrus was 85%, significantly higher than the double PG injection group, so the success rate of embryo transfer can be remarkably improved by utilizing recipient of spontaneous estrtus.
5. The frozen embryos were thawed by direct and programed methods, the pregnancy rate after embryo transfer was 45.22% and 47.64%, respectively. The result showed that the
embryos frozen by EG as freezing medium might be directly transferred after thawing in 35℃. 6. The recipient utilization rate and the pregnancy rate of nonpregnancy cows were improved after adjusting the reproductive functions with Chinese traditional medicine before synchronization. The utilization rate of parturient recipient cows treated by GnRH combined with PG during 20 to 25 days postpartum was closed to that of the natural recovered parturient cows, the interval between parturition and synchronization could be shortened by treatment of GnRH with PG. 7. The in vitro maturation rate, fertilization rate and blastocyst rate of oocytes from follicles with diameter of 3 to 6 mm were higher than that from others. Adding EGF 10 ng/mL to maturation medium benefitted the nuclei and cytoplasm maturation. In the process of the oocyte maturation, at least three-layer granule cells were necessary, and it could improve the sperm capacitation and fertilization. 8. In the sperm capacitation processes, the treatment time of Percoll method is shorter and the number of survival sperm is higher than that of swim-up method, but frequent polysperm was occurred. While swim-up method recovered few sperm but sperm viability is higher than Percoll method. Adding caffeine to capacitation medium had no significant effect on viability and fertilization capacity of sperm, even decrease the survival time of sperm. 9. The blastocyst rate was 12.0% and expanded blastocyst rate was 0% in protein-free medium (added PVA), the blastocyst rate and expanded blastocyst rate in media added BSA and FBS were 25.6%/28.0% and 10.0%/19.8%, respectively. Adding 2%Neaa and 1%Eaa to medium can significantly improve the development potential and the embryo quality. The embryos cultured in CR1aa+ 0.3%BSA medium for three days, then in SOFaa+5%FBS medium can get higher blastocyst rate and more embryo cells. Adding IGF-I to medium can improve the expanding potential of blastocyst. The co-culture system could improve the development potential of embryos (2.0% vs. 39.0%). 10. The fertilization rate of sexed sperm is slightly lower than that of the normal sperm when used for IVF, but there was no significant difference to the development potential of embryos between two groups. 127 sexed IVF embryos were transferred to 107 recipient cows, the pregnancy rate was 36.5%. 11. 998 in vivo embryos (including 136 fresh embryos and 835 thawed embryos) from normal sperm ,10 in vivo embryos from sexed sperm, 235 in vitro embryos from normal sperm and 116 in vitro embryos from sexed sperm were identified by consecutive and multiplex PCR. The pregnancy rate of identified in vivo fresh and thawed embryos from normal sperm was 41.03% and 37.72%,respectively, and from sexed sperm was 40%. The pregnancy rate of identified in vitro embryos from normal and sexed sperm was 27.78% and 25.00%, respectively. The coincident rate between the birth gender and identification
result was 97.85%. 12. Ovarian cysts are defined as anovulatory fluid-filled cystic structures ≧?25 mm in diameter that persist on the ovaries for more than 10 days. The cure rates of hormonal therapy with single GnRH injection, GnRH+PG injection, one treatment period of orally traditional Chinese medicine, GnRH+traditional Chinese medicine were 83.0%, 86.4%, 73.8% and 87.4%, respectively. The pregnancy rates by AI of cured cows of first estrus were 52.3%, 54.4%, 58.1% and 55.3%, respectively. 13. After two courses of treatment with the traditional Chinese medicine, the total cure rate of donor cows with hard tumidness formed from incomplete corpus luteum degeneration after superovulation was 91.3%. When cured cows were superovulated again, there was no significant difference of average available embryo number and available rate between cured cows and normal cows. Pregnant-urge perfusion fluid and hormonal therapy with PG injection for persistant corpus luteum had the best effect. The cure rates were 85.9% and 89.1% and the pregnancy rate by AI after two estruses of cured cows was 77.6% and 75.5%, respectively. 14. Four treatment groups of hypovaria were FSH+LH group, PMSG+hCG group, pregnant-urge perfusion fluid group and the traditional Chinese medicine taken group. The recovery rates were 75.6%, 63.8%, 73.8% and 82.5%, respectively, and the pregnancy rates of cured cows after two estruses were 62.9%, 58.2%, 65.7% and 67.3%, respectively. 15. The terramycin oil and pregnant-urge perfusion fluid were preferably for treatment of chronic endometritis in this experiment. The cure rates were 85.4% and 84.8%, and the pregnancy rates by AI of cured cows after two estruses were 83.9% and 82.0%, respectively. The cure rates of PG injection group and traditional Chinese medicine taken group were 76.3% and 79.4%, and the pregnancy rate by AI of cured cows after two estruses was 78.9% and 84.7%, respectively. 16. Three treatments were taken for prevention of embryonic loss. The pregnancy rate of the control group, P4 supplementation group, hCG group and the traditional Chinese medicine taken group after AI was 53.3%, 56.1%, 62.1% and 58.3%, respectively; the rate after embryo transfer was 43.3%, 49.2%, 53.3% and 55.3%, respectively. The pregnancy rates of the treated groups were improved.
引文
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